Prog. Lipid Res. Vol. 30, No. 2/3, pp. 219-230, 1991 Printed in Great Britain. All rights reserved

0163-7827/91/$0.00+ 0.50 © 1991 PergamonPress pie

SYNTHESIS AND SECRETION OF NASCENT LIPOPROTEIN PARTICLES NASSRIN DASHTI* Lipoprotein and Atherosclerosis Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, U.S.A.

CONTENTS I. II. III. IV.

ABe~aaONS INTRODUCTION Hel~A~CAND Iwrv.snNALSv.ca~-nONoF NAson,rr LIPOPROTEINS NUTmTIONALR~ULATXONoF LIPOI'ROT~NS~TH~IS AND SECaE'nON STUDmSIN HUMANHEPATOBLASTOMAHepG2 ANDADL~OC~CXNOMACaco-2 CELL LIN~S A. Studies in HepG2 cells I. Regulation of lipoprotein production by HepG2 cells (a) Effect of fatty acids on lipoprotein production by HepG2 cells Co) Effect of cholesterol on lipoprotein production by HepG2 cells (c) Effect of insulin on lipoprotein production by HepG2 cells (d) Effects of oleate, cholesterol and insulin on the levels of apolipoprotein mRNAs in HepG2 cells 2. Studies on the isolation and characterization of lipoprotein particles secreted by HepG2 cells (a) Isolation and characterization of ApoB-containing lipoprotein particles produced by HepG2 cells Co) Isolation and partial characterization of apolipoprotein A-containing lipoprotein particles produced by HepG2 cells B. Studies in Caco-2 cells I. Effect of oleate on the production of lipoproteins by Caco-2 cells 2. Production of APOA- and ApoB-containing lipoprotein particles by Caco-2 cells ACr~OWWJX~E~'TS ReFrJ~NCES

219 219 220 220 222 222 222 222 223 223 224 224 224 225 226 226 227 228 228

ABBREVIATIONS Apo--apolipoprotein VLDL--very low density fipoproteins LDL--low density lipoproteins HDL---high density lipoproteins VHDL--very high density lipoproteins LP--lipoprotein LCAT--lecithin:cholesterol acyltransferase anti--antibody

I. I N T R O D U C T I O N

Although numerous in vivo and in vitro studies have been conducted on the production of hepatic and intestinal lipoproteins in many animal species including the rat, rabbit, guinea pig and nonhuman primates, the lack of an adequate experimental model has hindered similar studies in humans. However, recent studies on the characterization of human hepatoblastoma cell line HepG2 and colonic adenocarcinoma cell line Caco-2 have shown that these cell lines represent useful models for studying the chemical nature and metabolism of human hepatic and intestinal lipoproteins, respectively. The human-derived *Corresponding address: Oklahoma Medical Research Foundation, 825N.E. 13th Street, Oklahoma City, OK 73104, U.S.A. 219

220

N. D~m'1

HepG2 and Caco-2 cell lines have been shown to retain many of the normal biochemical functions of human liver parenchymal and intestinal cells, respectively, including the ability to synthesize and secrete all major human plasma apolipoproteins (Apo) and lipoproteins. These cell lines have successfully been used for establishing the regulatory role of lipids and hormones in the synthesis and secretion of apolipoproteins and lipoproteins and for studying the catabolism of plasma lipoproteins. HepG2 cells, and to a lesser extent, Caco-2 cells have been used to investigate the chemical nature of ApoB- and ApoA-Icontaining lipoproteins. The following discussion will summarize information regarding the regulation of hepatic and intestinal lipoprotein production and then will focus on lipoprotein metabolism in HepG2 and Caco-2 cells. II. H E P A T I C A N D I N T E S T I N A L S E C R E T I O N OF NASCENT LIPOPROTEINS

