Lipoprotein lipid and protein synthesis in experimental nephrosis and plasmapheresis. I: Studies in rat in vivo

The incorporation of L-4,5-[³H]leucine into the ultracentrifugally separated apolipoproteins of very low, low, and high density lipoproteins (VLDL, LDL, HDL) and into serum albumin was found three-to four-fold higher in nephrotic than in normal rats one hour after intravenous injection. Incorporation of leucine into the circulating lipids was negligible. Increases of similar magnitude were obtained in the incorporation of simultaneously injected 1,5[¹⁴C] citrate into the lipids of VLDL, LDL, and HDL of nephrotic rats. Of the citrate carbons incorporated into serum and liver lipids, the proportion in cholesterol was higher in nephrotic rats when compared to normal rats. The incorporation of both precursors into total proteins and lipids of the liver vs. the incorporation into the lipoproteins was relatively lower in nephrotic than in control rats, indicating a preferential channeling into secretable products. The occurrence of enhanced new lipid synthesis in nephrosis was corroborated by the finding of markedly enhanced synthesis of lipoprotein-borne fatty acids and cholesterol from³H₂O. These results point out that while leucine is not an efficient in vivo precursor of lipoprotein lipids in nephrosis, de novo lipogenesis proceeds from other precursors. Similar trend of changes, though of smaller magnitude, was elicited in rats after double plasmapheresis, 18 hr apart, when measured 3 hr after the second plasma withdrawal. This indicates that the loss of circulating proteins either by direct removal or through kidney lesion stimulates the compensatory hepatic response involving excessive lipoprotein synthesis. Time-course studies showed that peak incorporation of leucine and citrate into the protein and lipid components of lipoproteins, respectively, as well as into serum albumin, occurred coincidentally 3 hr after the second plasmapheresis, suggesting an interdependence of the enhanced protein and lipid synthesis.     

Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside: Hepatic synthesis of lipoproteins and apolipoproteins

Hepatic synthesis of lipoproteins and apolipoproteins was investigated in male Wistar rats with severe nephrotic syndrome induced by puromycin aminonucleoside by incubating liver slices with a mixture of¹⁴C-amino acids. Labeled lipoproteins were separated by preparative ultracentrifugation from the incubation medium after the addition of carrier plasma. The incorporation of¹⁴C-amino acids into very low density lipoproteins (VLDL) (1.006 g/ml), low density lipoproteins (LDL) (1.006–1.063 g/ml) and high density lipoproteins (HDL) (1.063–1.210 g/ml) was increased in nephrotic liver 6.1-, 5.7- and 5.0-fold, respectively. The measurement of radioactivity associated to apolipoproteins isolated by SDS-PAGE documented an increased incorporation into apolipoprotein E (apoE) of nephrotic VLDL (33.1% vs 20% of the total radioactivity incorporated into VLDL apoproteins) and a markedly increased incorporation into apolipoprotein A-I (apoA-I) of nephrotic HDL (44.3% vs 16.3% of the total radioactivity incorporated into HDL apoproteins). In nephrotic liver, the total incorporation of amino acids into apolipoproteins (apoVLDL+apoLDL+apoHDL) was increased 12.6 times for apoA-I, 6.4 times for apoB, 5.0 times for apoE, 4.2 times for apoC+apoA-II and 2.5 times for apoA-IV. We suggest that, in nephrotic liver: (a) the synthesis of VLDL, LDL and HDL is increased, and (b) the total synthesis of apoA-I is selectively increased when compared to that of the other apolipoproteins. Preliminary reports of this work were presented at the Annual Meeting of the European Society for the Study of the Liver (Düsseldorf, September 13–15, 1979); at the 5th International Symposium on Atherosclerosis (Houston, November 6–9, 1979) and at the Annual Meeting of the Italian Society for the Study of the Liver (Rome, December 14–15, 1979).     

Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

Stearic acid modifies very low density lipoprotein lipid composition and particle size differently from shorter-chain saturated fatty acids in cultured rat hepatocytes

Stearic acid as compared to myristate, palmitate, or oleate is poorly incorporated into triacylglycerol, a major lipid component of very low density lipoprotein (VLDL). The present study investigated the effects of these fatty acids on VLDL metabolism in cultured rat hepatocytes. All fatty acids stimulated [2-³H] glycerol incorporation into VLDL lipids and secretion of [³H]-labeled VLDL by hepatocytes. However, the rate of [³H]-labeled VLDL secretion in the presence of nonlabeled stearate (12.8±0.7 pmol/mg protein/4h) was 46, 59, and 22% of that observed for those treated with myristate, palmitate, and oleate, respectively. [1-¹⁴C]Stearate as a substrate was also less effective than other labeled fatty acids to be incorporated into VLDL lipids. Of total VLDL lipids synthesized from [1-¹⁴C] stearate, triacylglycerol accounted for 78% as compared to 88–97% of that derived from palmitate, myristate, and oleate. The amounts of apoB100 and apoB48 were the same in hepatocytes treated with or without exogenous fatty acids. Similarly, the rate of apoB synthesis from [³⁵S] methionine was not affected by exogenous fatty acids. The treatment of cells with various saturated fatty acids increased the particle size of VLDL to different extents. The largest particles of VLDL, with a mean diameter of 79.3±11.9 nm, were seen in the cells treated with stearate, followed by those treated with palmitate and myristate (45.5±9.8 and 38.6±6.8 nm, diameter, respectively). Clearly, hepatocytes treated with stearate secrete less VLDL and produce larger VLDL particles than those treated with shorter-chain saturated fatty acids.     

Leucine as an in vitro precursor to lipids in rat sciatic nerve

The in vitro incorporation of leucine, isoleucine and pyruvate into lipids was compared and the possibility that leucine might serve as anin situ precursor to the correspondingiso fatty acids in the rat sciatic nerve was studied. The relative incorporation of¹⁴C from leucine into lipids vs. nonlipids was 20%, and the incorporation of label into total lipids from leucine was one-half that from pyruvate. The incorporation of label from leucine and pyruvate into sterols was nearly equivalent, but the incorporation of label into all other lipid classes from leucine was less than that from pyruvate, and the incorporation of label from isoleucine into lipids was much less in all cases. No detectable label from leucine was incorporated into brached chain fatty acids. It is concluded that leucine may be a substantial in vitro precursor to all major lipids in peripheral nerve, especially sterols. The possibility and significance of a leucine catabolic pathway in the cytosol in relation to availability of 3-hydroxy-3-methylglutaryl CoA for sterol biosynthesis is discussed.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Oxysterol mediated changes in fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells

We studied the actions of oxysterols on fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells. Cultures were labeled with [1-¹⁴C] arachidonate or [1-¹⁴C]oleate. During a 24-hr incubation, 25-or 22R-hydroxycholesterol enhanced the incorporation of label into triglycerides, concomitant with a reduction in the labeling of phospholipids. Cholestantriol or 20-hydroxycholesterol had the opposite effects. They caused a higher incorporation of radiolabel into phospholipids and a reduction of labeling of triacylglycerols. Similar changes were seen in cells labeled with [1-¹⁴C]acetate. Therefore, we conclude that oxysterols can promote changes in the distribution of fatty acids between neutral lipids and phospholipids through mechanisms that still need to be clarified.     

