Apolipoprotein C-II mRNA levels in primate liver. Induction by estrogen in the human hepatocarcinoma cell line, HepG2

We have demonstrated that physiological concentrations of 17 beta-estradiol increase nuclear estrogen-specific binding sites in the human hepatoma cell line HepG2 7- to 10-fold and the rate of accumulation of secreted apolipoprotein C-II (apo-C-II), 2.5-fold (Tam, S-P., Archer, T. K., and Deeley, R. G. (1985) J. Biol. Chem. 260, 1670-1675). Apo-C-II is the major activator of lipoprotein lipase, an enzyme which plays a key role in lipoprotein catabolism. In order to define more precisely the mechanism by which estrogen influences apo-C-II production, we have synthesized a triacontanucleotide DNA probe that is complementary to apo-C-II mRNA. We have used the probe both in Northern hybridization experiments and in DNA excess titrations to quantify apo-C-II mRNA in hormonally treated HepG2 cells and various primate tissues. These studies revealed that: 1) the concentration of apo-C-II mRNA in HepG2 cells is comparable with that present in human liver; 2) treatment of the cells with low levels of estrogen results in a doubling of the apo-C-II mRNA concentration; 3) the apo-C-II mRNA concentration in monkey liver is 60- to 70-fold greater than in the intestine and 2.5-fold higher than in human liver.     

A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.     

Very low density lipoprotein. Removal of Apolipoproteins C-II and C-III-1 during lipolysis in vitro.

In this study we have investigated the effects of very low density lipoprotein (VLDL) lipolysis on the removal of radiolabeled apolipoprotein C-II and apolipoprotein C-III-1 from in vitro lipolyzed lipoproteins. Lipolysis was carried out in vitro using lipoprotein lipase purified from bovine milk, and mixtures with or without plasma. Lipoproteins were isolated by ultracentrifugation and by gel filtration. Labeled apo-C-II and apo-C-III-1 distributed among plasma lipoproteins, predominantly VLDL and high density lipoprotein (HDL). Lipolysis induced transfer of apo-C-II and apo-C-III-1 from VLDL to HDL. The transfer was proportional to the extent of triglyceride hydrolysis, and similar for the two apoproteins. The apo-C-II/apo-C-III-1 radioactivity ratio did not change in either VLDL or the fraction of d greater than 1.006 g/ml during the progression of the lipolytic process. Similar observations were recorded while using plasma-devoid lipolytic systems. Gel filtration of incubation mixtures, on 6% agarose, revealed that the removal of labeled apo-C molecules from VLDL is not a consequence of either centrifugation or high salt concentration. These results suggest that there is no preferential removal of apo-C-II or apo-C-III-1 from lipolyzed VLDL particles. They further indicate that the ratio of apo-C-II to apo-C-III-1 does not regulate the extent of lipolysis of different VLDL particles, at least in VLDL isolated from normolipidemic humans.     

Intrahepatic assembly of very low density lipoproteins. Varied synthetic response of individual apolipoproteins to fasting

Hepatocytes obtained from rats fed for 3 days chow (control) or drinking water only (fasted) were used to examine how metabolic state affects lipogenesis, apolipoprotein synthesis, and the capacity to secrete de novo synthesized triacylglycerol. The secretion of triacylglycerol (mass and 3H-labeled via 3H2O incorporation) by both groups of cells was constant for 30 h. Moreover, cells from fasted rats secreted triacylglycerol at rates which were markedly reduced (mass -84%; 3H-labeled -91%). To assess the relative capacities of the two groups of hepatocytes to augment triacylglycerol secretion in response to stimulated lipogenesis, cells were incubated with increasing concentrations of glucose. Control cells responded to glucose by increasing equally the synthesis and secretion of [3H] triacylglycerol. When cells from fasted rats were challenged with glucose, triacylglycerol secretion was not increased. Rather, it accumulated intracellularly. Double-reciprocal plot analysis of the capacity to augment triacylglycerol secretion in response to glucose showed that cells from fasted rats had a greater than 10-fold decrease in V'max. Moreover, fasting changed the synthesis and secretion of apolipoproteins selectively: secretion of low molecular weight apo-B was decreased 50%, large molecular weight apo-B was unchanged, and apo-E was increased 2-4-fold. Analysis of the lipoproteins from both groups of cells on Bio-Gel A-50m showed that the very low density lipoprotein secreted by cells from fasted rats was smaller. In addition, all of the increased de novo synthesized apo-E secreted by cells from fasted rats eluted after the triacylglycerol-rich lipoproteins. The combined data show that: 1) the synthesis of individual very low density lipoprotein apolipoproteins is independently regulated, and 2) the synthesis (availability) of apo-B determines the capacity of the hepatocyte to assemble/secrete triacylglycerol-rich very low density lipoprotein.     

