Time-course of utilization of [stearic or lignoceric acid]sphingomyelin from high-density lipoprotein by rat tissues

Utilization of stearic and lignoceric acids supplied by high-density lipoprotein (HDL) sphingomyelin to different tissues was followed for 24 h after rats were injected with HDL containing [[1-14C]stearic (18:0) or [1-14C]lignoceric (24:0) acid [Me-3H]choline]sphingomyelin. Both isotopes reached a maximum in tissue lipids 3-12 h after injection and were recovered mainly in the liver (30%) and small intestine (3%), whereas the other tissues contained approx. 1% or less of the injected dose. All the tissues were able to take up some intact sphingomyelin from HDL and hydrolyze it. In the lung and erythrocytes, the 3H:14C ratio of sphingomyelin remained unchanged throughout the studied period, while an increase in the isotopic ratio was observed in the kidney due to the 3H choline moiety re-used for synthesis of new sphingomyelin. Conversely, the isotopic ratio of sphingomyelin decreased in the liver, indicating a saving of the 14C-labelled fatty acids, especially 24:0. Furthermore, [24:0]ceramide in the liver remained at a high level (6% of the injected dose), whereas [18:0]ceramide decreased to 1%. When the tissues were examined 24 h after injection, the proportion of the 14C linked to sphingomyelin in the total 14C was always higher for both kinds of sphingomyelin than the molar proportion of sphingomyelin in the whole of lipid classes. However, in the majority of the extra-hepatic tissues, more [14C]18:0 than [14C]24:0 was recovered in sphingomyelin, and more 14C radioactivity from 18:0 than from 24:0 was redistributed in the other lipids. The choline moiety from both kinds of sphingomyelin was re-used to synthesize phosphatidylcholine, especially in the liver (up to 20% of the injected dose). All these results show that utilization of sphingomyelin from HDL by tissues normally occurs in vivo and that this phenomenon should be taken into account in the study of the phospholipid turnover of cell membranes. They also show that metabolism of sphingomyelin from HDL in the liver and other tissues is dependent on the sphingomyelin acyl moiety.     

Uptake and Utilization of Double-Labeled High-Density Lipoprotein Sphingomyelin in Isolated Brain Capillaries of Adult Rats

Isolated rat brain capillaries were incubated in the presence of high-density lipoprotein (HDL) containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin. This double-labeled sphingomyelin was taken up in a concentration-dependent manner. Cerebral capillary-associated sphingomyelin had a 3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hydrolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of incorporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of "pulse-chase" experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidyl-choline of capillaries.     

Divergent fate of unsaturated and saturated ceramides and sphingomyelins in rat liver and cells in culture

The metabolism of sphingomyelins and ceramides with defined labeled fatty acids was compared after injection in vivo or incubation with cultured cells. The liver was the major site of uptake of sphingomyelins and ceramides with 18:2 or 16:0 fatty acids, but with both sphingolipids a higher recovery of radioactivity was found with 16:0 species. The distribution of radioactivity among liver lipids showed that 1.5 h after injection of 18:2 sphingomyelin, only 21% of the label was found as sphingomyelin, and this value was 37% in the case of 16:0 sphingomyelin. There was a very marked difference in the metabolism of 18:2 and 16:0 ceramides. After injection of 18:2 ceramide only 14% of the radioactivity was recovered as sphingomyelin, and this value was more than 50% with 16:0 ceramide. [14C]18:2 ceramide was converted also to glucoceramide and hydrolyzed more extensively than 16:0 ceramide. These observations were extended to sphingomyelins and ceramides with other fatty acids, using Hep-G2 cells in culture. Significantly more radioactivity was recovered as labeled sphingomyelin after incubation with 16:0, 18:0, 20:0 and 24:0 sphingomyelins than with 18:1 and 18:2 sphingomyelins, while more labeled phosphatidylcholine and phosphatidylethanolamine were found with the unsaturated sphingomyelins. In analogy to the findings in vivo, in the Hep-G2 cells more 16:0, 18:0 and 24:0 ceramides were converted to sphingomyelin than 18:1 or 18:2 ceramides. These differences were also seen with cultured macrophages, in which a more marked reutilization for sphingomyelin formation was found with the saturated ceramide series. The sphingomyelin liposomes were tested also for their capacity to mobilize cholesterol, and a rise in plasma unesterified cholesterol occurred after injection of 18:2 sphingomyelin. Marked enhancement of cholesterol efflux from cholesterol ester-loaded macrophages was also seen with 18:1 and 18:2, 20:0 sphingomyelin in the presence of delipidated high-density lipoprotein. The present results demonstrate that the metabolic fate of sphingolipids is related to their fatty acid composition. While ceramides with saturated fatty acids are predominantly reutilized for sphingomyelin formation, those with unsaturated fatty acids undergo probably more rapid hydrolysis with liberation of fatty acids and channeling into glycerolipids.     

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.     

