Effects of cortisol on lipid and lipoprotein synthesis in rat hepatocytes]

Under in vivo conditions cortisol induces moderate hyperlipidemia followed by an increase in the phospholipid and triglyceride concentrations in the blood and a decrease of cholesterol; similar changes were observed in the liver. At all time intervals studied cortisol inhibits the phospholipid and cholesterol syntheses and decreases the specific radioactivities of the lipids in the mitochondrial fraction. The hormone has an inhibiting effect on the fatty acid synthesis at early postinjection stages. The phospholipid synthesis is increased after adrenalectomy and is then inhibited after injection of the hormone. A single injection of ACTH or cortisol causes suppression of phospholipid and cholesterol syntheses and a decrease in their specific radioactivities in the mitochondria. A similar effect is observed under stress conditions. In addition, the hormone inhibits the synthesis of lipoprotein apoproteins of very low and high densities. After 5 hours following the hormone injection the lipoprotein apoprotein synthesis in the liver is activated; the activation of apoprotein synthesis is also observed after adrenalectomy. However, the injection of the hormone to adrenalectomized rats decreases the apoprotein synthesis. It was shown that in blood serum cortisol affects the conversions of very low density lipoproteins into low density lipoproteins, thus providing for hyperlipidemia.     

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.     

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.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Catabolism of very low density lipoproteins in the rabbit. Effect of changing composition and pool size.

To determine the metabolic mechanism of hypercholesterolemia in rabbits produced by feeding cholesterol-rich diets, control and hypercholesterolemic rabbits were injected with I-labelled very low density lipoproteins (VLDL, d 1.006 g/ml) from control and/or hypercholesterolemic donors. Apolipoprotein B in VLDL decayed biphasically. The first phase occurred much more rapid than the second. 95% of the VLDL apolipoprotein B was catabolized via the first phase (t1/2 = 0.55 +/- 0.19 h) in normal rabbit with the immediate appearance of this radioactivity in intermediate density lipoproteins (IDL, d 1.006-1.025 g/ml) and low density lipoproteins (LDL, d 1.025-1.063 g/ml). The apolipoproteins C and E at the same time were transferred to high density lipoproteins where they decayed biphasically. The apolipoprotein B from hypercholesterolemic VLDL in the normal recipient disappeared at a similar rate as from normal VLDL via phase I; however, it was incompletely converted to IDL and LDL. Apolipoprotein B from normal VLDL in cholesterol-fed rabbits disappeared at a normal rate via phase I, but only 82% was catabolized by this phase. Hypercholesterolemic VLDL injected into the hypercholesterolemic recipient was less rapidly catabolized via phase I (T1/2 = 2.5 +/- 0.89 H) and only a small fraction was converted to IDL and LDL.     

Metabolic fate of rat and human lipoprotein apoproteins in the rat

The fate of (125)I-labeled apolipoproteins was studied in vivo in rats that had received intravenous injections of (125)I-labeled rat HDL and (125)I-labeled human HDL, LDL, and VLDL. Plasma decay curves of rat and human HDL were exponential with similar half-lives in the circulation (11-12 hr). After injection, low molecular weight apolipoproteins (apoLP-alanine of human HDL and fraction HS-3 of rat HDL) were found to redistribute to other lipoproteins, predominantly VLDL. Decay curves of individual HDL proteins were constructed after lipoprotein fractionation, delipidation, and polyacrylamide gel electrophoresis. It was found that the half-lives of the different HDL apoproteins were not identical. A major rat HDL protein (52% of total counts) had a circulating half-life (t((1/2))) of 12.5 hr. Two others had a t((1/2)) of 8-9 hr while the t((1/2)) of several others was 11-12 hr. The t((1/2)) of three well-characterized human HDL apoproteins, apoLP-glutamine I, apoLP-glutamine II, and apoLP-alanine, were 13.5, 9.0, and 15.0 hr, respectively. The fate of (125)I-labeled human VLDL and LDL apoproteins in rats was similar to that described previously in humans. After injection of (125)I-labeled human VLDL into rats, apoLP-glutamic acid and apoLP-alanine rapidly transferred to rat HDL and were lost thereafter from the circulation from both VLDL and HDL. The apoLDL moiety of human VLDL moved metabolically to the LDL density range (d = 1.019-1.063) through a lipoprotein of intermediate density (d = 1.006-1.019).     

