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.     

Effect of fish oil diet on hepatic lipid metabolism in nonhuman primates: lowering of secretion of hepatic triglyceride but not apoB

African green monkeys were fed diets containing either 11% (by weight) fish oil or lard for 2.5 yr. To test the hypothesis that fish oil decreases hepatic secretion of triglyceride (TG) and apoB, livers from these animals were perfused with a fatty acid mixture [85% (w/w) oleate containing [14C]oleate and 15% n-3 containing [3H]eicosapentaenoic acid (EPA)] at a rate of 0.1 mumol fatty acid/min per g liver. Liver perfusate was sampled every 30 min during 4 h of recirculating perfusion. The concentration of triglyceride was similar for livers of animals of both groups and there was no difference between groups in the extent of incorporation of [3H]EPA or [14C]oleate into hepatic TG. While the secretion rate for the mass of TG was less in the fish oil-fed group (8.3 +/- 2.5 vs 18.3 +/- 4.4 mg/h per 100 g liver, P less than 0.05), the apoB secretion rate was similar (0.92 +/- 0.15 vs 1.01 +/- 0.13 mg/h per 100 g liver). Significantly less [3H]EPA was incorporated into secreted TG in the fish oil group (0.4 +/- 0.1 vs 1.0 +/- 0.1% infused dose/h; P less than 0.01). The rate of secretion of [14C]TG was similar for both groups (1.3 +/- 0.3 vs 1.4 +/- 0.1% infused dose/h for fish oil and lard groups, respectively). No significant diet-related differences in [3H]TG or [14C]TG fatty acid specific activity were observed for perfusate TG or hepatic TG. After perfusion, livers from fish oil-fed monkeys contained significantly more [3H]EPA in hepatic phospholipid than livers from lard-fed monkeys (19.5 +/- 1.8 vs 11.4 +/- 1.7% infused dose; P less than 0.01) although hepatic phospholipid mass concentrations were similar. The liver phospholipids of the fish oil group were enriched in n-3 fatty acid mass and were relatively depleted of oleate and linoleate. We conclude that although apoB secretion was unaffected, dietary fish oil significantly decreased hepatic TG secretion through relatively poor utilization of EPA for the synthesis of TG destined for secretion in VLDL; at the same time, increased incorporation of [3H]EPA into hepatic phospholipid accompanied the decreased incorporation into secreted TG and these events may be coupled.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of hyperthyroidism on synthesis, secretion and metabolism of the VLDL apoproteins by the perfused rat liver

Livers from fed male Sprague-Dawley rats, made hyperthyroid by treatment with triiodothyronine (T3), were isolated and perfused in vitro. T3 (9.6 micrograms/day) was administered by osmotic minipump implanted intraperitoneally. Treatment with T3 for either 7 or 28 days reduced hepatic output of very-low-density lipoprotein (VLDL) and net synthesis of total associated apoproteins. After 7 days treatment, incorporation of [4,5-3H]leucine by livers from hyperthyroid rats into VLDL apo E was reduced while incorporation into apo B100, apo B48, and apo C's did not differ from euthyroid controls. The depressed incorporation of radioactivity into total VLDL protein was accounted for almost entirely on the basis of apo E. Incorporation of leucine into the total lipoprotein apo E isolated in the d less than 1.210 was also diminished by the hyperthyroid state, while that into apo B100, apo B48, and apo C in the total perfusate lipoprotein was similar to that of the euthyroid, as was found for the VLDL. Increased amounts of radioactive apo B100 and apo B48, however, were detected in the HDL fraction isolated from the medium perfusing livers from hyperthyroid rats. Hepatic uptake of VLDL protein and lipid was similar in euthyroid and hyperthyroid rats. Reduction of VLDL lipid and protein in the medium perfusing livers from T3-treated rats, therefore reflects hormonal action on synthesis and secretion, rather than uptake. Since the availability of apo B is thought to be required for secretion of VLDL, our observation suggests that synthesis of apo B is not depressed by treatment with T3 and that apoprotein synthesis is not a significant factor in the decreased output of VLDL by the liver, but that, as reported earlier, the lower output is a consequence of decreased synthesis of TG, the result of a diminished supply of hepatic glycero-3-phosphate in the hyperthyroid. The diminished amount of VLDL protein appears to be accounted for by the decreased quantity of apo E associated with a smaller VLDL particle secreted by livers from T3-treated rats.     

Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line

Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control cultures.     

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2;-8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2;-5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver.     