The liver and intestine are the major sites of biosynthesis, assembly, and secretion of plasma-destined lipoproteins. The secretion of lipoproteins by the liver and intestine occur both in the form of triglyceride-rich very low density lipoproteins (VLDL) and cholesterol-rich high density lipoproteins (HDL). Studies in several animal species 32,52'Ss,6s,sl,s9,u3 have demonstrated that VLDL are the major lipoproteins produced by the liver, although VLDL-independent; direct synthesis of low density lipoproteins (LDL) in the rat, 42 miniature pig, 59 nonhuman primates~s.62.6s,69 as well as in humans 63.9° has also been demonstrated. The nascent hepatic VLDL and LDL are triglyceride-rich and cholesteryl-ester poor with apolipoproteins B and E as the major and ApoC as only a minor protein constituent. 35'5BIt appears that the nascent VLDL begin to acquire the ApoC peptides during their passage through the Golgi apparatus? 5 The secretion of both spherical and discoidal HDL particles by perfused livers of rat, 4~ guinea pig, s2 and nonhuman primates, 6s and rat bepatocytes in suspension76 and in culture7 has been demonstrated. Whereas spherical HDL particles contain primarily apolipoproteins A-I and A-II, the discoidal particles are enriched in ApoE and are deficient in cholesteryl esters? m In addition to the liver, the enterocyte is a significant source of fasting plasma VLDL and postprandial chylomicrons and VLDL. s The presence of apolipoproteins A-I, A-IV, B and C peptides have been demonstrated in rat and human enterocytes,s There is little evidence for the active synthesis of ApDE in the intestine of most animal species, s Normal rat intestinal Golgi VLDL are triglyceride-rich and contain apolipoproteins A-I, A-IV and B) °9 It has been suggested that these triglyceride-rich lipoproteins gain ApoC peptides from HDL after secretion from the enterocytes,s On the other hand, studies by Alpers e t al. 3 have shown that in enterocytes, ApoB and ApoC-III are associated with particles corresponding to chylomicrons, VLDL, LDL and HDL indicating that these apolipoproteins could be associated with the lipoproteins in various stages of assembly. It appears ~16 that chylomicrons and VLDL are differentially assembled by the small intestinal enterocytes. III. N U T R I T I O N A L R E G U L A T I O N OF L I P O P R O T E I N S Y N T H E S I S AND SECRETION

The plasma concentration of lipoproteins are markedly influenced by the nutritional state of the animal. The effects of dietary cholesterol and fatty acids on plasma lipoproteins and their relevance to hyperlipidemia have been reviewed.~'s It is generally agreed that, in humans, increased dietary cholesterol and saturated fat results in elevated LDL cholesterol35.1°2On the other hand, substitution of polyunsaturated fat for saturated fat causes a reduction in LDL cholesterol, both in normal subjects and hyperlipidemic patients. 49'1°4It has also been demonstrated that, in humans, the co-3 fatty acids are more effective in lowering the plasma concentration of VLDL and LDL than co-6 fatty acids, s In addition, polyunsaturated fatty acids have been shown to induce compositional

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221

changes in LDL, producing particles which contain a lower percentage of cholesterol and a higher percentage of phospholipids. ~°3 Although saturated and polyunsaturated lipids produced similar size chylomicrons, the ApoB content of polyunsaturated linoleate chylomicrons was significantly lower than that of palmitate chylomicrons.97 Cholesterol-rich diets result in a marked hypercholesterolemia and increased serum concentration of apolipoproteins B and E in a variety of species)° In the human subjects, cholesterol feeding results in increased plasma LDL levels ~4as well as altered VLDL and LDL composition. 14'49't°3 In contrast to VLDL and LDL from normal rat liver perfusate which consisted of a complex lipoprotein B: E :C, 3s the corresponding particles from cholesterol-fed rats only contained the lipoprotein B: E, 35In hypercholesterolemic rats, the intestinal Golgi VLDL particles contain a higher level of cholesterol, relatively more ApoA-IV and less ApoB than their normal counterparts. ~°9 A decreased synthetic rate and increased fractional catabolic rate of LDL-APOB during polyunsaturated fat feeding appear to be general trends in both normal and hyperlipidemic subjects, 49 implicating the direct roles of liver and intestine in this process. This is supported by a variety of studies demonstrating the stimulatory effect of long chain fatty acids on the rate of secretion of both hepatic 21'33'73 and intestinal7° VLDL. The effect of fatty acids on ApoB secretion is, however, controversial. Studies in human subjects, ~ in liver perfusate of nonhuman primates, ¢'~ and in cultured rat 3m'gt and chicken~°5 hepatocytes have demonstrated that the increase in VLDL triglyceride production was not accompanied by similar changes in ApoB secretion. In contrast, Salam e t al. 99 have reported simultaneous stimulation by oleic acid in both triglycerides and ApoB secretion by perfused rat liver. High dietary fat has also been shown to cause an increase in circulating ApoE level in mice. 98 However, this does not appear to be due to increased hepatic synthesis since addition of fatty acid to rat cultured hepatocytes3t,9~ did not change ApoE synthesis. The intestinal secretion of apolipoproteins A-I and B in response to fatty acid administration is also controversial. Studies in the rat and in human subjects have demonstrated both increased47'95't°°'l°l and decreased2 synthesis and secretion of APOA-I by the intestine in response to dietary fat. An increase in ApoA-I synthesis in rat jejunal enterocytes in response to acute triglyceride administration was observed only following prolonged dietary triglyceride withdrawal, s8 In the rat, both increased97'm°°'"7and an unchanged29rate of ApoB secretion by the intestine subsequent to fat feeding have been reported. Similarly, both increased secretion5~ and decreased intracellular content94 of APOB have been observed in human subjects after fat feeding. In addition to ApoA-I and APOB, the intestinal APOE and APOA-IV which are associated with triglyceride-rich lipoproteins may also be influenced by dietary fat. The observed increase in human plasma Apo-IV concentration in response to fat feeding9 may reflect increased intestinal synthesis of ApoA-IV. 4'51 Cholesterol feeding has a marked effect on hepatic and intestinal lipoprotein synthesis. Studies by Kosykh e t al. 75 have demonstrated that exposure of human hepatocytes to cholesterol resulted in increased ApoB secretion. On the other hand, Davis and MaloneMcNea134 have reported the lack of effect of dietary cholesterol on the synthesis of apolipoproteins B and E by rat hepatocytes. In the rat intestine, cholesterol absorption was shown to be negatively correlated with jejunal APOB-48 synthesis,3° whereas a high cholesterol diet stimulated the APOE synthesis. H2 The above studies clearly demonstrate that the synthesis and secretion of lipoproteins are regulated by dietary fatty acids and cholesterol. Some of the variabilities in the results can be attributed to the numerous differences in the experimental details of the above studies. In addition, studies with human subjects have been mostly conducted in vivo. Investigations of the regulation of lipoprotein production by liver or intestine in intact animals are often complicated by activities of, and interaction with, other organ systems. The HepG2 and Caco-2 cell culture models representing human liver and intestine, respectively, thus provide potentially useful systems for studying the direct, long-term actions of individual fatty acids and cholesterol and hormones on lipoprotein production.

