Apolipoprotein synthesis and secretion in Hep G2 cells: effects of monensin and cycloheximide.

Hep G2 cells were used to study the relationship between apolipoprotein synthesis and secretion, as revealed by their interaction with agents modulating these processes. Cycloheximide inhibited the secretion of both apolipoproteins (apo) AI and B, but the reduction in apo AI secretion was evident at earlier times. Monensin also inhibited secretion of apo AI and apo B, but only apo AI accumulated intracellularly. Pulse-chase studies showed that, at concentrations of monensin that had no effect on total protein synthesis, apo B synthesis was specifically inhibited. Triacylglycerol synthesis was inhibited to the same extent as apo B synthesis, but this preceded the latter inhibition and unlike apo B there was an accumulation of intracellular triglyceride. These results suggest that distinctive mechanisms modulate the synthesis and secretion of apo AI and apo B, and that apo B synthesis can be specifically inhibited by mechanisms that initially block triglyceride production.     

The effect of HMG-CoA reductase inhibitor (CS-514) on the synthesis and secretion of apolipoproteins B and A-1 in the human hepatoblastoma Hep G2

Human hepaotoblastoma cells (Hep G2) were cultured with a competitive inhibitor of HMG-CoA reductase, CS-514. The synthesis of cholesterol was markedly inhibited after 1 h preincubation with CS-514. The synthesis and secretion of apolipoprotein (apo) B and A-1, however, were not affected. A long-term incubation (21–24 h) of cells with CS-514 did not change apo B synthesis and secretion, although a slight depression of apo A-1 synthesis was observed. Hep G2 cells were found to secrete LDL- and HDL-like lipoproteins which were poor in cholesterol when cells were incubated with the drug. These results suggest that the modulation of cholesterol synthesis affects neither the synthesis and secretion of apo B and A-1 nor the formation of lipoproteins.     

Expression of the human serum apolipoprotein AI and AII genes in Xenopus laevis oocytes. Lipid-associated secretion of gene products

The two major apolipoproteins of plasma high-density lipoproteins (HDL) are apolipoprotein AI (apo AI) and AII (apo AII). The apo AI and the correctly oriented apo CIII genes separated by 2.6 kb were obtained by fusion of two human lambda-genomic clones. The apo AII gene was isolated as a 3 kb clone. These apolipoprotein genes have been injected independently and together into Xenopus laevis oocytes and their expression studied. Both apolipoprotein genes were transcribed and translated into their preproforms and processed in Xenopus laevis oocytes to their proforms. They were secreted into the medium associated with newly synthesized phospholipids and neutral lipids as particles floating in the high-density lipoprotein range between 1.12 and 1.21 g/ml. Secreted apo AI is associated mainly with newly synthesized phosphatidylethanolamine and little triglyceride, apo AII with phosphatidylethanolamine, lysophosphatidylethanolamine and neutral lipids. Simultaneous injection of the apo AI and apo AII genes led to the secretion of both apoproteins which separated into two bands during CsCl-density gradient centrifugation. The heavier particles were associated with proapo AI and AII, phosphatidylethanolamine (greater than 90%) and traces of lysophosphatidylethanolamine as lipid components. Proapo AII was immunoprecipitated from the less dense fraction and found to be mainly associated with lysophosphatidylethanolamine. Radiolabelled newly synthesized apolipoproteins in secreted particles were characterized by immunoprecipitation after delipidation of the secreted lipoprotein particles. The oocyte-system proved very suitable for studies of the expression of serum apolipoprotein genes, the assembly of the apolipoproteins with specific lipids to lipoprotein particles and their secretion.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

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.     

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.     

Serum apolipoprotein AI synthesis in rat hepatocytes and its secretion as proform

Rat hepatocytes in monolayer or suspension culture synthesize serum lipoprotein AI. It is secreted into the serum-free culture medium. Synthesis and secretion processes were studied in the presence of radiolabelled amino acids. The synthesis product of the hepatocytes and the secretion product from the medium were isolated by immunoprecipitation with a mono-specific rabbit antiserum against rat apolipoprotein AI. The intracellular and secreted products were homogeneous and identical in polyacrylamide gel electrophoresis but had reduced electrophoretic mobility as compared to native apolipoprotein AI. They were submitted to automated Edman degradation. They were present in their proform, the N-terminus of which is extended by a hexapeptide. In the presence of rat serum the proform is proteolytically transformed into the mature form of apolipoprotein AI.     

