Lipoprotein metabolism in the ovariectomized rat

The hyperlipoproteinemia observed after ovariectomy in rats was previously shown to be associated with increased concentrations of cholesterol, triglycerides, and apolipoproteins B, E, and C. In the present study, it was shown that increases in low density lipoproteins and high density lipoproteins were almost entirely responsible for the changes in plasma lipids and apolipoproteins after ovariectomy. The size of the low density lipoproteins and high density lipoproteins isolated from the plasma of ovariectomized rats as determined by agarose chromatography appeared to be somewhat different from that of control rats. Specifically, the apolipoprotein B appeared to be associated with somewhat smaller particles, whereas the apolipoprotein E from those rats appeared to be associated with larger particles than that of control rats. To determine the mechanism for the increased plasma low density lipoproteins, apolipoprotein B pool sizes and turnover rates were calculated and compared. In addition to an increased mass of low density lipoproteins in ovariectomized rats, the turnover rate of low density lipoproteins was increased almost twofold, indicating an increased low density lipoprotein synthesis and catabolism in those animals. We postulate that the increased low density lipoprotein levels of ovariectomized rats are due to an initial increased production of low density lipoproteins, followed by an enhanced catabolism of low density lipoproteins to establish a steady state at higher plasma low density lipoprotein concentrations.     

Isolation of apolipoproteins A-I, A-II and E and their estimation by rocket immunoelectrophoresis in blood plasma of dis-alpha-lipoproteinemic patients

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.     

Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey.

O-(4-Diazo-3-[125I]iodobenzoyl)sucrose ([125I]DIBS), a novel labelling compound specifically designed to study the catabolic sites of serum proteins [De Jong, Bouma, & Gruber (1981) Biochem. J. 198, 45-51], was applied to study the tissue sites of degradation of serum lipoproteins. [125I]DIBS-labelled apolipoproteins (apo) E and A-I, added in tracer amounts to rat serum, associate with high-density lipoproteins (HDL) just like conventionally iodinated apo E and A-I. No difference is observed between the serum decays of chromatographically isolated [125I]DIBS-labelled and conventionally iodinated HDL labelled specifically in either apo E or apo A-I. When these specifically labelled HDLs are injected into fasted rats, a substantial [125I]DIBS-dependent 125I accumulation occurs in the kidneys and in the liver. No [125I]DIBS-dependent accumulation is observed in the kidneys after injection of labelled asialofetuin or human low-density lipoprotein. It is concluded that the kidneys and the liver are important sites of catabolism of rat HDL apo E and A-I.     

Effects of atorvastatin versus fenofibrate on apoB-100 and apoA-I kinetics in mixed hyperlipidemia

Kinetics of apo B and apo AI were assessed in 8 patients with mixed hyperlipidemia at baseline and after 8 weeks of atorvastatin 80 mg q.d. and micronised fenofibrate 200 mg q.d. in a cross-over study. Both increased hepatic production and decreased catabolism of VLDL accounted for elevated cholesterol and triglyceride concentrations at baseline. Atorvastatin significantly decreased triglyceride, total, VLDL and LDL cholesterol and apo B concentrations (-65%, -36%, -57%, -40% and -33%, respectively, P<0.05). Kinetic analysis revealed that atorvastatin stimulated the catabolism of apo B containing lipoproteins, enhanced the delipidation of VLDL1 and decreased VLDL1 production. Fenofibrate lowered triglycerides and VLDL cholesterol (-57% and -64%, respectively, P<0.05) due to enhanced delipidation of VLDL1 and VLDL2 and increased VLDL1 catabolism. Changes of HDL particle composition accounted for the increase of HDL cholesterol during atorvastatin and fenofibrate (18% and 23%, P<0.01). Only fenofibrate increased apo AI concentrations through enhanced apo AI synthesis (45%, P<0.05). We conclude that atorvastatin exerts additional beneficial effects on the metabolism of apo B containing lipoproteins unrelated to an increase in LDL receptor activity. Fenofibrate but not atorvastatin increases apo AI production and plasma turnover.     