222

N. DA.m'n IV. STUDIES IN HUMAN HEPATOBLASTOMA HepG2 AND ADENOCARCINOMA Caco-2 CELL LINES

A. Studies in HepG2 Celb The human hepatoblastoma cell line HepG2 has been shown to retain many of the normal biochemical function of human liver parenchymal cells. HepG2 cells maintain the morphological characteristics and epithelial cell shape comparable to those of liver parenchymal cells and secrete 17 major plasma proteins, r~ The synthesis and secretion of human plasma apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III and E, as integral components of lipoproteins, by HepG2 cells have been reported. 2s-~,37,5°,~,H4,12° The synthesis of lecithin:cholesterol acyltransferase (LCAT) n'74 and cholesteryl ester transfer protein 39'~°sby HepG2 cells have also been reported. In addition, we have demonstrated the presence of Apo(a) in HepG2 culture medium,as The HepG2 cells have been used to study the kinetics of synthesis and secretion of ApoB-100 z°,n which is the only form of ApoB secreted by these cells. 2°'~ The HepG2 cell line has successfully been used for establishing the regulatory role of lipids, mt'2s'36'9~'~'H4insulin2s,93 and estrogen5,6,n°,m in the synthesis and secretion of lipoproteins and ApoB gene expression. These studies have demonstrated that the HepG2 cells the lipid supply, in particular cholesterol) 6 plays an important role in initiating the synthesis of apolipoproteins, specifically ApoB. Several studies from this laboratory ms'm9and of others 13'4°'82 have demonstrated the secretion of distinct ApoA-I- and ApoB-containing lipoprotein particles. In addition to lipoproteins, the synthesis and secretion of several bile acids by HepG2 cells have been demonstrated.38'65'66 Numerous studies have been reported on the use of HepG2 cells for investigating the uptake and catabolism of plasma lipoproteins. These studies which include the removal of chylomicron remnants and p-VLDL, ~5'79VLDL, u LDL 27;s5'56 and HDL 26's7 have been summarized in a recent review by Javitt. ~ The focus of the following discussion is to summarize our studies on lipoprotein metabolism by HepG2 cells and the regulatory role of fatty acids and cholesterol in the production of lipoprotein particles.