Effect of protein depletion on the VLDL triacylglycerol secretion and apoprotein synthesis by the perfused liver from pregnant rats

The effect of protein depletion in the pregnant rat on the polyunsaturated fatty acid incorporation into very low density lipoproteins (VLDL) has been investigated. The apoprotein pattern of these particles was determined. In in vivo experiments the amounts of serum and liver triacylglycerol were determined. VLDL were isolated and their apo C concentration calculated. In in vitro experiments the radioactivity of [³H] leucine incorporated into VLDL apoproteins was measured. The results show that protein depletion during pregnancy promotes a drastic increase in serum and liver triacylglycerol. The VLDL isolated from these animals show an increase in the triacylglycerol/protein ratio and a decrease in their content of apo C. Meanwhile, a significant reduction in the [³H]leucine incorporation into apo C peptides by the perfused liver of protein depleted rats was detected. On the other hand, protein deprivation did not affect labeled linoleic and arachidonic acid incorporation into triacylglycerol of the newly secreted VLDL. Taking these results together, let us deduce that a defective VLDL is secreted by the liver of the protein depleted pregnant rats. The abnormal composition of these particles may influence its normal metabolism through their effects on lipoprotein lipase and this fact could affect the normal supply of polyunsaturated fatty acids to the fetus.     

Accumulation of neutral lipids by human skin fibroblasts: Differential effects of saturated and unsaturated fatty acids

The accumulation of neutral lipids by human skin fibroblasts grown in medium supplemented with fatty acids has been investigated. GM-10 cells incorporated exogenous fatty acids into both phospholipids and neutral lipids. More [¹⁴C] oleate, linoleate, or linolenate was incorporated into triacylglycerol than was [¹⁴C] palmitate or stearate. Supplementation of medium containing delipidized serum with unsaturated fatty acids resulted in far more stimulation of [¹⁴C] glycerol incorporation into triacylglycerol than did supplementation with saturated fatty acids. Palmitate- and stearate-fed cells incorporated sizable amounts of [¹⁴C] fatty acids and [¹⁴C] glycerol into diacylglycerol as well as triacylglycerol, especially at higher fatty acid concentrations. Increased oleate supplementation from 10–300 μM resulted in increased triacylglycerol synthesis and accumulation of discrete cytoplasmic lipid droplets; palmitate concentrations above 70 μm were toxic. Micrographs of the palmitate-fed cells showed electron translucent slits, suggesting solid depositions of saturated fat, rather than the discrete osmiophilic droplets found in oleate-fed cells. Although GM-10 cells can synthesize fully saturated triacylglycerols, these data suggest that in cells fed saturated fatty acids, solid depositions of neutral lipids may sequester diacylglycerols and thus limit triacylglycerol synthesis.     

Metabolic fate of sphingomyelin of high-density lipoprotein in rat plasma

The metabolic fate of high density lipoprotein (HDL) sphingomyelin in plasma was studied in rats over a 24-hr period after injection of HDL containing sphingomyelin which was¹⁴C-labeled in the stearic (18∶0) or lignoceric acid (24∶0) moiety and³H-labeled in the choline methyl groups. Decay of label in plasma followed three phases. The first two phases were similar for both isotopes and both types of sphingomyelin (t_1/2≃10 and 110 min). However, during the third phase (from 10 hr after injection),³H label disappeared more slowly than¹⁴C label from 18∶0 sphingomyelin, whereas the³H/¹⁴C ratio remained relatively constant when 24∶0 sphingomyelin was used. Intact, doubly-labeled 18∶0 sphingomyelin disappeared from HDL rapidly (t_1/2=38 min) by tissue uptake and by transfer to very low density lipoprotein (VLDL). VLDL contained up to 12% of the sphingomyelin 1 hr after injection. This is the first demonstration of a transferin vivo of sphingomyelin from HDL to VLDL. A similarly rapid transfer was also observedin vitro. Some nontritated, [¹⁴C]18∶0 or [¹⁴C]24∶0 sphingomyelin was redistributed more slowly into HDL. Doubly-labeled phosphatidylcholine appeared in VLDL and HDL within 1 hr after injection and reached 1.8 and 2.1% of the injected¹⁴C and³H in VLDL at 1 hr, and 4.8 and 6.9% in HDL at 3 hr, respectively.     

