Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

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.     

Structural and functional properties of human plasma high density-sized lipoprotein containing only apoE particles

To investigate the metabolism of HDL-apolipoprotein E (apoE) particles in human plasma, we isolated a fraction of plasma HDL-apoEs that lack apoA-I (HDL-LpE) from subjects with apoE3/3 phenotype by immunoaffinity. Plasma HDL-LpE had a particle size ranging from 9 nm to 18.5 nm in diameter and was characterized by two-dimensional nondenaturing gradient gel electrophoresis as having either gamma-, prebeta1-, prebeta2-, or alpha-electrophoretic mobility. HDL-LpE was also present in the medium of cultured human hepatoma cell lines and monocyte-derived macrophages. The majority of apoE3 was found as a monomeric form in HDL-LpE and floated at density d > 1.21 g/ml. Plasma levels of HDL-LpE in normolipidemic, CETP-deficient, and ABCA1-deficient subjects were 0.72 +/- 0.15 mg/dl (n = 12), 1.77 +/- 0.75 mg/dl (n = 3), and 0.55 +/- 0.11 mg/dl (n = 3), respectively. The ratio of HDL-apoE containing apoA-I to HDL-LpE was significantly higher 4 h after a fat load, representing a 35 +/- 9% increase (n = 3). Isolated plasma HDL-LpE3 was as effective as apoE3, reconstituted HDL particles, or apoA-I in promoting cellular cholesterol efflux. These results demonstrate that 1) plasma HDL-LpE may have hepatogenous and macrophagic origins; 2) HDL-LpE was preserved even with large reductions in apoA-I-containing lipoproteins; 3) HDL-LpE was active in the transfer of apoE to triglyceride-rich lipoproteins, and 4) HDL-LpEs efficiently take up cell-derived cholesterol.     

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)     

Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.

The purpose of this experiment was to characterize the high density lipoproteins (HDL) as a function of hydrated density. HDL was subfractionated on the basis of hydrated density by CsCl density gradient centrifugation of whole serum or the d 1.063-1.25 g/ml HDL fraction isolated from three men and three women. Apolipoprotein A-I and A-II quantitation by radial immunodiffusion showed that the A-I/A-II ratio varied with the lipoprotein hydrated density. The A-I/A-II molar ratio of HDL lipoproteins banding between d 1.106 and 1.150 g/ml was nearly constant at 2.2 +/- 0.2. In the density range 1.151-1.25 g/ml the A-I/A-II ratio increased as the density increased. On the other hand, in the density range between 1.077 and 1.105 the A-I/A-II ratio increased as the density decreased, ranging from 2.8 +/- 0.5 for the d 1.093-1.105 g/ml fraction to 5.6 +/- 1.3 for the d 1.077-1.082 g/ml fraction. The d 1.063-1.076 g/ml fraction and the d 1.077-1.082 g/ml fractions had comparable A-I/A-II ratios. Serum and the d 1.063-1.25 g/ml HDL fraction exhibited similar trends. The cholesterol/(A-I + A-II) ratio decreased as the density increased in all 12 samples (six serum and six HDL) examined. Gradient gel electrophoresis of the density gradient fractions showed that as the density increased from 1.063 to 1.200 g/ml the apparent molecular weight decreased from 3.9 x 10(5) to 1.1 x 10(5). HDL subfractions with the same hydrated densities had comparable molecular weights and A-I/A-II and cholesterol/(A-I + A-II) ratios when isolated from men or women. HDL contains subpopulations that differ in the A-I/A-II molar ratio.-Cheung, M. C., and J. J. Albers. Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.     

Cholesterol metabolism in the genetically hypercholesterolemic (RICO) rat. II. A study of plasma lipoproteins and effect of dietary cholesterol

The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d greater than or equal to 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d less than or equal to 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cholesterolemia doubles in the two groups (SWCH, 1.32 +/- 0.10 mg/ml; RICOCH, 2.10 +/- 0.09 mg/ml). This hypercholesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SWCH) and 45% (in RICOCH) of the plasma cholesterol and are cholesterol-enriched compared with the lipoproteins observed in rats fed the base diet. In RICOCH, 24% of the plasma cholesterol is found in apoE-rich LDL2 (1.040 less than or equal to d less than or equal to 1.063), whereas in SWCH, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOCH shows an apoE enrichment of the d less than or equal to 1.063 lipoproteins and an apoA-I depletion of HDL compared to SWCH. These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.     