Regulation of apolipoprotein secretion by long-chain polyunsaturated fatty acids in newborn swine enterocytes

Long-chain polyunsaturated fatty acids (LC-PUFA) are important in the development of the immature nervous system, and adding these fatty acids to infant formula has been proposed. To determine the effect of n-3 LC-PUFA on apolipoprotein secretion and lipid synthesis in newborn swine enterocytes, differentiated IPEC-1 cells were incubated for 24 h with docosahexaenoic acid (DHA; 22:6) or eicosapentaenoic acid (EPA; 20:5) complexed with albumin at a fatty acid concentration of 0.8 mM or albumin alone (control) added to the apical medium. Oleic acid (OA; 18:1) was used a control for lipid-labeling studies. Both DHA and EPA reduced apolipoprotein (apo) B secretion by one-half, whereas EPA increased apo A-I secretion. The increased apo A-I secretion occurred primarily in the high-density lipoprotein fraction. These changes in apoprotein secretion were not accompanied by significant changes in synthesis. Modest decreases in apo B mRNA levels were observed for DHA and EPA, whereas there were no changes in apo A-I mRNA abundance. EPA reduced cellular triacylglycerol labeling by one-half, and DHA and EPA decreased cellular phospholipid labeling compared with OA. Labeled triacylglycerol secretion was decreased 75% by EPA, and DHA doubled labeled phospholipid secretion. If present in vivo, these effects should be considered before supplementing infant formula with these fatty acids.     

Cholesterol homeostasis in rat astrocytoma cells GA-1

Astrocytes play a key role in cholesterol metabolism in central nervous system. We have shown that fetal rat astrocytes in primary culture secrete cholesterol-rich HDL with the endogenous apolipoprotein (apo) E and generate cholesterol-poor HDL with exogenous apoE and apoA-I [Ito et al. (1999) J. Neurochem. 72, 2362]. In order to study these reactions in relation to the stage of cell differentiation, we examined generation of HDL by rat astrocytoma cells. Lack of apoE secretion was found in three astrocytoma cell lines, human T98G, rat C6, and GA-1 [Kano-Tanaka et al. (1986) Proc. Jpn. Acad. Ser. B 62, 109]. GA-1 produced apoE at very low level and therefore generated much less HDL by itself than the astrocytes in primary culture. In contrast, GA-1 interacted with exogenous apoE and apoA-I to produce cholesterol-rich HDL while the astrocytes produced cholesterol-poor HDL with these apolipoproteins. Cholesterol biosynthesis rate measured from mevalonate was higher and down-regulated more by LDL in the astrocytes than GA-1. On the other hand, the cellular cholesterol level, uptake of LDL, and cyclodextrin-mediated non-specific diffusion of cholesterol from cell surface were same between these two cells. Treatment of GA-1 with acidic fibroblast growth factor influenced neither the production of apoE nor the baseline lipid secretion, but increased the cholesterol synthesis from mevalonate and the magnitude of its down-regulation by LDL, and decreased cholesterol content in the HDL produced by exogenous apoA-I. In conclusion, suppression of apoE biosynthesis in the undifferentiated astrocytes GA-1 resulted in poor secretion of cholesterol-rich HDL and in turn more production of HDL with exogenous apolipoprotein. Cellular cholesterol homeostasis was altered accordingly.     