Antagonism of [3H]fatty acid incorporation into vimentin by sodium pyruvate: Pitfalls of protein acylation

In the course of studying possible fatty acid acylation of vimentin by cultured bovine lens epithelial cells, several potential pitfalls of protein-fatty acid acylation were recognized. Even exhaustive delipidation of vimentin with organic solvents failed to remove all noncovalently associated [³H]palmitate and [³H]myristate. Hydroxylamine treatment of vimentin, separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), failed to remove either palmitate or myristate derived radiolabel. Hydroxylamine treatment did remove palmitate label from a group of lower molecular weight proteins. The myristate radiolabel associated with vimentin recovered after SDS-PAGE and subjected to acid hydrolysis was shown due to incorporated [³H]amino acids, mainly glutamic acid, generated from the fatty acid. Adding excess sodium pyruvate to labeling media has been used by others to reduce the metabolic conversion of fatty acids to amino acids; however, no direct evidence in support of this antagonism was presented. We observed that inclusion of sodium pyruvate at between 5 and 20 mM in the labeling medium produced a dramatic decrease in incorporation of myristic acid radiolabel into vimentin. However, inclusion of even 20 mM pyruvate did not completely antagonize the metabolic conversion of fatty acid label to amino acids. Furthermore, the sodium pyruvate antagonism could be totally obscured if the exposure of X-ray film by fluorography was even slightly prolonged. The results illustrate the danger in assuming that solvent extraction totally delipidates proteins and that adding sodium pyruvate to labeling media prevents the transfer of fatty acid label to amino acids. Caution is necessary to conclude that radiolabel associated with specific proteins following incubation of cells with labeled fatty acid is due to covalent attachment of the fatty acid to the protein.     

The differential effect of eicosapentaenoic acid and oleic acid on lipid synthesis and VLDL secretion in rabbit hepatocytes

The suppression of plasma very low density lipoprotein (VLDL) triglyceride levels by dietary fish oils rich in polyunsaturated n−3 fatty acids has been attributed to decreased hepatic VLDL secretion. To investigate the effect of n−3 fatty acids on lipid metabolism and VLDL secretion in a tissue culture system, we incubated rabbit hepatocytes with oleic acid and eicosapentaenoic acid (EPA) and examined [³H]glycerol and [¹⁴C]fatty acid incorporation into hepatocyte triglyceride and phospholipid and into media VLDL. Glycerol incorporation studies showed that EPA failed to stimulate VLDL triglyceride secretion from hepatocytes as occurred with oleic acid (P<0.05). Oleic acid preferentially enhanced hepatocyte triglyceride synthesis while EPA stimulated significantly phospholipid synthesis (P<0.01). Varying the relative concentrations of oleic acid and EPA at a constant total fatty acid concentration corroborated preferential triglyceride synthesis from oleic acid. Synthesis shifted predominantly to phospholipids with increasing concentrations of EPA and lower levels of oleic acid. Incorporation of the [¹⁴C]fatty acids (800 μM) followed similar patterns: 87% of [¹⁴C]oleic acid was incorporated into hepatocyte triglyceride and 44% of [¹⁴C]EPA was assimilated in hepatocyte phospholipid (p<0.001). Fatty acids at trace concentrations (53 nM) showed a more divergent pattern of lipid incorporation: 60% of [¹⁴C]oleic acid was incorporated into triglyceride while 91% of [¹⁴CEPA was incorporated into phospholipid (p<0.001). We conclude that in primary rabbit hepatocyte culture, which appears to be a useful model to study lipid metabolism and VLDL secretion, EPA is avidly incorporated into phospholipid while oleic acid predominantly becomes esterified in triglyceride. In addition, EPA, unlike oleic acid, fails to stimulate hepatocyte VLDL secretion. These divergent effects on hepatocyte lipid metabolism are, at least in part, likely to be responsible for fish oil induced suppression of plasma triglycerides.     