Plasma lipoprotein changes attending the intravenous administration of Triton WR-1339 in normolipidemic dogs: preferential effect on high density lipoproteins

The nonionic detergent Triton WR-1339 was injected intravenously into normolipidemic dogs in a single dose of 150 mg/kg body weight followed by three other injections (75 mg/kg) on days 2, 6, and 12. The Triton produced a significant elevation of the plasma cholesterol of these animals, but not of their triglyceride levels, and profound changes of their plasma lipoproteins, particularly of the high density lipoprotein class. These changes were dependent on the concentration of Triton attained in plasma; when the levels were above 1.5 mg/ml, density gradient ultracentrifugation, electrophoretic, and chemical analyses indicated that an interaction between Triton and HDL had occurred. This interaction was attended by a gradual loss of the surface components of HDL, namely apoA-I, phospholipids, and unesterified cholesterol, and by the appearance of two cholesteryl ester-rich lipoproteins of d 1.019-1.024 g/ml and d 1.038-1.058 g/ml containing apoA-I and proteins with electrophoretic mobilities of apoB, apoE, and apoA-IV. At the time that these changes had occurred, the activities of the enzymes lecithin: cholesterol acyltransferase and post-heparin lipase were unaffected. When 125I-labeled apoA-I was injected intravenously into animals receiving Triton, the residence time of the radiolabeled protein in plasma increased from a control value of 3.1 days to 7.2 days. However, the apparent half-times of the radiolabeled apoA-I varied among the lipoprotein fractions it was associated with: d 1.119-1.159 g/ml, 5.28 days; d 1.019-1.024 g/ml, 7.55 days, and d 1.038-1.058 g/ml, 5.39 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-poor apolipoprotein A-I in Hep G2 cells: formation of lipid-rich particles by incubation with dimyristoylphosphatidylcholine

Apolipoprotein A-I is a major secretory product of the human hepatoma cell line, Hep G2; approx. 70% of apolipoprotein A-I was separated from the medium as lipid-poor apolipoprotein A-I in the d greater than 1.21 g/ml fraction while 30% was associated with high-density lipoproteins (HDL) of d 1.063-1.21 g/ml. The lipid-poor apolipoprotein A-I contains 50% proapolipoprotein A-I which is similar to the isoform distribution in Hep G2 preformed HDL. We tested the ability of lipid-poor apolipoprotein A-I from Hep G2 to form complexes with dimyristoylphosphatidylcholine (DMPC) vesicles at DMPC/apolipoprotein A-I molar ratios of 100:1 and 300:1. Lipid-poor apolipoprotein A-I was recovered in complex form while at a 300:1 ratio, 68.8 +/- 6.3% was recovered. On electron microscopy, the former complexes were small discs 16.9 nm +/- 4.5 S.D. in diameter while the latter were larger discs 21.4 +/- 4.4 nm diameter. Non-denaturing gradient gel electrophoresis of complexes formed at a 100:1 ratio had a peak in the region corresponding to 9.64 +/- 0.08 nm; these particles possessed two apolipoprotein A-I molecules. At the higher ratio, 300:1, two distinct complexes were identifiable, one which banded in the 9.7 nm region and the other in the 16.9-18.7 nm region. The former particles contained two molecules of apolipoprotein A-I and the latter, three molecules. This study demonstrates that lipid-poor apolipoprotein A-I which is rich in more basic isoforms forms discrete lipoprotein complexes similar to those formed by mature apolipoprotein A-I. It is further suggested that, under the appropriate conditions, precursor or nascent HDL may be assembled extracellularly.     

