Lipid and lipoprotein metabolism in Hep G2 cells

Lipid composition, lipid synthesis and lipoprotein secretion by the Hep G2 cell line have been studied with substrate and insulin supplied under different conditions. The lipid composition of Hep G2 cells was close to that of normal human liver, except for a higher content in sphingomyelin (P less than 0.005) and a lower phosphatidylcholine/sphingomyelin ratio. Most of the [14C]triacylglycerols secreted into the medium were recovered by ultracentrifugation at densities of 1.006 to 1.020 g/ml. The main apolipoproteins secreted were apo B-100 and apo A-I. Hep G2 mRNA synthesized in vitro the pro-apolipoproteins A-I and E. Triacylglycerol secretion was 7.38 +/- 1.04 micrograms/mg cell protein per 20 h with 5.5 mM glucose in the medium and increased linearly with glucose concentration. Oleic acid (1 mM) increased the incorporation of [3H]glycerol into the medium and cell triacylglycerols by 251 and 899%, with a concomitant increment in cell triacylglycerols and cholesterol ester. Insulin (1 mU or 7 pmol/ml) inhibited triacylglycerol secretion and [35S]methionine incorporation into secreted protein by 47 and 28%, respectively, with a corresponding increase in the cells. Preincubation of cells with 2.5-10 mM mevalonolactone decreased the incorporation of [14C]acetate into cholesterol 6.2-fold, indicating an inhibitory effect on HMG-CoA reductase. It is concluded that in spite of some differences between Hep G2 and normal human hepatocytes, this line offers an alternative and reliable model for studies on liver lipid metabolism.     

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.     

Metabolism of very low density lipoproteins--effect of sardine oil.

The effect of feeding fish oil on the metabolism of lipoproteins was studied in rats. Rats were fed diet containing 10% sardine or groundnut oil for 6 weeks. There was a significant decrease in the total cholesterol, phospholipids and triglycerides as well as the amount of the lipids associated with VLDL and LDL in serum in fish oil-fed rats. The synthesis and secretion of lipoproteins particularly apoB containing lipoproteins by primary cultures of hepatocytes from these rats were studied by 14(C)-acetate or 3(H)-leucine labelling. Primary cultures of hepatocytes derived from sardine oil-fed rats showed reduced incorporation of 3(H)-leucine into apoB containing lipoproteins secreted into the medium when compared to those fed groundnut oil, indicating a decreased synthesis and secretion of apoB. This was further confirmed by significantly lower incorporation of 14(C)-radioactivity into total and individual lipids of VLDL secreted into the medium, as well as that associated with different lipids in cell layer. The activity of lipoprotein lipase in adipose tissue and aorta was significantly higher in rats fed sardine oil which may cause an increased clearance of triglyceride-rich lipoproteins from circulation. These results indicate that the fish oil exerts hypolipidemic effect particularly by decreasing the synthesis and secretion of VLDL by liver and possibly by an increased clearance of triglyceride-rich lipoproteins from circulation.     

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.     

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 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.     

Production of apolipoproteins E and A-I by rat hepatocytes in primary culture

Hepatocytes were isolated from normal adult rat livers and cultured in a modified HI-WO/BA medium. A nearly confluent monolayer was established at the plating concentration employed. The hepatocytes synthesized ansd secreted albumin at rates similar to those observed in vivo. The cells secreted triacylglycerol in the absence of fatty acid substrate. Under these conditions the most abundant triacylglycerol molecular species contained 53 carbons. Incubation with oleic acid markedly increased triacylglycerol secretion predominantly in the form containing a total carbon number of 57. Approx. 80% of the secreted cholesterol was in the free form and this was unaffected by oleic acid. Employing monospecific antibodies constant rates of synthesis and secretion of apolipoproteins E and A-I were demonstrated by quantitative electroimmunoassay of the cell culture media. The rates of albumin, apolipoprotein E, and apolipoprotein A-I production were 1480, 170 and 60 microgram/h per g cell protein, respectively.     

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.     

A choline-deficient diet in mice inhibits neither the CDP-choline pathway for phosphatidylcholine synthesis in hepatocytes nor apolipoprotein B secretion

Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.     

Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacyglycerol acyltransferase activity

Microsomal monoacyglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fao rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [14C]acetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [14C]oleate into triacyglycerol about 1.7-fold within 2 h, but had little effect on [14C]oleate incorporation into phospholipid. These hormonally responsive rat-hepatoma/hepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway.     

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.     

Secretion of high and low molecular weight phosphorylated apolipoprotein B by hepatocytes from control and diabetic rats. Phosphorylation of APO BH and APO BL

Apolipoprotein B (apo B) phosphorylation was examined in primary cultures of hepatocytes from control and nonketotic streptozotocin diabetic rats. Following a 5-h incubation with ortho[32P]phosphate, media lipoproteins (d less than 1.063 g/ml) were isolated, and delipidated apolipoproteins were separated by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGGE), and gels were heat fixed. Autoradiographic bands corresponding to high (apo BH) and low molecular weight apo B (apo BL) were observed in media lipoproteins isolated from control rats, and these bands were more prominent in media lipoproteins isolated from diabetic rats. Apo B-specific activity was estimated from aqueous alcohol-precipitated radioactivity and apo B monoclonal immunoassay of isolated media lipoproteins. In lipoproteins secreted by hepatocytes of diabetic rats, the calculated apo B specific activity was between 18- and 31-fold greater than that secreted by hepatocytes of control rats, consistent with the SDS-PAGGE gel data. The increase in secretory 32P-labeled apo B from hepatocytes of diabetic rats was due, at least in part, to an increase in labeled phospho-tyrosine as determined by phosphoamino acid analysis. These data demonstrate that apo BH may be secreted as a phosphorylated protein and that apo B phosphorylation occurs on tyrosine as well as serine residues.     