Effect of nicotine on lipoprotein metabolism in rats

Nicotine, a major component of cigarette smoke, plays an important role in the development of cardiovascular disease and lung cancer in smokers. The effect of nicotine on lipoprotein metabolism was studied using rats as the experimental animal. There was a significant increase in the total cholesterol, phospholipids, and triglycerides as well as the amount of lipids associated with very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in sera of nicotine-treated rats. The incorporation of ³H labeled leucine into the apo B was found to be increased both in the medium and associated cells in the hepatocytes isolated from nicotine-treated rats indicating an increased synthesis and secretion of the apo B containing lipoproteins. This was further confirmed by the higher incorporation of ¹⁴C acetate into total and individual lipids of LDL and VLDL secreted into the medium as well as that associated with different lipids in the cell layer. The activity of lipoprotein lipase in extrahepatic tissues and plasma lecithin cholesterol acyltransferase activity were significantly lower in nicotine-treated rats. These results indicate that nicotine exerts hyperlipidemic effects particularly by increasing the synthesis and secretion of triglyceride-rich lipoproteins. Since nicotine is one of the major hazardous components present in cigarette smoke and tobacco, one can extrapolate that the deleterious effect exerted by nicotine on rats extends to cigarette smokers and those who use other forms of tobacco.     

Selectivity in incorporation,utilization and retention of oleic and linoleic acids by human skin fibroblasts

Fetal human fibroblasts were grown in culture medium containing 10% fetal bovine serum supplemented with [1-¹⁴C] linoleate or [1-¹⁴C] oleate. At all concentrations of exogenous fatty acids, the incorporation of oleate was greater than that of linoleate. With increased medium fatty acid concentrations, linoleate in triacylglycerol (TAG) could be increased from 13 to 75% of the total incorporated; at each concentration, relatively more linoleate than oleate was in TAG. When the cells were exposed to exogenous oleate/linoleate mixtures, the composition of the mixture determined the extent of incorporation of both fatty acids. When the mixture was primarily linoleate, scarce oleate was used preferentially for phospholipids (PL); no such specificity for scarce linoleate was observed. Addition of exogenous fatty acids resulted in a shift of previously incorporated¹⁴C fatty acids from phospholipid into TAG; retention of oleate in PL was greater than that of linoleate. Incorporation of oleate into phospholipids was also higher than that of linoleate from exogenous fatty acid mixtures which were 80% saturated. It is suggested that normal human fibroblasts have adapted to the low levels of exogenous polyunsaturated fatty acids in culture media by increased use of oleate in phospholipid. Even when the cells aresupplemented with linoleate, the preferential use of oleate in phospholipid groups is retained. Presented in part at the ASBC Meeting, Dallas, april 1979.     

Incorporation of lipids labeled with various fatty acids into the cytoskeleton of aggregating platelets

Earlier studies showed that during the first 20 to 25 seconds of aggregation induced by thrombin (0.1 U/mL) or adenosine diphosphate (ADP) (2μM) of rabbit or human platelets prelabeled with [³H]palmitic acid, labeled lipid became associated with the cytoskeleton (isolated after lysis with 1% Triton X-100, 5 mM EGTA [ethylene glycol-bis-(β-aminoethyl ether(N,N,N′,N′-tetraacetic acid] in the presence of 0.5 mM leupeptin and 50 mM benzamidine). In comparison with labeled lipid in intact platelets, the labeled lipid that was associated with the cytoskeleton was enriched in phospholipids and ceramide. To determine whether these effects were specific for lipids labeled with palmitic acid, we studied rabbit platelets in which lipids had been labeled by incubation of the platelets with pairs of¹⁴C- or³H-labeled palmitic, stearic, arachidonic, and linoleic acids. Examination of the distribution of label among the lipid classes of intact platelets showed that phospholipids contained most of the label. Under the conditions of limited, thrombin-induced aggregation used, labeled lipids were not lost from the platelets and the distribution of label among the lipid classes was essentially unchanged. There were major differences in the incorporation of labeled lipids into the cytoskeleton. The greatest incorporation (2.1 to 2.8% of the label in the platelets) was observed with palmitic acid-labeled lipids; by direct comparison, only 44% as much of the label of stearic acid-labeled lipids, 21% as much of the label of linoleic acid-labeled lipids, and only 6% as much of the label of arachidonic acid-labeled lipids was incorporated into the cytoskeleton. Thus the pool of phospholipid that is readily labeled with arachidonic acid appears to be selectively excluded from the cytoskeleton. Also noteworthy is the 4- to 5-fold enrichment of the cytoskeleton with labeled ceramide; an average of 16% of the label from stearic acid in the cytoskeleton was in ceramide. We suggest that ceramide and phospholipids that are readily labeled with saturated fatty acids are selectively incorporated into the cytoskeleton during the early stages of aggregation and may be specifically associated with the points of contact between platelets.     