Early changes in plasma lipoprotein structure and biosynthesis in cholesterol-fed rabbits

Plasma lipoproteins of d < 1.063 g/ml from rabbits fed a diet containing 1% cholesterol for 4 days showed changes in concentration and rates of flotation as determined by analytical ultracentrifugation. A marked increase in cholesteryl ester content of lipoprotein with d < 1.019 g/ml was the most prominent change in rabbits fed the diet for 21 days. Gel electrophoresis and immunochemical procedures demonstrated that in control and hypercholesterolemic rabbits there were some common apolipoproteins found in all lipoproteins with density < 1.063 g/ml. In control rabbits, there were also apolipoproteins specific to the lipoprotein fraction with d < 1.019 and to the fraction with d 1.019-1.063 g/ml. However, in rabbits fed the hypercholesterolemic diet for 21 days, the apolipoproteins characteristic of fraction 1.019-1.063 were the most abundant in the fraction with d < 1.019 g/ml. Liver slices from rabbits fed the high cholesterol diet for 7 and 21 days incorporated more l-[(14)C]leucine into very low density and low density lipoproteins than controls. The results suggest that cholesterol feeding leads to an increase in biosynthesis of lipoproteins with d < 1.063 g/ml. The newly synthesized lipoprotein contains apolipoproteins similar to those found in controls but with a higher lipid-to-protein ratio. From the apoprotein composition, it is concluded that the very low density fraction present in cholesterol-fed animals is more structurally related to low density lipoproteins than to the very low density lipoproteins isolated from control animals.     

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.     

Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

Summary The analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [³⁵S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [³⁵S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included B _h , E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes. This investigation was supported in part by a research grant from the Southwest Foundation Forum, by program project HL 28972 from the National Heart, Lung and Blood Institute, Bethesda, MD, and by grants to R. V. H. from the National Institutes of Health (HL 15062), the American Heart Association, and the Louis Block Fund.     

Identification and partial characterization of discrete apolipoprotein B containing lipoprotein particles produced by human hepatoma cell line HepG2

The purpose of this study was to test the use of human hepatocarcinoma HepG2 cells as a model for studying the formation and secretion of human hepatic lipoproteins. To this end, we determined the rate of accumulation and percent composition of neutral lipids and apolipoproteins in the culture medium of HepG2 cells and isolated and partially characterized the apolipoprotein B (ApoB) containing lipoprotein particles. The rates of accumulation in the medium of HepG2 cells, grown in minimum essential medium during a 24-h incubation, of triglycerides, cholesterol, and cholesterol esters expressed as microgram/(g of cell protein X h) were 373 +/- 55, 167 +/- 14, and 79 +/- 10, respectively; the secretion rates for apolipoproteins B, A-I, E, A-II, and C-III were 372 +/- 36, 149 +/- 14, 104 +/- 13, 48 +/- 4, and 13 +/- 1 microgram/(g of cell protein X h), respectively. The major portion of ApoB was present in very low density lipoproteins (VLDL) and low-density lipoproteins (LDL) (84%), with the remainder occurring in high-density lipoproteins (HDL) (16%). Approximately 10-13% of ApoA-I and ApoA-II were present in VLDL and LDL, while 60% of ApoE occurred in HDL and 40% in VLDL and LDL. To separate ApoB-containing lipoproteins, secreted lipoproteins were fractionated by either sequential immunoprecipitation or immunoaffinity chromatography with antibodies to ApoB and ApoE. 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 350 A for LP-B and from 200 to 500 A 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 (50-70% of the total neutral lipid content); cholesterol and cholesterol esters were present in equal amounts. The LP-B:E particles contained 70-90% ApoB and 10-30% ApoE. The ApoB was identified in both types of particles as B-100. A time study on the accumulation of ApoB-containing lipoproteins showed that LP-B particles were secreted independently of LP-B:E particles.     

Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2

A total of six established human hepatoma-derived cell lines, including Hep3B, NPLC/PRF/5 (NPLC), Tong/HCC, Hep 10, huH1, and huH2, were screened for their ability to accumulate significant quantities of lipoproteins in serum-free medium. Only two cell lines, Hep3B and NPLC, secreted quantitatively significant amounts of lipoproteins. In a 24-h period the accumulated mass of apolipoproteins (apo) A-I, A-II, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 micrograms/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-II, B, and E was 0.45, 0.05, 0.32, and 0.68 micrograms/mg cell protein per 24 h, respectively. Twenty four-hour serum-free medium of Hep3B cells contained lipoproteins corresponding to the three major density classes of plasma; percent protein distribution among the lipoprotein classes was 4%, 41%, and 56% for very low density lipoprotein ("VLDL"), low density lipoprotein ("LDL"), and high density lipoprotein ("HDL"), respectively. NPLC was unusual since most of the lipoprotein mass was in the d 1.063-1.235 g/ml range. Hep3B "LDL", compared with plasma LDL, contained elevated triglyceride, phospholipid, and free cholesterol. Nondenaturing gradient gel electrophoresis revealed that Hep3B "LDL" possessed a major component at 25.5 nm and a minor one at 18.3 nm. Immunoblots showed that the former contained only apoB while the latter possessed only apoE. Like plasma VLDL, Hep3B "VLDL" particles (30.5 nm diameter) isolated from serum-free medium contained apoB, apoC, and apoE. "HDL" harvested from Hep3B and NPLC medium were enriched in phospholipid and free cholesterol and poor cholesteryl ester which is similar to the composition of HepG2 "HDL." "HDL" from Hep3B and NPLC culture medium on gradient gel electrophoresis had peaks at 7.5, 10, and 11.9 nm which were comparable to major components found in HepG2 cell medium. Hep3B cells, in addition, possessed a particle that banded at 8.2 nm which appeared to be an apoA-II without apoA-I particle by Western blot analysis. The cell line also produced a subpopulation of larger-sized "HDL" not found in HepG2 medium. NPLC "HDL" had a distinct peak at 8.3 nm which by Western blot was an apoE-only particle. Electron microscopy revealed that "HDL" harvested from Hep3B and NPLC medium consisted of discoidal and small, spherical particles like those of HepG2. The "HDL" apolipoprotein content of each cell line was distinct from that of HepG2. ApoA-II at 35% of apolipoprotein distinguishes Hep3B "HDL" from HepG2, which contains only 10%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased production of apolipoprotein B and its lipoproteins by oleic acid in Caco-2 cells

The production of lipids, apolipoproteins (apo), and lipoproteins induced by oleic acid has been examined in Caco-2 cells. The rates of accumulation in the control medium of 15-day-old Caco-2 cells of triglycerides, unesterified cholesterol, and cholesteryl esters were 102 +/- 8, 73 +/- 5, and 11 +/- 1 ng/mg cell protein/h, respectively; the accumulation rates for apolipoproteins A-I, B, C-III, and E were 111 +/- 9, 53 +/- 4, 13 +/- 1, and 63 +/- 4 ng/mg cell protein/h, respectively. Whereas apolipoproteins A-IV and C-II were detected by immunoblotting, apoA-II was absent in most culture media. In contrast to an early production of apolipoproteins A-I and E occurring 2 days after plating, the apoB expression appeared to be differentiation-dependent and was not measurable in the medium until the sixth day post-confluency. In the control medium, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL), and lipid-poor very high density lipoproteins (VHDL) accounted for 12%, 46%, 18%, and 24% of the total lipid and apolipoprotein contents, respectively. The triglyceride-rich VLDL contained mainly apoE (75%) and apoB (23%), while the protein moiety of LDL was composed of apoB (59%), apoE (20%), apoA-I (15%), and apoC-III (6%). The cholesterol-rich HDL contained mainly apoA-I (69%) and apoE (27%). In the control medium, major portions of apolipoproteins B and C-III (93-97%) were present in LDL, whereas the main parts of apoA-I (92%) and apoE (76%) were associated with HDL and VHDL. Oleate increased the production of triglycerides 10-fold, cholesteryl esters 7-fold, and apoB 2- to 4-fold. There was also a moderate increase (39%) in the production of apoC-III but no significant changes in those of apolipoproteins A-I and E. These increases were reflected mainly in a 55-fold elevation in the concentration of VLDL, and a 2-fold increase in the level of LDL; there were no significant changes in HDL and VHDL. VLDL contained the major parts of total neutral lipids (74-86%), apoB (65%), apoC-III (81%) and apoE (58%). In the presence of oleate, the VLDL, LDL, HDL, and VHDL accounted for 76%, 15%, 3%, and 6% of the total lipoproteins, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatocyte damage induced by carbon tetrachloride: inhibited lipoprotein secretion and changed lipoprotein composition

