Stimulation by oleic acid of incorporation of L-[4,5-3H]leucine into very low density lipoprotein apoprotein by the isolated perfused rat liver

Livers from normal fed or fasted (24h) rats were perfused in vitro to determine whether fatty acid affects the biosynthesis of very low density lipoprotein (VLDL) apoprotein. Oleate stimulated VLDL triacylglycerol output and increased incorporation of L-[4,5-3H]leucine into VLDL apoprotein in both the fed and fasted groups. The increased incorporation of [3H]leucine was mainly into VLDL-apoprotein E. The total mass of VLDL apoprotein secreted was also stimulated by oleate proportionately. These data suggest that fatty acids may stimulate hepatic synthesis and/or secretion of the VLDL apoproteins and that apo E, may be required for the formation and secretion of triacyl-glycerol in the VLDL.     

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.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.     

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.     

Regulated biosynthesis and divergent metabolism of three forms of hepatic apolipoprotein B in the rat

Studies using rat livers perfused with recycled, serum-containing medium plus [3H]leucine revealed that secreted VLDL contain three forms of apolipoprotein B (apoB), B-48, B-95, and B-100, all synthesized by the liver. The B-48/(B-95 + B-100) [3H]leucine incorporation ratio ranged from 0.22 to 3.25 with livers of rats fed different diets, and the ratio was positively correlated with the triglyceride secretion rate in most of the livers. Generally, as more triglyceride was secreted, a greater proportion was packaged with B-48, which is the apoB form most rapidly cleared from the circulation. Together, these findings suggest a mechanism for regulating plasma triglyceride levels. [3H]Leucine incorporation into apoA-I also was positively correlated with the triglyceride secretion rate. Secretion of newly synthesized B-48 was delayed relative to all other apolipoproteins. There was little segregation of any of the three apoB forms into any of five subfractions of secreted VLDL separated on the basis of Sf value; only the smallest VLDL (Sf 20-100) were slightly enriched in B-95 and B-100. Less than 5% of newly synthesized apoB appeared in perfusate LDL. The B-100/B-95 [3H]leucine incorporation ratio was 3.3 with perfused livers of fed rats but only 1.6 in post-surgical, relatively fasted rats in vivo, suggesting physiologic regulation also of the relative amounts of the two large apoBs produced. With recycled serum-free perfusate, as opposed to serum-containing medium, there was hepatic reuptake of nascent VLDL, indicated by the reuptake of newly synthesized apoE and all three forms of apoB, and not other apolipoproteins. Divergent metabolism of B-100 and B-95 in the rat was evident from the following results: a) B-95 disappeared more rapidly from recycled, serum-free liver perfusate; b) B-100 disappeared more rapidly from the circulation in vivo; c) plasma lipoprotein fractions of increasing density between d less than 1.019 and d 1.072 g/ml contained increasing proportions of B-95 over B-100. In summary, these results show that hepatic VLDL production in the rat involves the biosynthesis of three forms of apoB, that the relative amounts produced are regulated by physiologic variables, and that there is divergent metabolism of the VLDL particles into which these different apoB forms, either individually or in combination, become incorporated.     

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.     

Effects of thyroid status and fasting on hepatic metabolism of apolipoprotein A-I