1. Regulation of Lipoprotein Production by HepG2 Cells (a) Effect of fatty acids on lipoprotein production by HepG2 cells. We have demonstrated 25that in HepG2 cells grown in basal (control) medium triglycerides were the major neutral lipids and accounted for 57%, while unesterified cholesterol accounted for 31%, and cholesteryl esters for 12% of the total neutral lipids. The low concentration of cholesteryl esters in the culture medium, in comparison to that in human plasma, reflects the low activity of LCAT in the culture medium.74 ApoB was the major apolipoprotein produced by HepG2 cells under control experimental condition, followed by ApoA-I, ApoE, ApoA-II and ApoC-III. The large molecular weight form of ApoB (ApoB-100) was the only form of this apolipoprotein in HepG2 culture medium3° The major lipoprotein density class produced by HepG2 in the control medium was LDL. Both VLDL and LDL were triglyceride-rich which accounted for 62-66% of the total neutral lipids. ApoB was the major apolipoprotein of both VLDL and LDL and accounted for 51% and 84% of the total apolipoprotein contents of VLDL and LDL, respectively. The addition of oleate to culture medium resulted in a 2-fold increase in the accumulation of triglycerides in the culture medium, without significantly altering total cholesterol production. The increase in triglyccrides, however, was not accompanied by parallel changes in the secretion of apolipoproteins, specifically ApoB3 5 Addition of oleate caused a marked increase in the production of lipoproteins, mainly in the form of VLDL which was increased 4-fold and contained a higher percentage of ApoB and ApoE. The increase in VLDL production, in response to fatty acid substrate observed in our laboratory, is in agreement with studies reported by other investigators demonstrating the accumulation of cytoplasmic lipid storage droplets in the cells n4 and increased VLDL secretion~ by HepG2 cells incubated with fatty acids.

Nascent fipoproteinparticles

223

The inability of oleate to stimulate the net accumulation of apolipoproteins, specifically ApoB in HepG2 culture medium observed in our laboratory, is consistent with studies by Ellsworth et al., 3~ Thrift et al. "( and Craig et al. ~6 Similar results have been obtained from studies in human subjects, u in liver perfusate of nonhuman primates t°~ and cultured rat hepatocytes.3t'91 On the other hand, studies by PuUinger et al. 93 and Moberly et al. 86 have shown enhanced APOB secretion by addition of oleate to HepG2 cells. Although these variable results may possibly b e explained by differences in the culture conditions, fatty acid concentration, and as demonstrated recentlyI the age of the cells, the exact factors responsible for these variations require further investigation. Many studies conducted in human subjects have shown that high dietary intake of saturated fat leads to increased concentration of LDL and ApoB. ~4,s5 Conversely, substitution of polyunsaturated fatty acids, specifically (.0-3 fatty acids for saturated fat in the diet results in decreased LDL cholesterol and ApoB. ~ 5 Using HepG2 cells, Stein et al.l°7 demonstrated that cholesterol release into the serum-free culture medium was significantly lower in cells supplemented with linoleic acid, when compared to palmitic or oleic acid. Furthermore, the secretion of VLDL triglycerides and APOB by HepG2 cells has been shown to be markedly inhibited by eicosapentaenoic acid in comparison to oleic and linoleic acid. Hs'H9 Thus, the above studies clearly establish the responsiveness of HepG2 cells to exogenous fatty acids. (b) Effect o f cholesterol on lipoprotein production by H e p G 2 cells. To examine whether the changes in the dietary cholesterol affect the synthesis and secretion of ApoB-containing lipoproteins, HepG2 cells were incubated with exogenous cholesterol supplied in various forms. Cells were preincubated for 24 hr with plasma LDL at concentration providing 100/~g cholesterol/nil of medium. After removing the preincubation medium and rinsing the monolayers, the secretion of lipoproteins into lipid-free medium during the following 17 hr incubation was determined. Results showed that this preincubation with LDL had no significant effect on cellular unesterified cholesterol, but it caused a 2-fold increase in the cholesteryl ester content. This was reflected in a significant increase of 2-fold in the concentration of cholesteryl esters in the medium; the tdglyceride content was also increased by 44%. Whereas APOA-I and APOA-II were not altered, cholesterol caused a 2-fold increase in the secretion of both APOB and APOE. To eliminate the possibility that the observed changes may partially be due to residual exogenous LDL, cells were also incubated with 25-hydroxycholesterol (10/~g/ml of medium). As reported previously, 17 25-hydroxycholesterol was highly effective in raising the cellular cholesteryl esters by 3-fold and this was associated with a 3-fold increase in the concentration of cholesteryl esters and a 2- to 3-fold increase in that of APOB in the culture medium. 17There was also a moderate increase (38%) in APOE concentration in the medium. These results demonstrate that, in contrast to oleate, cholesterol stimulates the secretion of ApoB-containing lipoproteins by HepG2 cells. Similar elevation in the accumulation of cholesteryl esters in HepG2 cells and culture medium37as well as increased secretion of APOB into the medium37,¢ have been observed after addition of various forms of cholesterol. The increased secretion of ApoE by cholesterol-rich liposomes reported by Craig et al. 16 is consistent with our results. However, in contrast to our studies 17and those by Craig et al., 16 Fuki et al. 46 did observe an increase in APOB secretion by pure cholesterol. The exact reason for this difference remains to be established. (c) Effect o f insulin on lipoprotein production by H e p G 2 cells. We have used HepG2 cells to assess the effect of insulin on lipoprotein production. Results demonstrated25 that incubation of HepG2 cells with insulin caused a significant decrease in the secretion of neutral lipids and apolipoproteins, particularly triglycerides and ApoB. In addition to a 60-68% reduction in the total concentration of VLDL and LDL, insulin altered the composition of these lipoproteins by producing particles that had significantly lower content of triglycerides, contained less APOB, and were deficient in APOE. There were no major changes in the concentration or composition of HDL particles. The increased