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.     

Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters

Male rats with thoracic duct cannulae were intubated with corn oil or fatty acid methyl esters and the lymph was collected over the next 2-72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate - polyacrylamide - glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas--liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phosphatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B-100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from corn oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.     

Alpha-adrenergic suppression of very-low-density-lipoprotein triacylglycerol secretion by isolated rat hepatocytes.

The effect of adrenaline on triacylglycerol synthesis and secretion was examined in isolated rat hepatocytes. Cells were incubated with 0.5 mM-[1-14C]oleate, and the accumulation of triacylglycerol and [14C]triacylglycerol was measured in the incubation medium. Triacylglycerol appearing in the medium was present in a form with properties similar to very-low-density lipoproteins. Triacylglycerol, [14C]triacylglycerol and [14C]phospholipid contents of hepatocytes were also determined. Addition of 10 microM-(-)adrenaline decreased accumulation of glycerolipid in the incubation medium and also decreased cellular [14C]phospholipid content. Prazosin abolished these effects, whereas propranolol did not. The hormone did not affect cellular triacylglycerol content or rates of incorporation of [1-14C]oleate into cell triacylglycerol. The effect of adrenaline on the removal of newly secreted triacylglycerol and the secretion of synthesized glycerolipid was also examined. The catecholamine did not affect rates of removal of newly secreted triacylglycerol. Adrenaline did inhibit the secretion of pre-synthesized lipid by the cells, as assessed by the appearance of radiolabelled triacylglycerol from hepatocytes that had been preincubated with [1,2,3-3H]-glycerol. Adrenaline did not affect rates of fatty acid uptake by hepatocytes, but did stimulate oxidation of [1-14C]oleate, principally to 14CO2.     

Replacement of soybean oil by fish oil increases cytosolic lipases activities in liver and adipose tissue from rats fed a high-carbohydrate diets

Several studies have demonstrated that fish oil consumption improves metabolic syndrome and comorbidities, as insulin resistance, nonalcoholic fatty liver disease, dyslipidaemia and hypertension induced by high-fat diet ingestion. Previously, we demonstrated that administration of a fructose-rich diet to rats induces liver lipid accumulation, accompanied by a decrease in liver cytosolic lipases activities. In this study, the effect of replacement of soybean oil by fish oil in a high-fructose diet (FRUC, 60% fructose) for 8 weeks on lipid metabolism in liver and epididymal adipose tissue from rats was investigated. The interaction between fish oil and FRUC diet increased glucose tolerance and decreased serum levels of triacylglycerol (TAG), VLDL-TAG secretion and lipid droplet volume of hepatocytes. In addition, the fish oil supplementation increased the liver cytosolic lipases activities, independently of the type of carbohydrate ingested. Our results firmly establish the physiological regulation of liver cytosolic lipases to maintain lipid homeostasis in hepatocytes. In epididymal adipose tissue, the replacement of soybean oil by fish oil in FRUC diet did not change the tissue weight and lipoprotein lipase activity; however, there was increased basal and insulin-stimulated de novo lipogenesis and glucose uptake. Increased cytosolic lipases activities were observed, despite the decreased basal and isoproterenol-stimulated glycerol release to the incubation medium. These findings suggest that fish oil increases the glycerokinase activity and glycerol phosphorylation from endogenous TAG hydrolysis. Our findings are the first to show that the fish oil ingestion increases cytosolic lipases activities in liver and adipose tissue from rats treated with high-carbohydrate diets.     

Postnatal development of hepatocellular apolipoprotein B assembly and secretion in the rat.

In this study, we explored the paradox that in suckling rats the serum concentration of LDL is high although the liver secretes only minimal quantities of VLDL, the presumed precursor of LDL. Freshly isolated hepatocytes and hepatocytes in primary culture obtained from adult (90 days old) and suckling (17 days old) rats were used to investigate the synthesis and secretion of apolipoprotein B (apoB) and lipids as well as the density profile of secreted apoB-containing lipoproteins. Furthermore, the effects of dexamethasone and oleate on apoB biogenesis were investigated in primary cultures of hepatocytes from adult and suckling rats. Hepatocytes from suckling rats were unable to assemble mature VLDL but secreted apoB as primordial lipoprotein particles in the LDL-HDL density range. Intracellular degradation of apoB was also reduced in hepatocytes from suckling rats compared with that in hepatocytes from adults. The immaturity in VLDL assembly and apoB degradation of hepatocytes from suckling rats could be overcome by treating the cultures with dexamethasone plus oleate or dexamethasone alone. The lower microsomal triacylglycerol transfer protein (MTP) mRNA concentrations in hepatocytes from suckling rats in comparison with hepatocytes from adult rats were not reflected in lower MTP activity levels. Furthermore, dexamethasone plus oleate treatment had no effect on MTP activity although VLDL assembly and secretion were clearly stimulated. We conclude that, during the suckling period of the rat, serum LDL is directly produced by the liver. This is a result of impaired hepatic VLDL assembly, which is a consequence of low triglyceride synthesis and an inefficient mobilization of bulk lipids in the second step of VLDL assembly.     

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.     

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 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.