Metabolism of apolipoprotein (apo)A-I was studied in normal and chow-fed hyperthyroid rats, in 24-h fasted untreated male rats, and in rats after thyroparathyroidectomy (TXPTX). Rats were made hyperthyroid by administration of T3 (9.6 micrograms/day) or T4 (30 micrograms/day) with an Alzet osmotic minipump. Hyperthyroidism produced a similar two- to threefold elevation in plasma levels of apoA-I in male or female animals. During treatment with T3, plasma levels of T3 ranged from 200 to 400 ng/dl and did not correlate with plasma apoA-I levels. The net mass secretion and synthesis ([3H]leucine incorporation) of apoA-I by perfused livers from male hyperthyroid rats was elevated, while secretion of albumin was not different than that of euthyroid rats. Furthermore, the incorporation of [3H]leucine into total perfusate and hepatic protein was not altered by hyperthyroidism. The effect of thyroid hormone on apoA-I synthesis, therefore, does not appear to be a general effect on protein synthesis. After longer periods of treatment (28 days) with T3 (9.6 micrograms/day), hepatic apoA-I production decreased from that observed after 7 or 14 days of treatment, yet plasma apoA-I concentrations remained elevated. Plasma T3 decreased from 100 ng/dl to 40 ng/dl, in the hypothyroid rat resulting from TXPTX, but the plasma concentration of apoA-I did not change during the 2-week experimental period. The net secretion of apoA-I by livers from hypothyroid animals was depressed and albumin was uneffected compared to the euthyroid. Overnight fasting of euthyroid rats did not alter hepatic apoA-I secretion or plasma apoA-I levels, although under fasting conditions we had reported that hepatic output of apoB and E of VLDL is depressed. The addition of oleic acid to the perfusion medium, sufficient to stimulate VLDL production, did not affect net hepatic secretion of apoA-I by livers from euthyroid, hyperthyroid, or hypothyroid rats. In summary, hepatic synthesis of apoA-I appears to be controlled independently of other apo-lipoproteins and secretory proteins (albumin). Hepatic apoA-I synthesis is sensitive to thyroid status, increased in the hyperthyroid and decreased in the hypothyroid state. The specific stimulation of hepatic synthesis and secretion of apoA-I in the hyperthyroid state, however, tends to normalize over an extended period, perhaps from compensatory effects of a hormonal nature.     

Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.     

The effect of sex on the uptake of very low density lipoprotein triglyceride fatty acid from the plasma of the rat in vivo.

Radioactive very low density lipoprotein (VLDL) was prepared by perfusion in vitro of livers isolated from normal fed male and female rats with [1-¹⁴C] oleate or [9, 10-³H] oleate, respectively. These VLDL, whose properties differed due to sex, were mixed. Aliquots of the mixture were injected intravenously into fasted male and female rats and the decay of the radioactivity (¹⁴C and ³H) was measured. Disappearance of radioactivity from plasma triglyceride was more rapid in female animals. Plasma half-life of ¹⁴C and ³H was 42.5±3.7 and 49.7±4.4 minutes, respectively, when the VLDL mixture was injected into male rats. The corresponding values in female rats were 28.3±1.1 and 30.7±1.7, respectively. These data suggest strongly that the rate of utilization of VLDL triglyceride fatty acid is more rapid in the female than in the male, and that the properties of the VLDL particles are of less importance than innate sex differences in the recipient for the rate of clearance of triglyceride fatty acids.     

Bile acids inhibit secretion of very low density lipoprotein by rat hepatocytes

The influence of taurocholate on very low density lipoprotein (VLDL) triacylglycerol synthesis and secretion was studied by isolated rat liver-parenchymal cells. The incorporation of [3H]glycerol into cell-associated and VLDL triacylglycerols were measured after incubation in medium containing 0.75 mM oleate. Taurocholate caused a maked decrease in VLDL [3H]triacylglycerol secretion from the hepatocytes: 50-150 microM taurocholate inhibited secretion of VLDL [3H]triacylglycerols by 70-90%. Similar results were obtained when the mass of secreted VLDL triacylglycerols was measured. Taurocholate caused a decreased secretion of VLDL [3H]triacylglycerols after 15-30 min incubation. A higher amount of cellular triacylglycerols was found in taurocholate-supplemented cells. Furthermore taurocholate did not change the intracellular lipolysis of triacylglycerols. These results suggest that bile acids interfere more probably with the assembly and/or secretion of VLDL-particles and not with earlier stages of VLDL formation, e.g. triacylglycerol synthesis.     