Polymorphisms of apolipoproteins A-IV and E in a Turkish population living in Germany

Human apolipoproteins (apo) E and apo A-IV are polymorphic with significantly different allele frequencies among different ethnic groups. Whereas the variation at the apo E gene locus affects plasma cholesterol levels in all populations studied so far and is associated with longevity in Caucasians, the influence of the common apo A-IV polymorphism on plasma lipoproteins has not been unanimously accepted. We have therefore determined the common apo E and apo A-IV polymorphisms by isoelectric focusing, calculated the respective allele frequencies and studied their effects on plasma lipoproteins in a random sample of 240 nonrelated Turkish subjects (141 males, 99 females) living in Germany and originating from central and eastern Anatolia. When compared with the German population and other Caucasians in Europe a prominence of the apo ɛ3 allele frequency (0.885) was accompanied by a decrease in the frequencies of both the apo ɛ2 allele (0.048) and the apo ɛ4 allele (0.067). Thus, the Turkish population studied here clustered with populations mainly from southern Europe and Japan, which have low ɛ2 and ɛ4 allele frequencies. Also, the frequency of the A-IV-1 allele was higher (0.967) and that of the A-IV-2 allele lower (0.033) in the Turkish subjects studied than in other populations. At an average level of total cholesterol of 194.5 ± 45 mg/dl, no significant influence of the A-IV alleles on plasma lipoproteins was seen. However, apo E and apo B differed significantly between apo E phenotypes, with high levels of apo E and low levels of cholesterol and apo B in carriers of the ɛ2 allele, and vice versa for the ɛ4 allele. The average cholesterol excess for the ɛ2 allele was –7.95 mg/dl, for the ɛ3 allele, –1.34, and for the ɛ4 allele, +14.15 mg/dl. Thus, despite the unusual frequency distribution of the apo E alleles, their effects on plasma lipoproteins are within the range reported for other populations in Europe. Received: 10 April 1995 / Revised: 25 March 1996     

Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes

We have investigated the binding of human apolipoprotein A-IV (apo A-IV) to human hepatocellular plasma membranes. Addition of increasing concentrations of radiolabeled apo A-IV to hepatic plasma membranes, in the presence and absence of a 25-fold excess of unlabeled apo A-IV, revealed saturation binding to the membranes with a KD of 154 nM and a binding maximum of 1.6 ng/microgram of membrane protein. The binding was temperature-insensitive, partially calcium-dependent, abolished when apo A-IV was denatured by guanidine hydrochloride or when the membranes were treated with Pronase and decreased when apo A-IV was incorporated into phospholipid/cholesterol proteoliposomes. In displacement studies using purified apolipoproteins and isolated lipoproteins, only unlabeled apo A-IV, apo A-I and high-density lipoproteins effectively competed with radiolabeled apo A-IV for membrane binding sites. We conclude that human apo A-IV exhibits high-affinity binding to isolated human hepatocellular plasma membranes which is saturable, reversible and specific.     

Interaction of plasma lipoproteins and apolipoproteins with lipid bilayer membranes

It was shown that the interaction of lipoproteins (LP) with bilayer lipid membranes (BLM) resulted in some changes in the physical-chemical properties of the membranes. Adsorption of very low and low density lipoproteins (VLDL and LDL) at concentrations of 5-8 g protein/ml increased the surface potential difference and decreased transversal elasticity module of the bilayer. LP concentrations higher than the mentioned ones increased BLM conductance and caused instability and disruption of the membranes. The same effects were revealed for high density lipoproteins (HDL) at higher concentrations--15-20 micrograms protein/ml. The effect of apolipoproteins in the interaction of LP with BLM was investigated. It is proposed that apolipoproteins and especially apo B are the main factor which affects the nonreceptor interactions of LP with the membranes.     

Importance of apolipoproteins in lipid metabolism

Lipids, which serve as a source of energy and are an important constituent of cell membrane structure, are readily stored in the body. By definition they are insoluble in water. Specific proteins called apolipoproteins interact with lipids to form soluble lipid-protein complexes called lipoproteins. It is in this form that the major lipids — cholesterol, triglyceride and phospholipid — circulate in plasma. Unesterified fatty acids, another major lipid group, are bound to albumin in the circulation. The plasma lipoproteins are complex macromolecules composed of lipids, apolipoproteins and carbohydrates. The relative proportions of these components differ markedly between lipoprotein classes.