A comparative study of serum lipoproteins in rabbits fed a natural ingredient diet or low-fat, cholesterol-free, semipurified diets containing casein or isolated soy protein

In rabbits fed a cholesterol-free, semipurified diet containing isolated soy protein, the average total serum cholesterol level was similar to that of rabbits fed a natural ingredient (chow) diet. However, the cholesterol and protein levels in very low density (VLDL) and low density lipoproteins (LDL) tended to increase, while the levels in high density lipoproteins (HDL) were reduced to about half of those on the chow diet, with little change in the cholesterol to protein ratio. Substitution of casein for soy protein in the semipurified diet caused a four- to five-fold increase in total serum cholesterol and a doubling of lipoprotein protein, with an increase of 1.4- to 3.0-fold in the cholesterol to protein ratio of the different lipoprotein fractions. Analysis of the apoproteins (apo) of the plasma lipoproteins indicated that apo B, E, and C all tended to increase in the VLDL and LDL of rabbits fed the soy protein diet compared with those fed chow diet. The levels of each of the apoproteins were increased further by substituting casein for soy protein in the semipurified diet. In this case, apo E showed the greatest relative increase (2.7-fold) in VLDL, while apo B and E were increased to a similar extent (about 4-fold) in LDL. Apo C was approximately doubled in each of these fractions. The apo A content in HDL of rabbits fed the semipurified diets was about half that of rabbits fed chow diet. No marked changes were noted in the apo E or C content of HDL. Separation of isoforms of the soluble apoproteins showed variations between individual animals, but these variations seemed largely unrelated to diet. The results of these studies indicate that semipurified diets produce changes in the serum lipoprotein patterns of rabbits that are only partly due to the protein component of these diets.     

Apolipoprotein and lipid distribution between vesicles and HDL-like particles formed during lipolysis of human very low density lipoproteins by perfused rat heart

A study was undertaken to determine the relative association of lipid and apolipoproteins among lipoproteins produced during lipolysis of very low density lipoproteins (VLDL) in perfused rat heart. Human VLDL was perfused through beating rat hearts along with various combinations of albumin (0.5%), HDL2, the infranatant of d greater than 1.08 g/ml of serum, and labeled sucrose. The products were resolved by gel filtration, ultracentrifugation, and hydroxylapatite chromatography. The composition of the lipoprotein products was assessed by analysis of total lipid profiles by gas-liquid chromatography and immunoassay of apolipoproteins. A vesicle particle, which trapped and retained 1-2% of medium sucrose, co-isolated with VLDL and VLDL remnants by gel filtration chromatography but primarily with the low density lipoprotein (LDL) fraction when isolated by ultracentrifugation. The vesicle was resolved from apoB-containing LDL lipolysis products by hydroxylapatite chromatography of the lipoproteins. The vesicle lipoprotein contained unesterified cholesterol (34%), phosphatidylcholine and sphingomyelin (50%), cholesteryl ester (6%), triacylglycerol (5%), and apolipoprotein (5%). The apolipoprotein consisted of apoC-II (7%), apoC-III (93%), and trace amounts of apoE (1%). When viewed by electron microscopy the vesicles appeared as rouleaux structures with a diameter of 453 A, and a periodicity of 51.7 A. The mass represented by the vesicle particle in terms of the initial amount in VLDL was: cholesterol (5%), phosphatidylcholine and sphingomyelin (3%), apoC-II (0.5%), apoC-III (2.2%). The majority of the apoC and E released from apoB-containing lipoproteins was associated with neutral-lipid core lipoproteins proteins which possessed size characteristics of HDL. The vesicles were also formed in the presence of HDL and serum and were not disrupted by serum HDL. It is concluded that lipolysis of VLDL in vitro results in the production of VLDL remnants and LDL apoB-containing lipoproteins, as well as HDL-like lipoproteins. A vesicular lipoprotein which has many characteristics of lipoprotein X found in cholestasis, lecithin: cholesterol acyltransferase deficiency, and during Intralipid infusion is also formed. The majority of apolipoprotein C and E released from apoB-containing lipoproteins is associated with the HDL-like lipoprotein. It is suggested that the formation and stability of the vesicle lipoprotein may be related to the high ratio of cholesterol/phospholipid in this particle.     