Identification and characterization of rodent ABCA1 in isolated type II pneumocytes

ATP-binding cassette transporter A1 (ABCA1) promotes transfer of cholesterol and phospholipid from cells to lipid-free serum apolipoproteins. ABCA1 mRNA and protein expression in primary cultures of rodent type II cells was sensitive to upregulation with 5 microM 9-cis-retinoic acid (9cRA) and 6.2 microM 22-hydroxycholesterol (22-OH). The increase in ABCA1 protein levels was time dependent and was maximal after 16 h of exposure to 9cRA + 22-OH. Inducible ABCA1 was also found in transformed cell lines of lung origin: WI38/VA13, A549, and NIH-H441 cells. Stimulation of ABCA1 in rat type II cells by 9cRA + 22-OH resulted in a four- or fivefold enhancement of efflux of radioactive phospholipid or cholesterol, respectively, from the pneumocytes to apolipoprotein AI (apo AI), whereas cAMP (0.3 mM) had no effect. ABCA1-mediated lipid efflux to apo AI was independent of the surfactant secretion pathway, inasmuch as upregulation of ABCA1 resulted in a reduction of secretagogue-stimulated surfactant phospholipid release. These studies demonstrate the presence of functional ABCA1 in type II cells from the lung.     

Lipid, apolipoprotein, and lipoprotein synthesis and secretion during cellular differentiation in caco-2 cells

Summary Although Caco-2 cells are frequently employed for the study of enterocyte lipid metabolism, variable results have been reported regarding their ability to synthesize and secrete lipids and apolipoproteins. The major goal of this investigation is to examine the capacity of Caco-2 cells to elaborate and secrete lipids, lipoproteins, and apolipoproteins at different degrees of morphological and functional differentiation. Cells were cultured in medium with 5% fetal bovine serum (FBS), on permeable polycarbonate filters from 2 to 30 d in the presence of ¹⁴C-oleate or ³⁵S-methionine. Cellular differentiation, as assessed by morphology (light and electron microscopy), transepithelial resistance, free fatty acid flux, and sucrase activity, progressed steadily up to 20 d of culture. Caco-2 cells esterified oleic acid mainly into phospholipids, triglycerides (TG), and smaller amounts of cholesterol esters. Lipid synthesis began as early as 2 d, and TG secretion was enhanced with increased duration of culture. However, very low efficiency of lipid export was observed at all levels of differentiation, reaching a maximum of only 6% of intracellular lipids. VLDL and LDL were the dominant lipoproteins secreted, with HDL comprising <20% of the total. VLDL secretion increased, while LDL decreased, whereas the lipid composition of lipoproteins varied little with increasing duration of culture. Apoprotein B and A-I synthesis and secretion increased markedly from 11 to 20 d of culture. The ratio of apo B-100/B-48 decreased between 11 and 30 d, consistent with enhanced apo B editing of more mature enterocytes. Taken together, our data suggest that from 20 d of culture, Caco-2 cells are morphologically and functionally mature, capable of lipid esterification, and lipoprotein and apolipoprotein synthesis. However, despite their functional and morphological similarities to mature enterocytes, Caco-2 cells have a very limited lipid export capacity.     

Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem.

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.     

Simvastatin causes the formation of cholesterol-rich remnants in mice lacking apoE

Mice that lack apolipoprotein E (apoE) display a severe hypercholesterolemia, caused by the accumulation of apolipoprotein B-48 (apoB-48)-carrying remnants of chylomicrons and very-low-density lipoproteins in the plasma. Statins are potent inhibitors of cholesterol synthesis that, when administered to mice lacking apoE, cause paradoxical further increases in plasma cholesterol levels. In the present study, we examined the mechanisms responsible for this phenomenon. ApoE-deficient mice fed a chow diet containing simvastatin developed, as anticipated, an enhanced increase in plasma cholesterol and a decrease in plasma triglycerides. Fractionation of the plasma lipoproteins by FPLC revealed that the lipid changes were confined to the lipoprotein remnants. Western blot analysis of the remnants from the untreated and simvastatin-treated mice showed no differences in their apoB-48 content, indicating that both groups of animals accumulated similar numbers of remnant particles in the plasma. Following the injection of Triton WR-1339, the simvastatin-treated mice accumulated in the plasma significantly more cholesterol and significantly less triglycerides than the untreated animals. These results indicate that the enhanced hypercholesterolemia observed in apoE-deficient mice treated with simvastatin is not the result of an increased number of remnant particles in circulation but is caused by synthesis and secretion into the plasma of lipoproteins that are enriched in cholesterol and depleted of triglycerides.     