Hyperlipidemia is a term used for increased concentrations of plasma cholesterol and/or triglycerides. Any one plasma lipid is present in several types of lipoproteins. Thus, hyperlipidemia implies the presence of hyperlipoproteinemia. The latter has important therapeutic implications. Most of the recent attempts at classification have been directed at the lipoprotein level of plasma lipid organization.

Decreased concentrations of lipids in plasma can be achieved by altering the rates of metabolism of lipoproteins. Decrease in lipoprotein synthesis, increased catabolism or impaired release from cells into the blood stream may all result in a decrease of plasma lipids. Drugs which affect one or more of these factors are used to treat hyperlipoproteinemia. In order to elucidate the mechanism of action of hypolipidemic drugs it is necessary to understand the lipoprotein defect at the molecular level. This requires a more detailed knowledge of lipoprotein metabolism than is presently available for most of the hyperlipoproteinemias.

This paper will review some of the generally accepted properties of the plasma lipoproteins, describe some difficulties which hamper the understanding of lipoprotein metabolism, and identify possible mechanisms by which drugs may affect lipoprotein metabolism.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

Apolipoproteins and amyloid fibril formation in atherosclerosis

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures. The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases. A number of plasma apolipoproteins, including apolipoprotein (apo) A-I, apoA-II, apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins. We review present knowledge of amyloid formation by apolipoproteins in disease, with particular focus on atherosclerosis. Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions. Additionally, we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis, and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.     

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci

We have tested for evidence of linkage between the genetic loci determining concentrations and composition of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes participating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple sequence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determining plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) locus on chromosome 6 (P = 0.02). Thus, these data indicate the simultaneous involvement of at least two different genetic loci in the determination of the levels of HDL and its associated lipoproteins.     

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.     

Lipoprotein composition as a component in the lipoprotein clearance defect in experimental diabetes

The hypertriglyceridemia associated with streptozotocin-induced diabetes in rats is largely reflected in the plasma lipoproteins of density less than 1.006 g/ml. Analysis of the plasma apolipoproteins of these rats indicated marked alterations in both the total levels and in the lipoprotein distribution of the major apolipoproteins. In whole plasma, diabetes was associated with significant increases in apolipoprotein (apo)-AIV, apo-AI, and apo-B (mainly in the intestinally derived apo-B240) and a marked decrease in apo-E. In the d less than 1.006 g/ml lipoprotein fraction (very-low-density lipoproteins (VLDL], there were significant increases in apo-B240, apo-AI, and apo-AIV and decreased levels of apo-E and the C apolipoproteins. The decrease in apo-C was primarily due to lower levels of apo-CII, and the ratio of the lipoprotein lipase inhibitor, apo-CIII, to the lipoprotein lipase activator, apo CII, was significantly increased over that in controls. The comparative clearance of triglycerides of VLDL particles from control and diabetic rat plasma was tested in recirculating heart perfusion in vitro. During 45-min perfusions of hearts from control donor rats, lipolysis of triglycerides of VLDL from diabetic rats was only 63-64% of that using plasma VLDL from control rats. Perfusion of hearts from diabetic rats with VLDL from control rats gave lipolysis values of only 53% of that obtained with normal hearts. Where both the VLDL and hearts were obtained from diabetic rats, lipolysis was 23% of that observed when both the lipoprotein and the organ were from control rats. The data suggest that in addition to depressed lipoprotein lipase activity in the tissue from diabetic rats, there are also major compositional changes in circulating lipoproteins which may contribute to defective triglyceride clearance from the circulation.     