Secretion of lipids, apolipoproteins, and lipoproteins by human hepatoma cell line, HepG2: effects of oleic acid and insulin

The aim of this study was to determine the effect of oleic acid and insulin on the secretion of lipoproteins by HepG2 cells grown in minimum essential medium. Triglycerides were the major neutral lipid (57% of total) and apoB was the predominant apolipoprotein (56% of total) secreted by these cells. The addition of oleate resulted in a two-fold increase in the concentration of neutral lipids but only a slight to moderate increase in the apolipoprotein (A-I, A-II, B, and E) levels. The secretion of very low density lipoproteins (VLDL) was stimulated by 425%, low density lipoproteins (LDL) by 77%, and high density lipoproteins (HDL) by 68%. Whereas neutral lipid composition of LDL was unchanged, the VLDL particles contained a significantly higher percentage of triglyceride and lower percentages of cholesterol and cholesteryl esters compared with VLDL secreted in the absence of oleate. Oleate had no significant effect on the composition of apolipoproteins in VLDL, LDL and HDL. In basal medium, 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 their composition by producing particles that had a 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. Insulin had a similar but less pronounced effect on the concentration and composition of lipoproteins secreted in the presence of oleate. The increased accumulation of triglycerides in the HepG2 cells concomitant with their reduced levels in the medium suggests that insulin may affect the secretion rather than synthesis of triglyceride-rich lipoproteins.     

Characterization of nascent lipoproteins produced by perfused rat liver: evidence of hepatic secretion and post-secretory modification of nascent lipoproteins

We characterized the lipoproteins produced by perfused rat liver in recirculating and non-recirculating systems. The apolipoprotein (apo) B of the perfusate very low density lipoprotein (VLDL) and low density lipoprotein (LDL) were labeled with a radioactive precursor amino acid in both systems, suggesting that newly synthesized apo B was secreted in association with VLDL and LDL. When the lipoproteins obtained from the non-recirculating perfusate were injected into rats in vivo, the half life of the VLDL was 13 min and most of it was converted to LDL, while that of the LDL was 5.2 h, indicating that the perfusate LDL was different from the VLDL with respect to its metabolic fate. These observations suggest that both VLDL and LDL are produced as independent primary products in the liver, although the majority of LDL is derived from VLDL in vivo. The nascent lipoproteins in the non-recirculating perfusate were richer in apo E than those in the recirculating perfusate, and a part of the apo E disappeared when the VLDL was added to the recirculating perfusate. The particle sizes of the VLDL and LDL were examined by electron microscopy, which revealed that those in the non-recirculating perfusate were more homogeneous and smaller than the plasma counterparts, while those in the recirculating perfusate were more heterogeneous and their mean diameter was closer to that of the plasma lipoproteins, than in the case of non-recirculating perfusate. These observations suggest that apo E secreted with the nascent lipoproteins may be picked up by the liver just after secretion, causing the heterogeneity in size, as observed in the case of plasma lipoproteins.     

Isolation and analysis of lipoproteins secreted by rat liver hepatocytes

A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.     

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)     

Concentration and composition of serum lipoproteins in the fetal rat]

1. Concentration and composition of the "very low density lipoproteins" (VLDL), "low density lipoproteins" (LDL) and "high density lipoproteins" (HDL) and of non-floatable lipids of fetal rat serum (day 22 of pregnancy) were determined by ultracentrifugation, thin-layer chromatographic separation of the floated lipids and quantitation of the lipid and protein moiety. 2. The concentration of VLDL is in the fetal rat by one order of magnitude lower, and that of LDL, 5fold higher than in the adult animal; the concentration of HDL in fetal serum amounts to 60% of the value of adult animals. 3. The composition of LDL and HDL of fetal serum does not differ from that in the serum of adult animals; in contrast, the fetal VLDL have a higher proportion of protein and cholesterol and a lower proportion of triglycerides than the VLDL of adult serum. The electrophoretic mobility of the fetal VLDL is lower than that of adult VLDL.     