224

N. D~SHTI

accumulation of triglycerides in HepG2 cells concomitant with their reduced levels in the medium suggested that insulin may affect the secretion rather than the synthesis of triglyceride-rich lipoprotein particles. (d) Effects of oleate, cholesterol and insulin on the levels of apolipoprotein mRNAs in HepG2 cells. We have recently reported the concentrations of mRNA for apolipoproteins A-I, A-II, B and E as influenced by oleate and insulin, z3 These studies have demonstrated that the lack of any significant effect of oleate on the accumulation of ApoB in the culture medium was paralleled with its unchanged mRNA level. 23These data confirm our previous study demonstrating that increased triglyceride secretion by HepG2 cells in response to fatty acid provision was not accompanied by similar changes in apolipoprotein, specifically ApoB synthesis.25 Furthermore, these data 23 demonstrated that inhibition of ApoB secretion upon addition of insulin to HepG2 cell was not due to the decrease in its mRNA level. Thus, the unchanged ApoB mRNA concentration under conditions that modulate ApoB output demonstrated by our studies,23and reported by others, 86'93'H9implicates that, in addition to possible transcriptional regulation, the changes in mRNA translability and/or post-translational modification of ApoB, resulting in altered assembly and secretion of ApoB-containing lipoproteins, may be operative in HepG2. Recent studies from this laboratory 17have established that the enhanced secretion of ApoB caused by incubation of HepG2 cells with 25-hydroxycholesterol is, in part, due to increase in the level of ApoB mRNA. In contrast to studies by Monge et aL87we have not detected any increase in the level of ApoA-I mRNA by cholesterol.

2. Studies on the Isolation and Characterization of Lipoprotein Particles Secreted by HepG2 Cells Most of the reported studies on the hepatic lipoproteins have been carried out using major lipoprotein density classes including VLDL, LDL and HDL. Ultracentrifugal fractionation is a very convenient procedure for the isolation of relatively large amounts of lipoproteins of any desirable hydrated density range, but it is also a procedure most likely to result in the formation of artifacts77 by causing dissociation of apolipoproteins from lipoprotein particles. To surmount this adverse effect of centrifugation, we have undertaken studies on the identification, isolation and characterization of lipoprotein particles secreted by, and accumulated in, the culture medium of HepG2 cells using sequential immunoprecipitation and immunoaffinity chromatography. (a) Isolation and characterization of ApoB-containing lipoprotein particles produced by HepG2 cells. To separate ApoB-containing lipoproteins, secreted lipoproteins were fractionated either by sequential immunoprecipitation or immunoaffinity chromatography with antibodies to ApoB and ApoE. ~s Results showed that 60-70% of ApoB occurred in the culture medium as lipoprotein B (LP-B) and 30-40% as lipoprotein B:E (LP-B:E). Both ApoB-containing lipoproteins represent polydisperse systems of spherical particles ranging in size from 100 to 350A for LP-B and from 200 to 500A for LP-B:E. LP-B particles were identified in VLDL, LDL and HDL, while LP-B: E particles were only present in VLDL and LDL. The major neutral lipid of both ApoB-containing lipoproteins was triglyceride which accounted for 50-70°/$ of the total neutral lipid content; cholesterol and cholesteryl esters were present in equal amounts (Table 1).~s The LP-B:E particles contained on the average 70% ApoB and 30% ApoE (Table 1). 18 The ApoB was identified in both types of particles as ApoB-100. A time study on the accumulation of ApoB-containing lipoproteins showed that LP-B particles were secreted independently of LP-B: E particles. ~s The rates of production of LP-B and LP-B: E by HepG2 cells appear to be influenced by nutrients and hormones. Our unpublished studies have shown that our previously reported25 decrease in total ApoB by insulin might be due to the reduced level of LP-B and ultimate increased accumulation of the other ApoB-containing lipoprotein,