Transient expression of wild type and mutant human apolipoprotein AI in COS cells

A human apolipoprotein AI (apo AI) minigene and two mutants were cloned into the vector pUHD10-1 for expression studies in COS cells under the control of the strong CMV (cytomegalovirus) enhancer and the own apo AI promoter. In the mutated apo AI minigene (mutant M1) the positions of the triplets of Gln(-2)-Gln-1 at the C-terminus of the prosequence were exchanged against Gln(-8)-Ala-7, the recognition site of the signal peptidase of the wild type human apo AI. The prosequence has been deleted in mutant M2 and the presequence linked directly to the N-terminus of the mature apo AI form. We report here on expression studies in COS cells, a cell line, which does not express apo AI. They were transfected by electroporation with pUHD10-1 constructs, which contain a) the wild type apo AI minigene and b) the two mutant apo AI minigenes with mutations described above. The following results were obtained: a) the wild type and mutant apo AI constructs were efficiently transcribed and translated in COS cells, b) the expression of the wild type preproapo AI minigene in COS cells led to the secretion of proapo AI (29 kDa), that of the mutant (M2) gene, devoid of the prosequence of mature apo AI (28.4 kDa), whereas the product of mutant gene M1 (31 kDa) with the recognition site of the signal peptides transposed to the C-terminus of the prosequence remained uncleaved within the COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of apolipoprotein secretion in very-low-density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone.

The effects of dexamethasone (a synthetic glucocorticoid) and insulin on the secretion of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were investigated. Rat hepatocytes in monolayer culture were preincubated for 15 h in the presence or absence of combinations of 100 nM-dexamethasone and 2 nM-, 10 nM- or 50 nM-insulin. Dexamethasone increased [3H]oleate incorporation into secreted triacylglycerol by 2.7-fold and the mass of triacylglycerol secreted by 1.5-fold. Insulin alone decreased these parameters and antagonized the effect of dexamethasone. Dexamethasone increased the secretion of [3H]leucine in apolipoprotein (apo) E, and in the large (BH) and small (BI) forms of apo B in VLDL by about 7.1-, 3.6- and 4.0-fold respectively. Insulin alone decreased the secretion of these 3H-labelled apolipoproteins in VLDL. However, 2 nM-insulin with dexamethasone increased the secretion of 3H-labelled apo BH and apo BL by a further 0.8- and 3.2-fold respectively; 50 nM-insulin decreased the secretions of apo E, apo BH and apo BL in VLDL. Similar effects for dexamethasone or insulin alone were also obtained for the masses of apo E and apo BL + H secreted in VLDL. Albumin secretion was not significantly altered by either dexamethasone or insulin alone, but in combination they stimulated by 2.1-2.6-fold. Insulin or dexamethasone alone had little effect on the secretion of apolipoproteins in the HDL fraction. However, dexamethasone plus 2 nM-insulin increased the incorporation of [3H]leucine into apo AI, apo AH plus apo C, apo AIV and apo E of HDL by about 1.8-, 1.6-, 1.7- and 2.0-fold respectively. The apo E in the bottom fraction represented about 69% of the total 3H-labelled apo E secreted. The responses in the total secretion of apo E from the hepatocytes resembled those seen in HDL. The interactions of insulin and dexamethasone are discussed in relation to the general regulation of lipoprotein metabolism, the development of hyperlipidaemias and the predisposition to premature atherosclerosis.     

Effect of pravastatin on serum lipids, apolipoproteins and lipoprotein (a) in patients with non-insulin dependent diabetes mellitus.

In 43 patients with non-insulin dependent diabetes mellitus (NIDDM) associated with hypercholesterolemia, the effect of pravastatin, a potent HMG CoA-reductase inhibitor, on serum lipids, apolipoproteins and lipoprotein (a) was examined. After 1 to 3 months administration of 10 mg per day of pravastatin, the serum levels of total cholesterol, triglycerides and low-density lipoprotein cholesterol (LDL-C) were significantly decreased, while the serum level of high density lipoprotein cholesterol (HDL-C) was significantly increased in patients with NIDDM. The levels of apolipoproteins B (apo B) and E were significantly decreased, while apolipoprotein AI (apo A-I) was not changed by the administration of pravastatin. The atherogenic indices (LDL-C/HDL-C and apo B/apo A-I) were significantly decreased by the administration of this drug. The serum lipoprotein (a), which was increased in the diabetic patients, was not affected by the pravastatin treatment. Plasma glucose and hemoglobin A1c levels were not affected by the treatment. We concluded that pravastatin is a potentially useful agent in the treatment of hypercholesterolemia in patients with NIDDM.     