Lipoprotein secretion in lean and obese Zucker female rats in vivo and in a single-pass-perfused liver preparation

The plasma lipoprotein composition as well as lipoprotein synthesis and secretion were studied in vivo and in a single-pass-perfused liver preparation in lean and obese Zucker rats. Compared with their lean littermates the levels in the plasma of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) + low density lipoprotein (LDL) and high density lipoprotein (HDL) were increased 4-, 2- and 2.5 fold, respectively, in obese rats. In these rats both VLDL and IDL + LDL were enriched in triglycerides, while the HDL were enriched in cholesterol. Although the VLDL and IDL + LDL protein concentrations were the same in lean and obese rats, the HDL protein concentration was 3-fold greater in the obese rats. Both the lean and obese rats incorporated similar amounts of [14C]leucine into total liver protein. However, obese rats incorporated 2.5-fold and 6-fold more [14C]leucine into VLDL and HDL in vivo, 2.7-fold and 1.7 fold more [35S]methionine in VLDL and HDL present in the perfusate, than did lean rats. The perfusate [35S]S-labelled apoproteins (apo-B100, B48; apo-E, apo-AI, apo-AIV and apo-C) were separated by gel electrophoresis and identified by autoradiography. Incorporation of [3H]glycerol into liver, VLDL, IDL + LDL and HDL triglycerides was 2-, 48-, 13- and 1.5-fold higher in obese than in lean rats, respectively. The [3H]-labelled triglycerides in VLDL and IDL + LDL present in the perfusate was 5.4-fold and 4.4-fold more in obese rat. There was no difference in the incorporation of [3H]glycerol into triglycerides of perfusate HDL between the two genotypes of rats. Thus, the hypertriglyceridaemia observed in obese Zucker rats results from very high synthetic rates of both the lipid and protein moieties of plasma lipoproteins. Before this study, no report of the simultaneous triglycerides and protein synthesis in vivo and in a single-pass-perfused liver preparations had been reported.     

A rapid electrophoretic technique for identification of subunit species of apoproteins in serum lipoproteins

The denaturing solvent tetramethylurea (TMU) delipidates and quantitatively liberates the apoproteins of human serum high-density lipoprotein (HDL) in soluble form while virtually the whole apoprotein of human lowdensity lipoprotein (LDL) is precipitated. A fraction of the apoprotein of very low density lipoprotein (VLDL) which appears to represent its content of LDL-like protein (apo B) is precipitated by this reagent, while the remaining apoprotein species are liberated in soluble form.The dissociation of the soluble apoproteins from lipid by TMU obviates the need for time-consuming delipidation by organic solvents, permitting immediate electrophoretic analysis in polyacrylamide gels. Bands are observed with mobilities corresponding to those of all the major soluble polypeptide species isolated from serum lipoproteins by ion-exchange chromatography. The apparent distribution of these elements in the different classes of lipoproteins is in agreement with findings of studies employing chromatographic methods. The predominant apoprotein of HDL, which has been identified immunochemically in VLDL, appears to comprise less than 1% of the apoprotein of VLDL from normal serum.     

Metabolsim of apoB and apoC lipoproteins in man: kinetic studies in normal and hyperlipoproteininemic subjects.