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TABLE 1. Neutral Lipid and Apofipoprotein Composition of ApoB-containing Lipoprotein Particles Isolated From HepG2 Cell Culture Medium by Immunoprecipitation or Immunoaifmity Chromatography Neutral lipids TG Lipoprotein particles

Isolation procedure

LP-B:E LP-B LP-B:E LP-B

Immunoprecipitation Immunoprecipitation Immunoaffmity chromatography Immunoaffmity chromatography

Apolipoproteins

FC

CE

B

E

% 63.2 71.1 65.2 68.4

17.6 13.6 19.2 19.5

% 19.2 15.3 15.6 12.1

69.3 100.0 70.5 100.0

30.6 ND* 29.5 ND

Values are average of four different experiments. *ND--not detectable. TG--triglycerides; FC--free cholesterol;CE--cholesteryl esters.

i.e. LP-B:E. These results have also indicated that the increase in total ApoB after addition of 25-hydroxycholesterol was reflected in the preferential increase in LP-B production. These studies provide further support for the hypothesis that the production of distinct ApoB-containing fipoprotein particles is differentially regulated by nutrients and hormones. (b) Isolation and partial characterization of apolipoprotein A-containing lipoprotein particles produced by HepG2 cells. In continuation of our studies on lipoprotein particles produced by HepG2 cells, we have isolated and partially characterized two types of apolipoprotein A-I-containing particles, lipoprotein A-I (LP-A-I) and lipoprotein A-I:A-II (LP-A-I:A-II). 19 The ApoB-containing lipoproteins were removed by either affinity chromatography on concanavalin A or immunoaffinity chromatography on an anti-ApoB immunosorber as previously described. ~9As shown in Table 2, both fractionation procedures were equally effective in separating the ApoB-containing lipoproteins from ApoA-containing lipoproteins. The cholesterol-rich unretained fractions from either the concanavalin A column or anti-ApoB immunosorber which showed the absence of ApoB (Table 2), as determined by electroimmunoassay, were fractionated on an immunosorber with monoclonal antibodies to ApoA-II (pan anti-ApoA-II). The retained fraction contained apolipoproteins A-I, A-II, and E, while the unretained fraction contained apolipoproteins A-I and E only (Table 3). ApoC peptides were not detected probably because their concentrations were too small to be quantified by electroimmunoassay which was employed in these studies. Both ApoA-containing particles were characterized by high percentage (53-60%) of cholesterol (Table 3)J 9 To verify the presence of LP-A-I:E, the anti-ApoA-II unretained fraction was chromatographed on an anti-ApoA-I immunosorber. The protein moiety of the retained fraction consisted of 70% ApoA-I and 30% ApoE (Table 4). To exclude the possible presence of lipoprotein A-II particles (LP-A-II), the anti-ApoA-II retained fraction was chromatographed on an anti-ApoA-I immunosorber. The near complete recovery of apolipoproteins A-I and A-II in the retained fraction (Table 4) indicated that ApoA-II was only present in association with ApoA-I. Studies TABLE2. Neutral Lipid and Apolipoprotein Composition of ApoB- and ApoA-containing Lipoprotein Particles Produced by HepG2 Cells Neutral lipids TG Fraction

Lipoproteins particles

ConA R "PanB" R ConA UR "PanB" UR

ApoB-containing particle ApoB-containing particle ApoA-containing particle ApoA-containing particle

54.6 52.4 24.0 24.0

Apolipoproteins

FC %

CE

30.6 29.4 61.8 59.6

14.8 18.3 14.2 16.4

A-I

A-II

B

E

79.4 71.8 0 0

14.1 26.2 40.9 41.7

% 6.6 2.0 41.7 45.7

0 0 17.2 12.7

After an 18 hr incubation, conditioned medium was removed, concentrated 15-fold, and appfied to either concanavalin A (ConA) or to an anti-ApoB immunosorber ("pan" 13). The concentrations of neutral lipids and apolipoproteins were measured in both the retained (R) and unretained (U) fractions. Values are means of 4 experiments.