Effect of chylomicron remnants on cholesterol metabolism in cultured rabbit hepatocytes: Very low density lipoprotein and bile acid production

The interrelationship between very low density lipoprotein (VLDL) secretion and bile acid production was studied in primary culture of rabbit hepatocytes. Chylomicron remnants (CR) were added to the cultures to study their effect on VLDL secretion and bile acid production. After 24 hr preincubation of cells with CR (10–50 μg protein/mL), intercellular neutral lipid content was increased 1.5–4-fold in a dose-dependent manner. Neutral lipid accumulation was accompanied by a 70–90% reduction of [¹⁴C]acetate incorporation into cholesterol, while no stimulation of [¹⁴C]oleate incorporation into cholesteryl esters was observed. Incubation of cells with CR increased secretion of free cholesterol, triacylglycerol and apoproteins B and E in VLDL. Stimulation of VLDL cholesterol secretion was accompanied by a reduction of taurocholic acid synthesis. These data demonstrate the existence of an inverse relationship between secretion of VLDL cholesterol and bile acid production under conditions of effective uptake of triacylglycerol-rich CR by hepatocytes.     

Incorporation of [1-13C]oleate into cellular triglycerides in differentiating 3T3L1 cells

Oleate is one of the most abundant dietary fatty acids, and much remains to be learned about its metabolism in fat cells. We studied the incorporation of exogenous [1-¹³C]-oleate into triglycerides (TG) in differentiating 3T3L1 preadipocytes using ¹³C NMR spectroscopy. The quantity of oleate incorporated into TG was found to increase as preadipocytes differentiated into fat cells. The ratio of unesterified [1-¹³C]oleate to total stored fatty acids was higher in less differentiated cells, and declined at later stages of differentiation as cells accumulated fatty acids through de novo synthesis. When added as the only exogenous fatty acid, oleate was largely esterified at the sn-2 position. When equimolar unlabeled linoleate was co-provided at the same time, the ratio of [1-¹³C]oleate esterified at the sn-1,3 position increased, implying competition between linoleate and oleate for esterification, especially at the sn-2 position. When cells pre-enriched with [1-¹³C]oleate (esterified to TG) were treated with isoproterenol, a lipolytic agent, most of the [1-¹³C]oleate was still found in TG, despite a high rate of lipolysis determined by measuring glycerol release. This implies extensive re-esterification of the oleate released by lipolysis.     

p-Chlorophenoxyisobutyrate enhanced retention of homologous lipoproteins by human aortic smooth muscle cells

Human aortic smooth muscle cells (SMC) specifically bind and take up indiscriminately both the lipid and protein moietics of homologous²⁵I-very low density lipoproteins (VLDL) and¹²⁵I-low density lipoproteins LDL). Sixty-five to 80% of absorbed lipids are incorporated into the cell lipids, preferentially into the phospholipid fraction. Twenty to 35% of the lipid bound and the protein moiety are eliminated from the cells. Half of the eliminated protein label is recovered as TCA soluble products. Five mM of p-chlorophenoxyisobutyrate (CPIB) raise the level of intracellular radioactivity derived from the lipid moieties of VLDL and LDL by about 40% via a reduced elimination. The processing of the protein moiety and lipoprotein binding to the cell surface are not affected by 5.0 mM of CPIB. CPIB lowers the incorporation of¹⁴C-acetate,¹⁴C-pyruvate, and³²phosphate radioactivity into fatty acids and phospholipids of aortic SMC. Five mM of CPIB reduce the overall palmitic acid synthesis by shifting from de novo synthesis to the mechanism of chain elongation, although the further elongation to saturated C₁₈–C₂₄ fatty acids is also depressed. The CPIB-enhanced retention of the lipid-derived lipoprotein radio-activity is interpreted as a compensatory mechanism providing cellular fatty acids which are deficient as a result of the CPIB inhibited synthetic processes.     