Estradiol-induced alteration of very-low-density lipoprotein assembly. Possible competition among apoproteins for incorporation into nascent very-low-density lipoprotein

The hypothesis that the apoprotein composition of nascent very-low-density lipoprotein (VLDL) secreted by the hepatocyte is determined by the relative rates of apoprotein synthesis and their affinities of binding to VLDL was tested using chick hepatocytes in monolayer culture. Chick cells were chosen for the study of lipoprotein assembly since estradiol treatment can be used to alter the composition of the apoprotein mixture synthesized by these cells. The secretion of apoprotein (apo) B by estradiol-treated hepatocytes was elevated 4.2-fold above the basal level measured in control cells. Furthermore, estradiol-treated cells secreted apo-II, a major VLDL apoprotein not synthesized prior to estradiol treatment, at a level equivalent to that of apo-B. However, no difference in the secretion of apo-A-I and other newly identified nascent VLDL apoproteins was detected. These changes in relative rates of apoprotein synthesis altered the composition of nascent VLDL secreted by control versus estradiol-induced cells from: apo-B, 22 to 40%; apo-II, 0 to 32%; apo-37 kDa, 14 to 6%; apo-A-I, 31 to 12%; apo-17 kDa, 10 to 4%; apo-9 kDa, 15 to less than 10%; and apo-6 kDa, 8 to less than 2%. To investigate the basis for the preferential incorporation of apo-B and apo-II into nascent VLDL, the relative affinities of the apoproteins for VLDL were compared by measuring their capacities to transfer from VLDL into other lipoprotein or nonlipoprotein density classes. Culture medium containing [3H]leucine-labeled VLDL was incubated with plasma deficient in lipoproteins of rho less than 1.006 g/ml. Within 30 min of incubation at 37 degrees C, 3H-labeled apo-A-I and apo-9 kDa exchanged between VLDL and high-density lipoprotein, whereas apo-37 kDa exchanged between VLDL and the rho greater than 1.21 g/ml fraction. Neither apo-B nor apo-II underwent transfer from nascent VLDL. These results suggest that the relative rates of input of apoproteins into the secretory pathway and their affinities of binding to the nascent VLDL particle determine their extent of incorporation into, and, thus, the apoprotein composition of secreted VLDL.     

The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver.

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor-product relationship. Analysis of the apoproteins of the Golgi VLDL by polacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1, showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent. The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.     

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)     

Lipid and lipoprotein synthesis in isolated and cultured hepatocytes from lean and obese Zucker rats

Hepatocytes were isolated by EDTA perfusion of livers from lean (Fa/-) and obese (fa/fa) Zucker rats. Triacylglycerol (TG) and sn-glycerol 3-phosphate were increased in fa/fa hepatocytes, but free fatty acids, cholesterol and phospholipid concentrations were similar in both groups. In spite of an identical fatty acid uptake rate, glycerolipid synthesis was higher in obese compared to lean rat hepatocytes, and this difference remained for at least 2-3 days of culture. Triacylglycerol mass secretion was 2-fold higher in obese than in lean rat hepatocytes. This was confirmed by the higher incorporation of labeled glycerol and oleic acid into the medium TG fraction floating at density 1.006 g/ml. Density gradient ultracentrifugation of [14C]oleate-labeled lipoproteins showed that fa/fa hepatocytes secreted more TG-rich lipoproteins, and that 87% of the label was in the VLDL fraction compared with 67% in the medium of Fa/- hepatocytes. Decreased utilisation of leucine for protein synthesis in obese rat compared to lean rat hepatocytes was associated with enhanced leucine oxidation to CO2. [35S]Methionine incorporation showed an identical cell protein synthesis rate. Autoradiography after PAGE separation of secreted apolipoproteins (apoBh, Bl, apoA-VI, apoE, apoA-I, apoC) showed an identical pattern in both cell types.     

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

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

Intrahepatic assembly of very low density lipoproteins. Varied synthetic response of individual apolipoproteins to fasting