The kinetics of apolipoproteins B and C were studied in 14 normal and hyperlipoproteinemic subjects after injection of exogenously (125)I-labeled very low density lipoprotein (VLDL) particles. Plasma radioactivities of apoB and apoC were determined over a period of 4 days in VLDL (d < 1.006) and total radioactivity in intermediate (IDL) (1.006 < d < 1.019), low (LDL) (1.019 < d < 1.063), and high (HDL) (1.063 < d < 1.21) density lipoproteins. The data were analyzed by the use of a model, developed mostly from these data, with the following results. The VLDL particle undergoes a series of incremental density changes, most likely due to a number of delipidation steps, during which apoB stays with the particle until the density reaches the IDL range. There is, however, a loss of apoC associated with these delipidation steps. In our normal subjects, all IDL apoB eventually becomes LDL. In our hyperlipemic subjects some of the apoB on IDL is also degraded directly. The apoC lost by VLDL and IDL recycles to HDL, and most of it is picked up again by newly synthesized VLDL. There is a slowdown of the stepwise delipidation process in all hyperlipemic individuals studied. Three additional features became apparent in the type III subjects. First, there is a significant increase (a factor of 2 compared to normal) in the apoB synthesis rate by way of VLDL; second, there is an induced direct apoB synthesis pathway by way of IDL (and/or LDL); third, a bypass of the regular stepwise VLDL delipidation pathway is induced by which VLDL particles lose apoC but none of their apoB, thereby forming a new particle with metabolic properties similar to LDL, but with a density still in the VLDL density range. Two type III patients treated with nicotinic acid and clofibrate showed a sharp decrease in their VLDL apoB synthesis rates. This was somewhat compensated by an increased IDL apoB synthesis rate. A type I patient on a medium chain triglyceride diet also showed a number of metabolic changes, including reduced VLDL apoB synthesis and the induction of considerable IDL and/or LDL apoB synthesis.     

Factors affecting the esterification of lipoprotein cholesterol by lecithin: cholesterol acyl transferase.

Lecithin: Cholesterol Acyltransferase (LCAT) esterified relatively small amounts of cholesterol from very low density lipoproteins (VLDL), low density lipoproteins (LDL) or high density lipoproteins (HDL) in the presence of 5% human serum albumin (HSA). On the other hand, in the presence of very high density (>1.225 g/ml) plasma fraction (F-4), the enzyme esterified cholesterol from VLDL at considerably higher rates than from LDL or HDL. VLDL together with some component present in the very high density plasma fraction (F-4) may thus provide a highly efficient complex resulting in a favorable configuration of substrate lipids for the enzyme.     

Lipolysis-induced degradation of apolipoproteins B and E of human very low density lipoproteins

We have found that in vitro lipolysis of human very low density lipoproteins (VLDL) by purified bovine milk lipoprotein lipase (LpL) promotes degradation of the apolipoprotein (apo) B moiety of VLDL. Analysis by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis showed that lipolysis of VLDL by purified LpL for 1 h at 37 degrees C induced the selective degradation of the high Mr apo-B (apo-B-100) from most hypertriglyceridemic VLDL and from a few normolipidemic VLDL into several small fragments with molecular weights ranging from 90,000-490,000. No detectable degradation of apo-B occurred in control VLDL when incubated without LpL. The apo-E moiety of VLDL from certain individuals was also degraded following lipolysis of VLDL, and the extent of degradation of apo-B and -E in VLDL was varied among the individual VLDL. The major degradation products of apo-E, identified from the gel, were 31,000- and/or 28,000-Da species. In contrast to the apo-E moiety of VLDL, purified apo-E was not degraded when incubated with LpL. Incubation of low density lipoproteins (LDL) with LpL showed only a minimal effect on the apoproteins of LDL. When high density lipoprotein (HDL) was included in the lipolysis mixture as an acceptor of lipolytic surface remnants, the apoproteins of HDL remained unaltered, while the apo-B moiety of VLDL remnants in the mixture was degraded. Inclusion of protease inhibitors in the lipolysis mixture prevented the degradation of apo-B, but the hydrolysis of VLDL-triglyceride was minimally affected. A selective degradation of apo-B in VLDL also occurred during lipolysis of VLDL when VLDL was perfused through rat hearts. These results suggest that conformational changes in apo-B and apo-E caused by VLDL lipolysis may increase the susceptibility of apo-B and apo-E to degradation by the proteases co-isolated with VLDL. The consequences of the lipolysis-induced degradation of apo-B and apo-E on changes in metabolic properties of VLDL remnants remain to be determined.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.