Interaction of model discoidal complexes of phosphatidylcholine and apolipoprotein A-I with plasma components. Physical and chemical properties of the transformed complexes

Conversion of model discoidal complexes of egg yolk phosphatidylcholine and apolipoprotein A-I, upon interaction with a source of lecithin:cholesterol acyltransferase (plasma d greater than or equal to 1.21 g/ml fraction or partially purified enzyme) and with different sources of substrate unesterified cholesterol (LDL, VLDL or cholesterol incorporated into complexes), was investigated by gradient gel electrophoresis, gel filtration, equilibrium density gradient ultracentrifugation, electron microscopy and chemical analysis. When the incubation mixture contained an inhibitor of lecithin:cholesterol acyltransferase, discoidal complexes with mean long dimension of approximately 10.5 +/- 1.9 nm were converted (within 1 h) predominantly to small round particles and were partially depleted of their phospholipid content. Upon electrophoresis the small particles showed peak maxima within the migration intervals of the human plasma ( HDL3b ) gge and ( HDL3c ) gge subpopulations with associated particle size ranges of 7.8-8.2 and 7.2-7.8 nm, respectively. Within 1 h, in the presence of activated enzyme, the complexes were again converted in major part to the small particles. However, further incubation resulted in an apparent single-step conversion to a larger major product with peak maximum occurring within the migration intervals of the ( HDL2a ) gge and the ( HDL3a ) gge subpopulations (particle size ranges 8.8-9.8 and 8.2-8.8 nm, respectively). Formation of an apolar core was indicated by detection of cholesteryl esters in the conversion product. The form in which the substrate unesterified cholesterol was introduced did not markedly influence the size properties of the final conversion product. With VLDL as source of substrate, considerable incorporation of triacylglycerol occurred in company with a lower level of cholesteryl esters, suggesting transfer of these lipids during formation of the apolar core. Incubation of complexes with a partially purified (3000-fold) preparation of lecithin:cholesterol acyltransferase yielded a product similar in properties to that when the d greater than or equal to 1.21 g/ml fraction was used. Our model discoidal complexes and their conversion products exhibit properties very similar to those of potential precursors to HDL as well as of mature HDL particles. Their further investigation shows promise of providing detailed insight into the possible origin and heterogeneity of human plasma HDL.     

Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.     

Inhibition of lipid transfer by a unique high density lipoprotein subclass containing an inhibitor protein

We have isolated from human plasma a unique subclass of the high density lipoproteins (HDL) which contains a potent lipid transfer inhibitor protein (LTIP) that inhibited cholesteryl ester, triglyceride, and phospholipid transfer mediated by the lipid transfer protein, LTP-I, and phospholipid transfer mediated by the phospholipid transfer protein, LTP-II. This HDL subclass not only inhibited cholesteryl ester transfer from HDL to LDL or VLDL, but also inhibited cholesteryl ester transfer from HDL to HDL. The inhibitor protein was isolated by sequential chromatography of human whole plasma on dextran sulfate-cellulose, phenyl-Sepharose, and chromatofocusing chromatography. Isolated LTIP had the following characteristics: an apparent molecular weight of 29,000 +/- 1,000, (n = 10) by sodium dodecyl sulfate gel electrophoresis, and an isoelectric point of 4.6 as determined by chromatofocusing. LTIP remained functional following delipidation with organic solvents. Antibody to LTIP was produced, and an immunoaffinity column of the anti-LTIP was prepared. Passage of human, rat, or pig whole plasma over the anti-LTIP column enhanced cholesteryl ester transfer activity in human (17%), pig (200%), and rat plasma (125%). The HDL subclass containing LTIP was isolated from whole human HDL (d 1.063-1.21 g/ml) by immunoaffinity chromatography. The isolated LTIP-HDL complex was shown to: i) contain about 60% protein and 40% lipid, ii) have alpha and pre-beta electrophoretic mobility, iii) have particle size distribution somewhat smaller than whole HDL, about 100,000 daltons, as determined by gradient gel electrophoresis, and iv) contain only a small amount of apoA-I (less than 5%) and a trace amount of apoA-II. Assay of ultracentrifugally obtained lipoprotein fractions revealed that approximately 85% of the total functional LTIP activity was in the d 1.063-1.21 g/ml HDL fraction. Furthermore, immunoblot analysis of whole plasma by nondenaturing gradient gel electrophoresis revealed that LTIP was found predominantly in particles in the size range of HDL. This unique HDL subclass may play an important role in the regulation of plasma lipid transfer and metabolism.     