Hepatocytes obtained from rats fed for 3 days chow (control) or drinking water only (fasted) were used to examine how metabolic state affects lipogenesis, apolipoprotein synthesis, and the capacity to secrete de novo synthesized triacylglycerol. The secretion of triacylglycerol (mass and 3H-labeled via 3H2O incorporation) by both groups of cells was constant for 30 h. Moreover, cells from fasted rats secreted triacylglycerol at rates which were markedly reduced (mass -84%; 3H-labeled -91%). To assess the relative capacities of the two groups of hepatocytes to augment triacylglycerol secretion in response to stimulated lipogenesis, cells were incubated with increasing concentrations of glucose. Control cells responded to glucose by increasing equally the synthesis and secretion of [3H] triacylglycerol. When cells from fasted rats were challenged with glucose, triacylglycerol secretion was not increased. Rather, it accumulated intracellularly. Double-reciprocal plot analysis of the capacity to augment triacylglycerol secretion in response to glucose showed that cells from fasted rats had a greater than 10-fold decrease in V'max. Moreover, fasting changed the synthesis and secretion of apolipoproteins selectively: secretion of low molecular weight apo-B was decreased 50%, large molecular weight apo-B was unchanged, and apo-E was increased 2-4-fold. Analysis of the lipoproteins from both groups of cells on Bio-Gel A-50m showed that the very low density lipoprotein secreted by cells from fasted rats was smaller. In addition, all of the increased de novo synthesized apo-E secreted by cells from fasted rats eluted after the triacylglycerol-rich lipoproteins. The combined data show that: 1) the synthesis of individual very low density lipoprotein apolipoproteins is independently regulated, and 2) the synthesis (availability) of apo-B determines the capacity of the hepatocyte to assemble/secrete triacylglycerol-rich very low density lipoprotein.     

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.     

Effect of ethanol on hepatic phosphatidate phosphohydrolase: dose-dependent enzyme induction and its abolition by adrenalectomy and pyrazole treatment

Protein synthesis, measured as the incorporation of [¹⁴C]valine into cell proteins and into proteins secreted into the medium, and albumin production were studied in isolated rat liver hepatocytes. Protein synthesis was substantially higher in cells from fed rats than in cells from fasted rats. Addition of carbohydrates or amino acids increased protein synthesis in cells from fasted rats, whereas no effect was seen in cells from fed rats. Addition of oleate had no effect on protein synthesis. Ethanol inhibited protein synthesis in cells from fasted rats, whereas no or only small effect was seen in cells from fed rats. Simultaneous addition of carbohydrates diminished the inhibitory effect of ethanol, whereas addition of oleate increased the inhibitory effect of ethanol. It is suggested that the rate of protein synthesis in cells from fasted rats could be restricted by lack of precursors for synthesis of nonessential amino acids. The effect of ethanol is explained by an inhibition of gluconeogenesis.     

The effect of sex on the quantity and properties of the very low density lipoprotein secreted by the liver in vitro.

Livers from normally fed male and female rats were perfused in vitro with different amounts of oleate, and the production and properties of the very low density lipoprotein (VLDL) were studied. The mobility of the VLDL in the zonal ultracentrifuge was dependent on the uptake of free fatty acid and on the sex of the animal from which the liver was obtained. A higher proportion of the VLDL secreted by livers from females displayed a more rapid mobility in the zonal ultracentrifuge and, in addition, contained less phospholipid and cholesterol per mole triglyceride than the VLDL from the male, suggestive of larger size of the VLDL secreted by livers from the female rats. Such differences were diminished when the VLDL was compared at equal output of triglyceride but unequal uptake of free fatty acid. These data suggest that the properties of the VLDL are only secondarily modulated by sex, and primarily result from differences in the capacities of livers from either male or female rats to synthesize triglyceride for transport as VLDL. The quantity of triglyceride secreted, regardless of sex, may be an important determinant of both size and number of the VLDL particles. The incorporation of endogenous hepatic fatty acid into VLDL triglyceride was diminished in livers from both sexes by increased uptake of oleate. The greater output of VLDL triglyceride by livers from female animals was dependent on both exogenous and endogenous fatty acids when relatively small quantities of exogenous oleate were available for uptake by the liver. The proportion of palmitate and oleate in the phospholipid of the VLDL secreted by livers from male rats decreased and the content of arachidonate increased with increasing uptake of oleate; no differences were observed in the composition of the phospholipid fatty acids among the various experimental female groups, although these contained more stearate and less oleate and linoleate compared to the male groups. The change of fatty acid composition of the VLDL phospholipid may reflect inclusion of specific types of phospholipid in the VLDL structure for transport of triglyceride from the liver under particular conditions.     

Effect of D-galactosamine in vitro on [U-14C]palmitate oxidation, triacylglycerol synthesis and secretion in isolated hepatocytes

Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.