226

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T~eLe 3. Neutral Lipid and Apolipoprotein Composition of ApoA-containing Lipoproteins Isolated from HepG2 Cell Culture Medium by lmmunoatfmlty Chromatosraphy Neutral lipids TG Fraction

Lipoprotein particles

ConA UR A-II UR ConAUR A-II R

A-I-containing particles A~I:A-II-containing particles

FC

Apolipoproteins CE

A-I

A-II

%

E

%

25.6 + 2.8

59.04- 4.1

15.44- 1.7

59.54- 2.5

0

40,1 4- 2.6

30.14-3.7

53.44-5.1

16.54-1.9

39.4+1.8

26.04-2.6

34.64-2.6

Values are mean 4-S.E. of 10 experiments. Experimental conditions are described in the legend to Table 2.

from other laboratories using nondenaturing gradient gel analysis45 and immunoaffinity chromatography 13 have also demonstrated the presence of distinct subpopulations of ApoA-I-containing lipoproteins, some of which contain ApoA-II while others are devoid of ApoA-II. The testing of ApoA-containing lipoprotein particles with polyclonal antisera to ApoA-IV failed to reveal the presence of A p o A - I V ) 9 This observation which is consistent with that reported by Cheung et al. ~3 demonstrated that ApoA-IV is not associated with either LP-A-I:A-II or LP-A-I particles but it is part of a separate lipoprotein family (LP-A-IV). The above studies have demonstrated the production of five distinct lipoprotein particles by HepG2 cells including LP-B, LP-B-E, LP-A-I, LP-A-I:A-II and LP-A-IV, each characterized by the presence of specific apolipoprotein(s). Studies are now in progress to further fractionate the nascent lipoprotein particles using an immunosorber with monoclonal antibodies to ApoA-I, ApoC-III and ApoE. B. Studies in Caco-2 Cells

The best characterized human colonic adenocarcinoma cell line is Caco-2 in which the in vitro differentiation extends to the expression of structural characteristics and functional

properties 92 typical of the small intestinal enterocyte. The synthesis and secretion of human apolipoproteins A-I, B, E and C peptides as integral components of lipoprotein particles by Caco-2 cells have been demonstrated. 22'6~'115In addition, we have demonstrated s3 the secretion of Apo(a) by Caco-2 cells. This cell line has been proven to be a useful model for studying intestinal cholesterol metabolism43.ssand regulation by fatty acid of intestinal lipoprotein synthesis and secretion. 22'**'~°'61's6 1. Effect of Oleate on the Production of Lipoproteins by Caco-2 Cells

We have utilized the newly developed human colonic adenocarcinoma Caco-2 cell line to study the properties of nascent lipoprotein particles of intestinal origin." All experiments were conducted with 10-14-days postconfluence cells, at which time the differentiation-dependent expression of their functional properties typical of small intestinal enterocytes is maximal. 92 Triglycerides were the major neutral lipid accumulated in the culture medium while cholesteryl esters accounted for only 12% of the total neutral TAeLe4. Apolipoprotein Composition of ApoA-I-containing Lipoprotein Particles Isolated from HepG2 Culture Medium by Immunoafl~nityChromatography A-I Fraction

Lipoprotein particles

ConA UR: A-II-UR: A-I R ConA UR: A-II-R: A-I R

LP-A-I and LP-A-I: E LP-A-I:A-II and LP-A-I: A-II: E

Apofipoproteins A-II

E

% 70.0

0

30.0

51.2

25.6

23.3

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227

iipids." Caco-2 cells synthesized and secreted most of major apollpoproteins, namely A-I, B, CoIII and E." The presence of ApoA-IV and ApoC-II was also detected by immunoblotting. Although small amounts of ApoA-II were detected in some conditioned media, in the majority of experiments, the cells did not produce this apolipoprotein. Addition of oleic acid bound to albumin resulted in a dose-dependent increase in the lipoprotein production reaching maximum at 0.8 ram fatty acids." Oleate caused a 12-fold stimulation in the secretion of triglycerides and an 8-fold elevation in the concentration of cholesteryl esters while unesterified cholesterol was increased by 61%." The most interesting effect of oleate was on the rate of APOB accumulation in the culture medium. Whereas the accumulation of apolipoproteins A-I, A-II and E in the media was relatively unaffected by oleate, there was a 2- to 4-fold increase in the production of ApoB by this fatty acid. 22 There was also a moderate increase of 33% in the accumulation of ApoC-III in the medium in response to fatty acid provision. The addition of oleate to the culture medium stimulated the secretion of VLDL by 59-fold and LDL by 2.6-fold. 22 In the control system, the major part of neutral lipids, ApoB and APOC-III were present in LDL, whereas most of the ApoA-I and ApoE were found in d > 1.063 g/ml lipoproteins. In oleate-supplemented medium, the major part of neutral lipids and apolipoproteins B, C-III and E were recovered in VLDL. 22 Similar to that in liver, the intestinal synthesis and secretion of ApoB in response to fatty acid administration is also controversial. In the rat, both increased 97,1°°,mt7 and an unchanged 29 rate of ApoB secretion by the intestine subsequent to fat feeding have been reported. Similarly, both increased secretion 5~ and decreased intracellular content 94 of ApoB have been observed in human intestine after fat feeding. Controversy also exists with regard to fatty acid-induced changes in ApoB secretion by Caco-2 cells. Our studies 22 demonstrating enhanced secretion of ApoB by oleate in Caco-2 cells are consistent with studies by Traber et al. It5 and Moberly et al. s6 but are at variance with results reported by Hughes et al. 6° The exact reasons for these differences remain to be established. Consistent with observation in human intestine, 71 Caco-2 cells produce both ApoB-48 and ApoB-100. 67'7sIt has been suggested ~7that the extent of editing of ApoB mRNA in Caco-2 cells is related to the differentiation state and that the secretion of ApoB is switched from ApoB-100 in undifferentiated cells to APOB-48 in differentiated cells. 2. Production of A p o A - and ApoB-containing Lipoprotein Particles by Caco-2 Cells