Apolipoprotein B synthesis in rat small intestine: regulation by dietary triglyceride and biliary lipid

Apolipoprotein B (apoB) synthesis rates have been determined, in vivo, in rat enterocytes. Following intralumenal administration of a pulse of [3H]leucine, newly synthesized apoB was quantitated by specific immunoprecipitation and compared to [3H]leucine incorporation into total, trichloroacetic acid-insoluble protein. ApoB synthesis rates were determined after acute administration of either 0.1 or 1 g of triglyceride to fasting animals. No differences were found at any time from 90 min to 6 hr after challenge and values were not different from the basal values established in fasted controls. Animals rechallenged with triglyceride after 8 days' intake of fat-free chow also failed to demonstrate a change in intestinal apoB synthesis rate. By contrast, enterocyte content of apoB appeared to fall, temporarily, with the onset of active triglyceride flux. Groups of animals were then subjected to external bile diversion for 48 hr, a maneuver designed to remove all lumenal sources of lipid. Jejunal apoB synthesis rates fell by 43% (from 0.76% +/- 0.14 to 0.43% +/- 0.12, P less than 0.001), a change that was completely prevented by continuous replacement with 10 mM Na taurocholate. The suppression of jejunal apoB synthesis, induced by prolonged bile diversion, was reversed after 14 hr, but not 8 hr, of intralumenal perfusion with 10 mM Na taurocholate. The addition of micellar fatty acid-monoolein to the perfusate for 4 hr produced no further change in apoB synthesis. Ileal apoB synthesis rates fell by 70% (from 0.61% +/- 0.15 to 0.18% +/- 0.10, P less than 0.001) following 48 hr external bile diversion, a change that was only partially prevented by continuous bile salt replacement. These results suggest that jejunal apoB synthesis demonstrates bile salt dependence but not regulation by acute triglyceride flux. The data further suggest that key aspects of the regulation of apoB synthesis by cellular lipid flux may be mediated independently in jejunal and ileal enterocytes.     

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.     

Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.     

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

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.     

The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes

Hepatocytes obtained from rats fed a choline-deficient diet for 3 days were cultured in a medium +/- choline (100 microM) or methionine (200 microM). We investigated how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis, and lipoprotein secretion. The mass of triacylglycerol and phosphatidylcholine secreted was increased about 3-fold and 2-fold, respectively, by the addition of either choline or methionine to the cultured cells. Similarly, a 3-fold stimulation in the secretion of [3H]triacylglycerol and [3H]phosphatidylcholine derived from [3H]oleate was observed after the addition of choline or methionine. Fractionation of secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of triacylglycerol and phosphatidylcholine from choline-deficient cells was mainly due to impaired secretion of very low density lipoproteins (VLDL) (but not high density lipoproteins (HDL)). Fluorography of L-[4,5-3H]leucine-labeled lipoproteins showed a remarkable inhibition of VLDL secretion by choline deficiency. The addition of choline or methionine stimulated the synthesis of phosphatidylcholine and increased the cellular phosphatidylcholine levels to that in normal cells. While there was little effect of choline on the synthesis and amount of cellular phosphatidylethanolamine, the addition of methionine diminished cellular phosphatidylethanolamine levels. Choline deficiency did not change the rate of incorporation of L-[4,5-3H]leucine into cellular VLDL apolipoproteins, nor the rate of disappearance of radioactivity from L-[4,5-3H]leucine-labeled cellular apoB, apoE, and apoC. These results suggest that hepatic secretion of VLDL, but not HDL, requires active phosphatidylcholine biosynthesis. Secondly, the inhibitory effect of choline deficiency on VLDL secretion can be compensated by the methylation of phosphatidylethanolamine.     

Contributions of fatty acid and sterol synthesis to triglyceride and cholesterol secretion by the perfused rat liver in genetic hyperlipemia and obesity

The relative importance of fatty acid synthesis in triglyceride secretion by perfused livers from lean (normal control) and obese Zucker rats was investigated. Livers from fed animals were perfused in a recirculating system with tritiated water and a constant infusion of oleic acid. Triglyceride secretion was 5 times greater and cholesterol secretion was 35% greater in the obese rat livers. The very-low-density lipoprotein hypersecreted by perfused livers from obese rats contained more apolipoprotein B and exhibited an increased B-48/B-100 ratio. Apo-B was also elevated in the hypertriglyceridemic plasma of obese rats in both fed and fasting states. The very-low-density lipoprotein isolated therefrom was likewise characterized by an increased B-48/B-100 ratio. Ketogenesis was depressed 40% in the obese rat livers and increased hepatic malonyl-CoA was implicated in this alteration. The de novo synthesis and secretion of newly synthesized cholesterol was moderately increased in the perfused livers from obese rats. Tritium incorporation into fatty acids was 15 times greater in the obese genotype. Most of the synthesized fatty acids remained in the liver and were recovered after perfusion in triglyceride and phospholipids. Newly synthesized fatty acids accounted for only 3 and 15% of the triglyceride secreted by the lean and obese rat livers, respectively. A large portion of the secreted triglyceride fatty acids was derived from endogenous liver lipids. When the turnover of newly synthesized fatty acids in these pools was considered, the contribution of de novo fatty acid synthesis to triglyceride secretion was estimated to be 9% in the lean and 44% in the obese rat livers. Therefore, the altered partition of free fatty acids (Fukuda, N., Azain, M. J., and Ontko, J. A. (1982) J. Biol. Chem. 257, 14066-14072) and increased fatty acid synthesis are both major determinants of the hypersecretion of triglyceride-rich lipoproteins by the liver in the genetically obese Zucker rat.     

Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Apoproteins of chylomicrons, very low density lipoprotein (VLDL), and a low density + high density fraction secreted by proximal and distal rat small intestine into mesenteric lymph were examined during triglyceride (TG) absorption. Apoprotein output and composition were determined and the turnover rates of labeled non-apoB (soluble) apoproteins in lipoprotein fractions were measured after an intraluminal [(3)H]leucine pulse during stable TG transport into lymph. The output of VLDL apoproteins exceeded that of chylomicrons during the absorption of 45 micro mol of TG per hour. More [(3)H]leucine was incorporated into VLDL than into chylomicrons and the decay of newly synthesized VLDL apoproteins was more rapid than that of chylomicrons, in part due to higher concentrations of apoA-I and apoA-IV with a rapid turnover rate. Chylomicrons from proximal intestine contained more apoA-I and less C peptides than chylomicrons from distal intestine. Ninety percent of [(3)H]leucine incorporated into soluble apoproteins was in apoA-I and apoA-IV, but little apoARP was labeled. The turnover rate of apoA-I and apoA-IV differed significantly in the lymph lipoproteins examined. Although total C peptide labeling was small, evidence for intestinal apoC-II formation and differing patterns of apoC-III subunit labeling was obtained. [(3)H]Leucine incorporation and apoprotein turnover rates in lipoprotein secreted by proximal and distal intestine were similar. The different turnover rates of apoA-I and apoA-IV in individual lipoproteins suggest that these A apoproteins are synthesized independently in the intestine.-Holt, P. R., A-L. Wu, and S. Bennett Clark. Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.     

Expression of apolipoprotein B-100 in isolated human small intestine epithelium.

Apolipoprotein B-48 (apoB48) is synthesized in the small intestine and becomes a component of chylomicrons (CM). Apolipoprotein B-100 (apoB100) is synthesized in liver and becomes a component of both very low density lipoprotein (VLDL) and low density lipoprotein (LDL). To evaluate whether apoB100 is present in the human small intestine, we performed immunohistochemical staining using anti-apoB100 monoclonal antibody (mAb). Jejunal samples stained positive and the granular staining was noted in the supranuclear region of epithelial cells. We also identified apoB100 expression in the epithelial cells by immunoblotting and dot-blotting of PCR-amplified cDNA. In order to exclude submucosal stroma contaminated with blood, we used isolated epithelium from human small intestine obtained by a crypt isolation technique. The results indicate that not only apoB48, but also apoB100 are expressed in human small intestine epithelium. The expression of apoB100 suggests that the dietary VLDL may be synthesized in human small intestine epithelium and converted into LDL, which might play an important role in atherosclerosis.     

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.     

The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins.

Previously, based on distinct requirement of microsomal triglyceride transfer protein (MTP) and kinetics of triglyceride (TG) utilization, we concluded that assembly of very low density lipoproteins (VLDL) containing B48 or B100 was achieved through different paths (Wang, Y. , McLeod, R. S., and Yao, Z. (1997) J. Biol. Chem. 272, 12272-12278). To test if the apparent dual mechanisms were accounted for by apolipoprotein B (apoB) length, we studied VLDL assembly using transfected cells expressing various apoB forms (e.g. B64, B72, B80, and B100). For each apoB, enlargement of lipoprotein to form VLDL via bulk TG incorporation was induced by exogenous oleate, which could be blocked by MTP inhibitor BMS-197636 treatment. While particle enlargement was readily demonstrable by density ultracentrifugation for B64- and B72-VLDL, it was not obvious for B80- and B100-VLDL unless the VLDL was further resolved by cumulative rate flotation into VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). BMS-197636 diminished B100 secretion in a dose-dependent manner (0.05-0.5 microM) and also blocked the particle enlargement from small to large B100-lipoproteins. These results yield a unified model that can accommodate VLDL assembly with all apoB forms, which invalidates our previous conclusion. To gain a better understanding of the MTP action, we examined the effect of BMS-197636 on lipid and apoB synthesis during VLDL assembly. While BMS-197636 (0.2 microM) entirely abolished B100-VLDL(1) assembly/secretion, it did not affect B100 translation or translocation across the microsomal membrane, nor did it affect TG synthesis and cell TG mass. However, BMS-197636 drastically decreased accumulation of [(3)H]glycerol-labeled TG and TG mass within microsomal lumen. The decreased TG accumulation was not a result of impaired B100-VLDL assembly, because in cells treated with brefeldin A (0.2 microgram/ml), the assembly of B100-VLDL was blocked yet lumenal TG accumulation was normal. Thus, MTP plays a role in facilitating accumulation of TG within microsomes, a prerequisite for the post-translational assembly of TG-enriched VLDL.     

Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat.

The corpulent JCR:LA-cp rat (cp/cp) is a useful model for study of the metabolic consequences of obesity and hyperinsulinemia. To assess the effect of hyperinsulinemia on VLDL secretion in this model, we measured rates of secretion of VLDL in perfused livers derived from cp/cp rats and their lean littermates. Livers of cp/cp rats secreted significantly greater amounts of VLDL triglyceride and apolipoprotein, compared with lean littermates. The content of apoB, apoE, and apoCs in both perfusate and plasma VLDL was greater in the cp/cp rat, as was the apolipoprotein (apo)C, apoA-I, and apoA-IV content of plasma HDL. Triglyceride content was also greater in cp/cp livers, as was hepatic lipogenesis and expression of lipogenic enzymes and sterol regulatory element binding protein-1 (SREBP-1). Hepatic mRNAs for apoE, and apoA-I were higher in livers of cp/cp rats. In contrast, the steady state levels of apoC-II, apoC-III, and apoB mRNAs were unchanged. Thus, livers of obese hyperinsulinemic cp/cp JCR:LA-cp rats secrete a greater number of VLDL particles that are enriched in triglyceride, apoE, and apoC. Greater secretion of VLDL in the cp/cp rat in part results from higher endogenous fatty acid synthesis, which in turn may occur in response to increased expression of the lipogenic enzyme regulator SREBP-1c.     

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.     

Apolipoprotein B-100: immunolocalization and synthesis in human intestinal mucosa

Despite the evidence that the human small intestine produces two separate species of apoB mRNA encoding for B-100 and B-48, there is a paucity of data concerning the expression of the latter form in this organ. Using a high resolution immunogold approach, with specific polyclonal antibodies and a panel of monoclonal antibodies (2D8, 3A10, 4G3), both forms of apoB (B-48 and B-100) were revealed over enterocytes of pediatric intestinal samples. Intense labeling was observed over microvilli, apical smooth membrane vesicles, multivesicular bodies, the basolateral membrane, as well as the trans Golgi region. Only low labeling was found over the rough endoplasmic reticulum (rER). Similar patterns of apoB distribution characterized both duodenal and jejunal regions. The presence of labeling over the Golgi apparatus and rER suggests a synthetic activity of both forms of apoB by the epithelial cells. To test this hypothesis, human intestine was incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation for apoB. Immunoprecipitates contained radioactivity mainly in apoB-48 with relatively small amounts in apoB-100 when examined by NaDodSO4-polyacrylamide gel electrophoresis. These findings were further supported by the biochemical determination of apoB-100 and apoB-48 in chylomicron particles isolated from thoracic duct lymph of a human donor. Taken together, our data suggest that the human intestine is able to synthesize and to express the apoB-100.     

Measurement of apolipoprotein B synthesis in perfused rat liver using stable isotopes: [15N]hippurate as a measure of the intracellular [15N]glycine precursor enrichment

Rat livers were perfused by the nonrecirculating technique with medium containing [15N]glycine and sodium benzoate. At various times, the isotopic enrichment of hepatic free glycine, hepatic glycyl-tRNA, and perfusate hippurate was measured by GLC-MS. After 60 min, these parameters had reached approximately maximal values. At 90 min, the perfusate hippurate had a 30% greater enrichment of 15N than the intracellular glycine or glycyl-tRNA. Hippurate enrichment was half that of the medium glycine. The rat livers secreted apolipoprotein B (B-100 plus B-48) at a rate of 22 micrograms/g per h. From the 15N enrichment and the secretion rate, an intrahepatic pool size of 86 micrograms/g of apoB was calculated. From the minimal intracellular transit time of 30 min, an apoB fractional synthetic rate (FSR) of 2 pools/h was indicated, whereas the FSR estimated from the 15N-enrichment was 0.26/h. A possible explanation for the discrepancy is that apoB may recycle within the hepatocyte. On the basis of the present experiments, when hippurate enrichment is used as a measure of the enrichment of intrahepatic glycine in in vivo studies with 15N-labeled glycine, a correction should be applied, under normal metabolic circumstances, of approximately 20-30%.