Interaction of unilamellar liposomes with serum lipoproteins and apolipoproteins

The effect of rat whole blood plasma, serum, serum lipoproteins, and apolipoproteins on the stability of unilamellar liposomes prepared with French pressure cell was evaluated by measuring the release of entrapped carboxyfluorescein and by electron microscopy. In the absence of serum components, dye escaped very slowly (hours) from egg phosphatidylcholine and phosphatidylcholine-cholesterol (43 mol % cholesterol) vesicles without apparent change in liposomal structure. This slow release was both temperature- and size-dependent. serum and some of its constituents induced a far more rapid (seconds) loss of entrapped dye from phosphatidylcholine liposomes, associated with structural changes. For equal masses of protein the order of potency of this induced activity was: free apolipoproteins (apo A-I, apo E) > isolated lipoproteins (HDL and VLDL) > whole serum or whole plasma. Substantial activity was found in three preparations of bovine serum albumin. This activity could be attributed to small and variable amounts of contaminating lipoprotein-like particles and apolipoprotein A-I. Induced release of dye from liposomes by apolipoproteins was usually associated with rapid formation of discs although other structures were sometimes formed. Purified rat apolipoproteins A-I and E appeared to interact identically with liposomes to induce dye release. This effect was progressively impaired for both apoproteins by increasing amounts of cholesterol and was completely inhibited when liposomes contained 37 mol % cholesterol.     

Responses of plasma apolipoproteins to gonadectomy and androgen substitution in male rats

To obtain information on testosterone effects on plasma apolipoproteins, the amount and composition of apo-proteins in lipoproteins of 5 density classes (VLDL, LDL, HDL2b, HDL2a, HDL3) was estimated in 3 groups of adult male rats: normal control rats, castrated rats, and rats injected daily with testosterone propionate (200 micrograms/day) for one week after castration. Apoproteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis after ultracentrifugation of plasma, and determined colorimetrically. Total amount of apoprotein carried in LDL (d = 1.006-1.063 g/ml) and HDL2b (d = 1.063-1.100 g/ml) was higher in castrated than in control rats, but was not significantly different from controls in testosterone substituted rats. LDL apo B and HDL2b apo E were higher in castrated than in normal rats; control levels were observed in androgen substituted rats. Except for a greatly increased relative amount of HDL2b apo E, and a decreased percentage of HDL2b apo A-I in castrated rats, there were no significant alterations by castration of apoprotein composition of the lipoproteins. The results raise the question whether the androgenic state might affect processes related to the effects of plasma LDL apo B and HDL apo E.     

Characterization of disulfide-linked heterodimers containing apolipoprotein D in human plasma lipoproteins.

Human plasma apolipoprotein (apo) D is a glycoprotein with an apparent molecular weight of 29,000 M(r). It is present, mainly, in high density lipoproteins (HDL) and very high density lipoproteins (VHDL). Western blot analysis of HDL and VHDL using rabbit antibodies to human apoD revealed major immunoreactive bands at 29,000 and 38,000 M(r), with minor bands ranging from 50,000 to and 80,000 M(r). Only the 29,000 M(r) band corresponding to apoD remained when the electrophoresis was conducted under reducing conditions, demonstrating that apoD is cross-linked to other proteins via disulfide bonds. The broad pattern of immunoreactivity was also observed under nonreducing conditions when the blood was collected into a solution of sulfhydryl-trapping reagents, or when these reagents were added to the isolated lipoproteins. These results indicated that the disulfide bonds were not the result of disulfide exchange during the experimental procedures. On the basis of amino acid sequencing and reactions to antibodies, the 38,000 M(r) band was identified as an apoD-apoA-II heterodimer. The apoD-apoA-II was also demonstrated in plasma. In both HDL and plasma, the apoD-apoA-II heterodimer constituted the major form of apoD. Disulfide-linked heterodimers of apoD and apoB-100 were also found in low and very low density lipoproteins, and in whole plasma. It is concluded that a fraction of human apoD, like other cysteine-containing apolipoproteins, exists as a disulfide-linked heterodimer with other apolipoproteins in all major human lipoprotein fractions.     