The effect of penicillin on fatty acid synthesis and excretion inStreptococcus mutans BHT

Treatment of exponentially growing cultures ofStreptococcus mutans BHT with growth-inhibitory concentrations (0.2 μg/ml) of benzylpenicillin stimulates the incorporation of [2-¹⁴C] acetate into lipids excreted by the cells by as much as 69-fold, but does not change the amount of¹⁴C incorporated into intracellular lipids. At this concentration of penicillin cellular lysis does not occur. The radioactive label is incorporated exclusively into the fatty acid moieties of the glycerolipids. The increase in the radioactive content of the extracellular lipids reflects an actual net increase in the total fatty acid content as determined by a chemical assay. During a 4-hr incubation in the presence of penicillin, the extracellular fatty acid ester concentration (per mg cell dry weight) increases 1.5 fold, even though there is no growth or cellular lysis. No change is observed in the intracellular fatty acid ester content. An indication of the relative rate of fatty acid synthesis was most readily obtained by placingS. mutans BHT in a buffer containing¹⁴C-acetate. Under these nongrowing conditions free fatty acids are the only lipids labeled, a factor which simplifies the assay. The addition of glycerol to the buffer causes all of the nonesterified fatty acids to be incorporated into glycerolipid. The cells excrete much of the lipid whether glycerol is present or not. Addition of penicillin to the nongrowth supporting buffer system does not stimulate the incorporation of [¹⁴C]-acetate into fatty acids. However, if cells are exposed to penicillin in a growth-supporting medium and then are transferred to the nongrowing buffer system containing no penicillin, the previously exposed cells retain the ability to incorporate [¹⁴C]-acetate into fatty acid at a higher rate than untreated cells over a prolonged period of time. The stimulation of [¹⁴C]-acetate into fatty acids in this system parallels but is not dependent on the stimulation by penicillin of the incorporation of [¹⁴C]-glycerol into glycerolipid and lipoteichoic acid synthesis previously demonstrated by our laboratory. The material of this paper is part of a thesis to be submitted by J.L.B. in partial fulfillment of the requirements for the Ph.D. degree from the Department of Biochemistry, Temple University.     

Acetate and mevalonate labeling studies with developingCuphea lutea seeds

Cuphea seeds contain large amounts of medium chain (C₈ to C₁₄) fatty acids, mainly as triacylglycerols. The biosynthesis of these lipids was studied in vivo by incubating developingCuphea lutea seeds with labeled acetate. Incorporation of label into triacylglycerols and into medium chain fatty acids occurred principally during the period of endogenous lipid deposition, but some label was encountered in these products even during seed dehydration. At this later stage palmitate and oleate were the dominant labeled fatty acids. During the period of rapid endogenous lipid deposition acyl lipids other than triacylglycerols were minor labeled components. The labeling patterns were consistent with the Kennedy pathway for triacylglycerol biosynthesis. The fatty acid composition of the acyl-CoA pool was similar to the total lipid fatty acid composition, but the acyl-ACP pool contained relatively more short chain acyl groups. Squalene was labeled from acetate throughout the period of seed development, but labeled sterols were not detected. Using [2-¹⁴C]mevalonic acid lactone as substrate, squalene was the principal labeled product. Small amounts of label were found in free sterols. However, in terms of mass, free sterol dominated over squalene. The possibility of two independent sites of isoprenoid biosynthesis in the developing embryo is discussed.