Changes of lipoprotein secretion and composition in response to CCl4 treatment were studied in monolayer cultures of rat primary hepatocytes. (1) CCl4 decreased secretion of very low density lipoproteins (VLDL) by about 85%, while high density lipoprotein (HDL) secretion was less affected (about 40%). The effect was concentration-dependent. (2) CCl4 significantly inhibited secretion of VLDL- and HDL-associated triglycerides and cholesterol esters. VLDL- and HDL-associated cholesterol was not affected, while secretion of phospholipids was increased. (3) Hepatocytes secreted the apolipoproteins B48, B100, E, C, and A-I. CCl4 reduced secretion of apoproteins associated with VLDL by almost 20%, and by about 75% when associated with HDL. The de novo synthesis of apolipoproteins was attenuated by CCl4. (4) CCl4 caused variations in the apolipoprotein composition in VLDL and HDL. CCl4 intoxication of the liver affected the morphology and/or function of the lipoproteins, which drastically impaired their ability to act as transport vehicles for lipids from the liver to the circulation.     

Apolipoproteins of high, low, and very low density lipoproteins in human bile

We tested the hypothesis that apolipoproteins, the protein constituents of plasma lipoproteins, are secreted into bile. We examined human gallbladder bile obtained at surgery (N = 54) from subjects with (N = 44) and without (N = 10) gallstones and hepatic bile collected by T-tube drainage (N = 9) after cholecystectomy. Using specific radioimmunoassays for human apolipoproteins A-I and A-II, the major apoproteins of high density lipoproteins, for apolipoproteins C-II and C-III, major apoproteins of very low density lipoproteins, and for apolipoprotein B, the major apoprotein of low density lipoproteins, we found immunoreactivity for these five apolipoproteins in every bile sample studied in concentrations up to 10% of their plasma values. Using double immunodiffusion, we observed complete lines of identity between bile samples and purified apolipoproteins A-I, A-II, or C-II. Using molecular sieve chromatography, we found identical elution profiles for biliary apolipoproteins A-I, A-II and B and these same apolipoproteins purified from human plasma. When we added high density lipoproteins purified from human plasma to lipoprotein-free solutions perfusing isolated rat livers, we detected apolipoproteins A-I and A-II in bile. Similarly, when we added low density lipoproteins purified from human plasma to lipoprotein-free solutions perfusing isolated livers of rats treated with ethinyl estradiol in order to enhance hepatic uptake of low-density lipoproteins, we found apolipoprotein B in bile. These data indicate that apolipoproteins can be transported across the hepatocyte and secreted into bile.     

Serum Lp(a) lipoprotein concentrations in insulin dependent diabetic patients with microalbuminuria.

OBJECTIVE--To compare the serum concentrations of lipoproteins and apolipoproteins in insulin dependent diabetic patients with and without microalbuminuria. DESIGN--Cross sectional study. SETTING--Paediatric and medical outpatient clinic at a university hospital. PATIENTS--76 insulin dependent diabetic patients: 41 with microalbuminuria (20 males, 21 females) and 35 controls without microalbuminuria (18 males, 17 females). The two groups were similar with respect to age, duration of disease, and haemoglobin A1c concentrations before the study. MAIN OUTCOME MEASURES--Serum concentrations of Lp(a) lipoprotein, total cholesterol, high density lipoprotein cholesterol, very low density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides, and apolipoproteins A-I, A-II, and B. RESULTS--Median serum Lp(a) lipoprotein concentration was 10.0 mg/100 ml in the microalbuminuric group and 4.9 mg/100 ml in the control group (p = 0.007). 17 (41%) of the microalbuminuric patients and five (14%) of the control patients had Lp(a) lipoprotein values above the upper quartile of a normal population. Median serum triglycerides concentrations in the microalbuminuric and control groups were 1.15 mmol/l and 0.88 mmol/l respectively (p = 0.03). Median very low density lipoprotein cholesterol concentration was 0.52 mmol/l in the microalbuminuric group and 0.40 mmol/l in the control group (p = 0.03). No significant differences in serum concentrations of total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, or apolipoproteins A-I, A-II, and B were found between the groups. CONCLUSIONS--Serum concentrations of Lp(a) lipoprotein are twice as high in insulin dependent diabetic patients with microalbuminuria as in those without microalbuminuria. Increased concentrations of Lp(a) lipoprotein might partly explain the increased morbidity and mortality from cardiovascular disease observed among patients with diabetic nephropathy.     