A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins

A new apolipoprotein, termed apolipoprotein J (apoJ), was purified from human plasma by immunoaffinity chromatography. ApoJ is a glycoprotein consisting of disulfide-linked subunits of 34-36 and 36-39 kDa. Each subunit is glycosylated and has a pI range of 4.9-5.4. ApoJ exists in the plasma associated with high density lipoproteins (HDL) and specifically with subclasses of HDL which also contain apoAI and cholesteryl ester transfer protein activity. Immunoaffinity purified apoJ-HDL subclasses have apparent molecular masses of 80, 160, 240, 340, and 520 kDa, as determined by gradient gel electrophoresis. By negative staining electron microscopy, apoJ-HDL range in diameter from 5 to 16 nm. Fractionation of plasma by vertical gradient density centrifugation revealed apoJ-HDL in HDL2 (d 1.063-1.125 g/ml) with the majority overlapping HDL3 (d 1.125-1.21 g/ml) and very high density lipoprotein (d 1.21-1.25 g/ml). The bimodal density distribution of apoJ-HDL suggests that these subclasses have a unique metabolic relationship and may play a role in the transport of cholesterol from peripheral tissues to the liver.     

Effects of triiodothyronine and propylthiouracil on plasma lipoproteins in male rats

Hyperalphalipoproteinemia, characterized by increased plasma concentrations of apoA-I and of HDL lipid and protein, was observed in rats treated with triiodothyronine (T(3)) for 7 days. The increase in the plasma HDL apoproteins was general for apoC, apoE plus A-IV, and apoA-I, as determined by isoelectric focusing. Hypotriglyceridemia, characterized by decreased concentrations of VLDL and apoB, was also observed in the hyperthyroid state. Although in the mildly hypothyroid animals (propylthiouracil-treated), hepatic metabolism of free fatty acid is shifted toward esterification to triglyceride and VLDL formation, as we reported previously, plasma HDL and apoA-I concentrations were not different from control plasma values, while the d 1.006-1.063 g/ml (IDL + LDL) lipoprotein fraction tended to be increased. In general, the proportion of apoE in the (IDL + LDL) fraction of the hypothyroid rat was greater than in controls and hyperthyroid animals, while the proportion of apoE tended to be lower in VLDL from both hypo- and hyperthyroid rats than in VLDL from controls. An enhanced release of apoA-I by perfused livers isolated from rats treated with T(3) was also observed; this enhanced output of apoA-I may explain, in part, the hyperalphalipoproteinemia observed in these rats. The depressed net output of apoA-I in vitro by perfused livers from rats treated with propylthiouracil (PTU) was not expressed in a statistically significant diminished plasma concentration of HDL or apoA-I in the intact animals. Treatment with T(3) also resulted in modification of the content of essential fatty acids in various lipid classes. Linoleic acid residues were significantly reduced and arachidonic acid content was increased in plasma phospholipids and esterified cholesterol in T(3)-treated rats. However, the relative fatty acid composition of unesterified fatty acids and triglyceride fatty acids was not altered by T(3) treatment. PTU treatment had no effect on fatty acid distribution in any of the plasma lipids. Secretion of biliary lipids was increased in perfused livers from T(3)-treated rats, while treatment with PTU did not affect release of lipids in the bile. These observations suggest a regulatory role for thyroid hormones that determine concentration and composition of plasma HDL and other lipoproteins.-Wilcox, H. G., W. G. Keyes, T. A. Hale, R. Frank, D. W. Morgan, and M. Heimberg. Effects of triiodothyronine and propylthiouracil on plasma lipoproteins in male rats.     

Regulation of apoprotein synthesis and secretion in the human hepatoma Hep G2. The effect of exogenous lipoprotein

The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.     