The isolation and characterization of simple and complex lipoprotein particles secreted by Caco-2 cells were carried out either by immunoprecipitation or immunoaffinity chromatography as described for HepG2 cells. The ApoB-containing lipoprotein particles were isolated by immunoprecipitation a s d e s c r i b e d , ms Results showed that 66% of the total APOB in the culture medium occurred as LP-B, and 34% as LP-B:E (unpublished data). We have also isolated and partially characterized ApoA-I and ApoB-containing llpoprotein particles by immunoaffinity chromatography. In these studies, Caco-2 cells were grown in medium containing 10% fetal calf serum for 15 days. The maintenance medium was removed, cells were washed twice with phosphate-buffered saline and incubated in serum-free medium for 15-18 hr. The conditioned medium was collected and was centrifuged at 4°C for 30 rain at 2000 rpm to remove cells and debris. After addition of preservatives, 'g the medium was concentrated 20-fold and all ApoB-containing lipoproteins were removed by immunoaffinity chromatography on an anti-ApoB immunosorber ("pan B") as previously described. ~s,19The triglyceride-rich retained fraction from "pan B" immunosorber contained 77% APOB, 22% APOE and trace amounts of ApoC-III (unpublished results). The cholesterol-rich unretained fractions from anti-ApoB immunosorber, which showed the absence of APOB and contained 62% APOA-I, 37% ApoE and small amounts of APOC-III, as determined by electroimmunoassay, were fractionated on an immunosorber with monoclonai antibodies to APOA-I (pan anti-ApoA-I) since Caco-2 cells do not secrete ApoA-II. The retained fraction contained apofipoproteins A-I (76%) and E (24%) while the unretained fraction contained only trace amounts of ApoE. ~ L R 30/2/3.--J

228

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These results demonstrate that, in addition to LP-B and LP-B:E, Caco-2 cells secrete LP-A-I:E and possible LP-E. Thus, the above studies have provided the following information: (1) HepG2 and Caco-2 cells synthesize and secrete all the lipid and apolipoprotein constituents of human plasma lipoproteins; (2) in the absence of added lipids, ApoB-containing LDL are the major lipoproteins produced by HepG2 and Caco-2 cells; (3) consistent with observations in normal human liver, HepG2 cells produce only ApoB-100, whereas similar to human intestine, Caco-2 cells produce both ApoB-48 and ApoB-100; (4) addition of oleate stimulates the production of lipoproteins, specifically VLDL, by both HepG2 and Caco-2 cells; (5) in the absence of exogenous lipids, at least two types of ApoB-containing lipoproteins, namely LP-B and LP-B:E, are produced by HepG2 and Caco-2 cells; (6) HepG2 cells synthesize and secrete at least two types of cholesterol-rich ApoA-Icontaining lipoprotein particles, one with ApoA-II (LP-A-I:A-II) and one without ApoA-II (LP-A-I); and (7) Caco-2 cells also produce cholesterol-rich ApoA-I-containing lipoprotein particles (LP-A-I) which contain ApoE but are devoid of ApoA-II. The effects of dietary fatty acids and cholesterol on the synthesis and secretion of simple and complex ApoA-I- and ApoB-containing lipoprotein particles by HepG2 and Caco-2 cells remain to be established. Acknowledgements--Tiffs investigation was supported by a research grant HL-23181 from the National Institutes of Health and, in part, by research grant HR7-032 from the Oklahoma Health Research Program, and by the resources of Oklahoma Medical Research Foundationl

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