Relative importance of the LDL receptor and scavenger receptor class B in the beta-VLDL-induced uptake and accumulation of cholesteryl esters by peritoneal macrophages

We investigated the mechanism of beta-very low density lipoprotein (beta-VLDL)-induced foam cell formation derived from peritoneal macrophages from control mice and low density lipoprotein (LDL) receptor-deficient mice to elucidate the role of the LDL receptor in this process. The LDL receptor appeared to be of major importance for beta-VLDL metabolism. Consequently, the accumulation of cholesteryl esters in LDL receptor(-)(/)- macrophages is 2.5-fold lower than in LDL receptor(+)(/)(+) macrophages. In the absence of the LDL receptor, however, beta-VLDL was still able to induce cholesteryl ester accumulation and subsequently we characterized the properties of this residual beta-VLDL recognition site(s) of LDL receptor(-)(/)- macrophages. Although the LDL receptor-related protein is expressed on LDL receptor(-)(/)- macrophages, the cell association of beta-VLDL is not influenced by the receptor-associated protein, and treatment of the macrophages with heparinase and chondroitinase was also ineffective. In contrast, both oxidized LDL (OxLDL) and anionic liposomes were able to inhibit the cell association of (125)I-labeled beta-VLDL in LDL receptor(-)(/)- macrophages by 65%. These properties suggest a role for scavenger receptor class B (SR-B), and indeed, in the LDL receptor(-)(/)- macrophages the selective uptake of cholesteryl esters from beta-VLDL was 2.2-fold higher than that of apolipoproteins, a process that could be inhibited by OxLDL, high density lipoprotein (HDL), and beta-VLDL.In conclusion, the LDL receptor on peritoneal macrophages is directly involved in the metabolism of beta-VLDL and the subsequent foam cell formation. When the LDL receptor is absent, SR-B appears to mediate the remaining metabolism of cholesteryl esters from beta-VLDL.     

Abnormal apolipoprotein composition in alcoholic hepatitis

Alcoholic hepatitis leads to major derangements in lipoprotein metabolism. This study defines the characteristics of the abnormal high density lipoprotein and very low density lipoprotein in relation to the severity of the disease. In severely affected subjects very low density lipoprotein apolipoproteins were deficient in apolipoprotein E and apolipoprotein C. The concentration of high density lipoprotein was markedly reduced, although the proportion of high density lipoprotein 1 was substantially elevated when compared to normal subjects. High density lipoproteins were deficient in apolipoprotein AI and apolipoprotein AII but enriched in apolipoprotein E, apolipoprotein E complexes and apolipoprotein C, and contained a mixture of particles. The high density lipoprotein of subjects with alcoholic hepatitis contained a high proportion of material which bound to heparin affinity columns. This bound fraction contained a group of particles rich in apolipoprotein E, apolipoprotein E complexes and apolipoprotein C and was deficient in apolipoprotein AI and apolipoprotein AII. Examination by electron microscopy showed the presence of both discoidal and spherical particles, which varied in concentration according to the severity of the disease. Another fraction of high density lipoprotein, not bound to heparin, contained reduced amounts of apolipoprotein AI and apolipoprotein AII, consisted of disc-shaped particles and showed a higher esterified: free cholesterol ratio than the other high density lipoprotein fraction.     

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

Nature of the enhancement of lecithin-cholesterol acyltransferase reaction by various apolipoproteins

The effects of human apolipoproteins on the lecithin-cholesterol acyltransferase reaction were studied by using purified human lecithin-cholesterol acyltransferase and phosphatidylcholine-cholesterol vesicles. When the assay mixtures contained an optimal amount or excess of apo-A-I, the addition of apo-A-II, apo-C-II, apo-C-III1, or apo-C-III2 inhibited the enzymatic reaction. However, at suboptimal apo-A-I concentrations, the addition of low concentrations of these apolipoproteins exhibited activating effects. The relative activating effects were greater at lower apo-A-I levels. Under no circumstance did the combined activating effect of apo-A-I and other apolipoproteins exceed the maximum activating effect observed with the optimal level of apo-A-I alone. Since apo-A-II, apo-C-II, and apo-C-III did not show significant activating effects in the absence of apo-A-I, these apolipoproteins apparently did not act as true activator proteins for the enzymatic reaction. The activation of the enzymatic reaction by apo-A-I alone was shown to be due in part to the enhancement of the enzyme transfer between the substrate particles. The replacement of the transfer-enhancing effect of apo-A-I by apo-A-II, apo-C-II, or apo-C-III appears to be responsible for their apparent activating effects in the presence of suboptimal levels of apo-A-I. These apolipoproteins seemed to coexist with both the enzyme and apo-A-I on the substrate particles under the conditions when they showed the activating effect. However, at the concentrations inhibitory to the enzymatic reaction, these apolipoproteins displaced both the enzyme and apo-A-I from the phosphatidylcholine-cholesterol vesicles.