Structure and conformational analysis of lipid-associating peptides of apolipoprotein B-100 produced by trypsinolysis

Apolipoprotein B-100 (apo B-100) contains putative lipid-associating regions that are, in part, responsible for its overall structure in human plasma low-density lipoproteins. Some of these regions have been identified by reassembly of the total tryptic peptides of apo B-100 with bovine brain sphingomyelin, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and dimyristoylphos-phatidylcholine (DPMC). Although more than 500 tryptic peptides are predicted from the known number of arginines and lysines in apo B-100, significant amounts of only 13 peptides spontaneously associate with all three phospholipids. These peptides share some structural characteristics, as predicted by several algorithms, that distinguish them from the water-soluble apolipoproteins. Most apolipoproteins associate with lipids via amphipathic helices and are highly helical in native and reassembled lipoproteins. Analysis of all apo B-100 lipophilic peptides by circular dichroism and by use of a predictive algorithm reveals no evidence of amphipathic helices. Although the predictive algorithm suggested that the lipophilic peptides of apo B-100 contain the sequence determinants for -sheet, no spectroscopic evidence for this structure was found. We conclude that the lipophilic regions of apo B-100 liberated by trypsinolysis are highly hydrophobic, although their secondary structures do not fit any simple model.     

Folic acid supplementation delays atherosclerotic lesion development in apoE-deficient mice

Folic acid is a vitamin that when used as a dietary supplementation can improve endothelial function. To assess the effect of folic acid on the development of atherosclerosis, male apolipoprotein E-deficient mice fed a standard chow diet received either water (control group) or an aqueous solution of folic acid that provided a dose of 75 microg/kg/day, for ten weeks. At the time of sacrifice, blood was drawn and the heart removed. The study measured plasma homocysteine, lipids, lipoproteins, low-density lipoprotein (LDL) oxidation, isoprostane, paraoxonase, and apolipoproteins, and aortic atherosclerotic areas. In folic acid-treated animals, total cholesterol, mainly carried in very low-density and low-density lipoproteins, increased significantly, and homocysteine, HDL cholesterol, paraoxonase, and triglyceride levels did not change significantly. Plasma isoprostane and apolipoprotein (apo) B levels decreased. The resistance of LDL to oxidization and plasma apoA-I and apoA-IV levels increased with a concomitant decrease in the area of atherosclerotic lesions. The administration of folic acid decreased atherosclerotic lesions independently of plasma homocysteine and cholesterol levels, but was associated with plasma levels of apolipoproteins A-I, A-IV and B, and decreased oxidative stress.     

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.     

Identification of factors regulating lipoprotein lipase catalyzed hydrolysis in rats with the aid of monoacid-rich lipoprotein preparations(1)

To identify the substrate specificity and regulatory factors in lipoprotein lipase (LPL) catalyzed hydrolysis of triacylglycerol-rich lipoprotein, monoacid-rich lipoproteins were used to study the kinetic parameters of LPL. Feeding growing rats with diets rich in palmitic acid (16:0), oleic acid (18:1) or linoleic acid (18:2) for 10 days increased the corresponding acid content in the triacylglycerols of the lipoproteins. Force-feeding the monoacid-rich triacylglycerols, particularly 16:0 or 18:1, increased the respective fatty acid content in both chylomicrons and VLDLs. Major apolipoproteins and lipid compositions were essentially similar among all lipoproteins differing in monoacid species, except for apo A-IV. The Vmax of LPL for 16:0-rich chylomicrons and VLDLs were higher than for 18:1- or 18:2-rich lipoproteins. Order parameter (S), an indicator of the surface fluidity of lipoproteins, decreased with the chain length and unsaturation of monoacid in similar manner as the Vmax. The Vmax of LPL increased linearly (P < 0.05) with an increase in either the palmitic acid content of the lipoprotein triacylglycerols or order parameter (S) of the lipoproteins. The order parameter (S) and Vmax of LPL were higher in 16:0 triacylglycerol emulsions with apo B than with 18:1 or 18:2 triacylglycerols. The apo A-IV in triacylglycerol emulsions stimulated Vmax of LPLs in the presence of apo B and apo C-II. The binding of apo A-IV to 16:0 triacylglycerol emulsions was higher than to other triacylglycerol emulsions. These findings suggest that lipoprotein catalysis by LPL is modulated by the 16:0 level in the lipoprotein triacylglycerol, which affects the surface fluidity and apo A-IV content of lipoproteins.