Factors influencing interaction of human plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine

The effect of plasma components on the particle size distribution and chemical composition of human plasma low-density lipoproteins (LDL) during interaction with discoidal complexes of human apolipoprotein A-I and phosphatidylcholine (PC) was investigated. Incubation (37 degrees C, 1 h and 6 h) of LDL with discoidal complexes in the presence of the plasma ultracentrifugal d greater than 1.20 g/ml fraction (activity of lecithin-cholesterol acyltransferase inhibited) produces an increase in LDL apparent particle diameter two-to six-fold greater than that observed in the absence of the plasma d greater than 1.20 g/ml fraction. In incubation mixtures of LDL and discoidal complexes, both in the presence and absence of the plasma d greater than 1.20 g/ml fraction, the extent of LDL apparent particle diameter increase is: (1) approximately three-fold greater at 6 h than at 1 h, and (2) markedly greater for LDL with initially small (22.4-24.0 nm) major components than for LDL with initially large (26.2-26.8 nm) major components. The facilitation factor in the plasma d greater than 1.20 g/ml fraction is not plasma phospholipid transfer protein. Purified human serum albumin produces an apparent particle diameter increase comparable to the plasma d greater than 1.20 g/ml fraction. The discoidal complex-induced increase in LDL apparent particle diameter value by albumin is associated with an increase in phospholipid uptake by LDL and a decreased loss of LDL unesterified cholesterol. In preliminary experiments, high-density lipoproteins (HDL) reverse the apparent particle diameter increase originally induced by discoidal complexes. The presence of HDL (HDL phospholipid/LDL phospholipid molar ratio of 10:1) in the incubation (6 h) mixture of LDL and discoidal complexes also attenuates LDL apparent particle diameter increase. In vivo, the plasma LDL/HDL ratio may be a controlling factor in determining the extent to which phospholipid uptake and the associated change in LDL particle size distribution occurs.     

Distribution and characterization of the serum lipoproteins and apoproteins in the mouse, Mus musculus

Murine lipoproteins were separated into nine subfractions by a density gradient ultracentrifugal procedure. They were characterized by electrophoretic, immunological, chemical, and morphological analyses, and their protein moieties were defined according to charge, molecular weight, and isoelectric point. HDL predominated (approximately 500 mg/dl serum), the mode of its distribution being situated in the d 1.09-1.10 g/ml (F 1.21 approximately 4) region. Chemical analysis showed subfractions of d 1.085-1.136 g/ml to resemble human HDL3 closely, including the presence of apoA-I (Mr 25,000-27,000) as their major apolipoprotein. An apoA-II-like protein, of Mr 8400 (in monomeric form), was also tentatively identified. In electrophoretic mobility and chemical composition, the d 1.060-1.085 g/ml subfraction (approximately 10% of total HDL) was distinct and akin to human HDL2. ApoA-I represented approximately 60% of its complement of low molecular weight apoproteins. The density range used for separation of human HDL2 (d 1.066-1.100 g/ml) by gradient ultracentrifugation is inadequate in the mouse, and the d 1.060-1.085 g/ml interval is more appropriate. The 1.063 g/ml boundary for separation of mouse LDL from HDL was unsuitable. Immunological and electrophoretic studies revealed that alpha-migrating lipoproteins were present in the d 1.046-1.060 g/ml range, a finding consistent with their enrichment in apoA-I; apoE-, apoA-II-, and apoC-like proteins were also detected. These findings indicate the presence of HDL1 particles. Murine apoA-I and apoB-like proteins of higher (apoBH) and lower (apoBL) molecular weight were constituents of the d 1.033-1.046 g/ml fraction. Alternative techniques, such as electrophoresis in starch block, are therefore a prequisite for separation of apoB from alpha-migrating, apoA-I-containing lipoproteins in the low density range in mouse serum. The LDL class (d 1.023-1.060 g/ml) amounted to only approximately 20% of the total murine lipoproteins of d less than 1.188 g/ml (65-70 mg/dl serum). Particles were richer In triglyceride, larger in diameter (mean 244 A), and more heterogeneous than typical of man. VLDL (40-80 mg/dl serum) was triglyceride-rich (66% by weight) and similarly heterogeneous in size (mean diameter 494 A; range 270-750 A). ApoBH and apoBL were prominent in murine VLDL, and cross-reacted with an antiserum to human apoB. ApoE- and apoA-I-like proteins were also detectable in apoVLDL, as was a protein of 70,000-75,000 mol wt. The presence of murine apolipoproteins analogous to human apoB and apoE was confirmed by the immunological cross-reactivities of VLDL and LDL with monospecific antisera to the human proteins. The marked similarity of lipoprotein and apolipoprotein profile in the mouse and rat is notable. Since murine VLDL contains apoE and apoBL, this resemblance may extend to the metabolism of chylomicron remnants and hepatic VLDL in the two species.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Secretion of apolipoprotein A-I in lipoprotein particles following transfection of the human apolipoprotein A-I gene into 3T3 cells