Rat Leydig cells use apolipoprotein E depleted high density lipoprotein to regulate testosterone production

Rat HDL are known to increase testosterone production by cultured Leydig cells either following gonadotropin stimulation or cholesteryl ester depletion. However, rat HDL contain apolipoprotein E and have a high affinity for the members of the low density receptor family such as LDL receptor, LDL receptor related protein and VLDL receptor. In contrast with the adrenal cells, the contribution of apo A-I and apo E pathways in HDL cholesterol uptake has not been yet evidenced in rat Leydig cells. Recent data provided evidence that hCG stimulates scavenger receptor BI expression in testes. In order to investigate if testosterone production can be stimulated by apo E depleted HDL, we compared the level of testosterone stimulation by HDL with or without apo E first, in presence of saturating dose of hCG (1 IU/ml) and second, after depletion of cholesterol synthesis by pravastatin, an inhibitor of HMG-CoA reductase. In presence of hCG, HDL with or without apo E increased testosterone production respectively by 37 and 25%. Pravastatin at 100 g/ml inhibited the cholesterol synthesis and the testosterone production by 25% and decreased the cholesteryl content by 25%. The addition of HDL with or without apo E (50 g protein HDL/ml) completely overcame the depletion of cellular cholesteryl esters and the inhibition of testosterone production induced by pravastatin. In the presence of heparin, apo E depleted HDL overcame the testosterone production induced by pravastatin, indicating that uptake of HDL without apo E via a secretion of apo E by the cells themselves was not involved. Therefore, in absence of apo E, it is suggested that rat Leydig cells used HDL to regulate steroidogenesis via an apolipoprotein A-I pathway.     

Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver.

The effects of oleic acid on the biosynthesis and secretion of VLDL (very-low-density-lipoprotein) apoproteins and lipids were investigated in isolated perfused rat liver. Protein synthesis was measured by the incorporation of L-[4,5-3H]leucine into the VLDL apoproteins (d less than 1.006) and into apolipoproteins of the whole perfusate (d less than 1.21). Oleate did not affect incorporation of [3H]leucine into total-perfusate or hepatic protein. The infusion of oleate, however, increased the mass and radioactivity of the VLDL apoprotein in proportion to the concentration of oleate infused. Uptake of oleate was similar with livers from fed or fasted animals. Fasting itself (24 h) decreased the net secretion and incorporation of [3H]leucine into total VLDL apoprotein and decreased the output of VLDL protein by the liver. A linear relationship existed between the output of VLDL triacylglycerol (mumol/h per g of liver) and secretion and/or synthesis of VLDL protein. Net output of VLDL cholesterol and phospholipid also increased linearly with VLDL-triacylglycerol output. Oleate stimulated incorporation of [3H]leucine into VLDL apo (apolipoprotein) E and apo C by livers from fed animals, and into VLDL apo Bh, B1, E and C by livers from fasted rats. The incorporation of [3H]leucine into individual apolipoproteins of the total perfusate lipoprotein (d less than 1.210 ultracentrifugal fraction) was not changed significantly by oleate during perfusion of livers from fed rats, suggesting that the synthesis de novo of each apolipoprotein was not stimulated by oleate. This is in contrast with that observed with livers from fasted rats, in which the synthesis of the total-perfusate lipoprotein (d less than 1.210 fraction) apo B, E and C was apparently stimulated by oleate. The observations with livers from fed rats suggest redistribution of radioactive apolipoproteins to the VLDL during or after the process of secretion, rather than an increase of apoprotein synthesis de novo. It appears, however, that the biosynthesis of apo B1, Bh, E and C was stimulated by oleic acid in livers from fasted rats. Since the incorporations of [3H]leucine into the VLDL and total-perfusate apolipoproteins were increased in fasted-rat liver when the fatty acid was infused, part of the apparent stimulated synthesis of the VLDL apoprotein may be in response to the increased formation and secretion of VLDL lipid.