Apolipoprotein A-I (apoA-I) is the major protein constituent of plasma high density lipoproteins (HDL). To examine apoA-I processing and secretion, the human apoA-I gene (2.2-kilobase PstI-PstI fragment) linked to the mouse metallothionein promoter was transfected by electroporation into NIH 3T3 fibroblasts along with the plasmid pSV2 neo, which confers neomycin resistance. Transfected cells were selected for neomycin resistance and screened for the ability to produce apoA-I by enzyme-linked immunosorbent assay. In the absence of lipids in the medium, selected 3T3 cells secreted apoA-I, mainly in the proprotein form, at density greater than 1.25 g/ml. Following incubation of cells with lipids, and subsequent washing with lipid-free medium, apoA-I was recovered in the HDL region (1.063-1.21 g/ml) as well as in the 1.21 g/ml infranatant. Examination of the HDL fraction by electron microscopy revealed round particles, 10-21 nm in diameter. These data indicate that human apoA-I secreted by transfected 3T3 fibroblasts can assemble into lipoprotein particles under the appropriate conditions.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.     

Density distribution and physicochemical properties of plasma lipoproteins and apolipoproteins in the goose, Anser anser, a potential model of liver steatosis

The fractionation and physicochemical characterization of the complex molecular components composing the plasma lipoprotein spectrum in the goose, a potential model of liver steatosis, are described. Twenty lipoprotein subfractions (d less than 1.222 g/ml) were separated by isopycnic density gradient ultracentrifugation, and characterized according to their chemical composition, particle size and particle heterogeneity, electrophoretic mobility, and apolipoprotein content. Analytical ultracentrifugal analyses showed high density lipoproteins (HDL) to predominate (approximately 450 mg/dl plasma), the peak of its distribution occurring at d approximately 1.090 g/ml (F1.21 approximately 2.5). The HDL class displayed marked density heterogeneity, HDL1-like particles being detected up to a lower density limit of approximately 1.020 g/ml, particle size decreasing progressively from 17-19 nm at d 1.024-1.028 g/ml to 10.5-12 nm (d 1.055-1.065 g/ml), and then remaining constant (approximately 9 nm) at densities greater than 1.065 g/ml. HDL subfractions displayed multiple size species; five subspecies were present over the range d 1.103-1.183 g/ml with diameters of 10.5, 9.9, 9.0, 8.2, and 7.5 nm, four in the range d 1.090-1.103 g/ml (diameters 10.5, 9.9, 9.0, and 8.2 nm) and three over the range d 1.076-1.090 g/ml (diameters 10.5, 9.9, and 9.0 nm). ApoA-I (Mr 25,000-27,000) was the major apolipoprotein in all goose HDL subfractions, while the minor components (apparent Mr 100,000, 91,000, 64,000, 58,000, approximately 42,000, 18,000 and apoC-like proteins) showed marked quantitative and qualitative variation across this density range (i.e., 1.055-1.165 g/ml). The d 1.063 g/ml boundary for separation of goose low density lipoproteins (LDL) from HDL was inappropriate, since HDL-like particles were present in the density interval 1.024-1.063 g/ml, while particles enriched in apoB (Mr approximately 540,000) and resembling LDL in size (approximately 20.5 nm) were detected up to a density of approximately 1.076 g/ml. Goose LDL itself was a major component of the profile (90-172 mg/dl) with a single peak of high flotation rate (Sf approximately 10.5). The physicochemical properties and apolipoprotein content of intermediate density lipoproteins (IDL) and LDL varied but little over the range d 1.013-1.040 g/ml, presenting as two particle species (diameters 20.5 and 21 nm) of essentially constant chemical composition; LDL (d 1.019-1.040 g/ml) were separated from HDL1 by gel filtration chromatography and appeared to contain primarily apoB with lesser amounts of apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)