The uptake and metabolism of plasma lysophosphatidylcholine in vivo by the brain of squirrel monkeys

1. Adult squirrel monkeys were injected intravenously with doubly labelled lysophosphatidylcholine (a mixture of 1-[1-(14)C]palmitoyl-sn-glycero-3-phosphorylcholine and 1-acyl-sn-glycero-3-phosphoryl[Me-(3)H]choline; (3)H:(14)Cratio 3.75) complexed to albumin, and the incorporation into the brain was studied at times up to 3h. 2. After 20min, 1% of the radioactivity injected as lysophosphatidylcholine had been taken up by the brain. 3. Approx. 70% of the doubly labelled lysophosphatidylcholine taken up by both grey and white matter was converted into phosphatidylcholine, whereas about 30% was hydrolysed. 4. The absence of significant radioactivity in the phosphatidylcholine, free fatty acid and water-soluble fractions of plasma up to 30min after injection of doubly labelled lysophosphatidylcholine rules out the possibility that the rapid labelling of these compounds in brain could be due to uptake from or exchange with their counterparts in plasma. 5. The similarity between the (3)H:(14)C ratios of brain phosphatidylcholine and injected lysophosphatidylcholine demonstrates that formation of the former occurred predominantly via direct acylation. 6. Analysis of the water-soluble products from lysophosphatidylcholine catabolism revealed that appreciable glycerophosphoryl-[Me-(3)H]choline did not accumulate in the brain and that radioactivity was incorporated into choline, acetylcholine, phosphorylcholine and betaine. 7. The role of plasma lysophosphatidylcholine as both a precursor of brain phosphatidylcholine and a source of free choline for the brain is discussed.     

Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Arterio-venous differences of choline and choline lipids across the brain of rat and rabbit.

The concentration of unesterified choline in the plasma in the jugular vein of the rat (0.85 nmol/ml) was found to be three times that of the arterial supply to the brain (0.25 nmol/ml), indicating a higher efflux than uptake of unesterified choline by the brain. No such difference was found for the rabbit and no arterio-venous difference for phosphatidylcholine or lysophosphatidylcholine was observed in either species. No arterio-venous difference was found for choline in blood cells. The infusion of [Me-3H]choline into the circulation of the rat or rabbit indicated an uptake of radioactive choline by the brain and an efflux of non-radioactive choline. In the rabbit such an infusion produced a steady rise in the labelling of phosphatidylcholine and lysophosphatidylcholine in the plasma. When [14C2]ethanolamine was injected intraperitoneally into the rat there was a labelling of phosphatidylcholine, lysophosphatidylcholine and sphingomyelin in the plasma and cells of blood from the jugular vein and the arterial supply, as well as in the brain tissue. However, no labelling of unesterified choline in these tissues could be detected. Unesterified choline was shown to be liberated into the plasma when whole blood from the rat or man, but not the rabbit, was incubated for short periods at 30 degrees C.     

Exogenous [1-14C]lignoceric acid uptake by neurons,astrocytes and myelin,as compared to incorporation of [1-14C]palmitic and stearic acids

In order to compare the incorporation of several saturated fatty acids into the brain, radioactive palmitic, stearic and lignoceric acids were injected into mice. The radioactivity was measured in lipids from isolated neurons, astrocytes and myelin.Our data indicate that specific radioactivity of lignoceric acid after its injection was very high in neurons and astrocytes when comparing with serum lignoceric acid specific radioactivity: evidence of the uptake of exogenous lignoceric acid by brain cells and myelin is provided.The incorporation of exogenous palmitic acid into brain cells was much higher than the incorporation of exogenous stearic acid. We hypothesize that exogenous saturated fatty acid uptake is selective in relation with the acyl chain length and the intracerebral synthesis.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

Diacylglycerol synthesized in vitro from sn-glycerol 3-phosphate and the endogenous diacylglycerol are different substrate pools for the biosynthesis of phosphatidylcholine in rat lung microsomes

In microsomes of rat lung, labeled diacylglycerol was synthesized from sn-[3H]glycerol 3-phosphate, which had been added, and from the endogenous free fatty acids. In these microsomes containing biosynthesized [3H]diacylglycerol as well as endogenous nonlabeled diacylglycerol, the synthesis of phosphatidylcholine was measured from added [14C]CDPcholine. The incorporation of [methyl-14C]choline and of [3H]diacylglycerol into phosphatidylcholine showed an entirely different progress in the time-course of incubation. The 14C label of phosphatidylcholine increased continuously, whereas the 3H label remained constant after 2 min up to the end of the incubation period of 20 min. From this result we concluded that the diacylglycerols, synthesized in vitro from glycerol 3-phosphate over an incubation period of 20 min, constitute a separate substrate pool for the biosynthesis of phosphatidylcholine, and are not mixed with the endogenous diacylglycerol pool.     

In vivo metabolism of 3-ketoceramide in rat brain

Eight hours after intracerebral injection of a double-labeled 3-ketoceramide⁴, [1-¹⁴C]lignoceroyl 3-keto [1-³H]sphingosine, various brain sphingolipids were isolated. Free ceramide and the ceramide portions of nonhydroxy cerebroside and sphingomyelin were further fractionated into subgroups containing longer-chain or shorter-chain fatty acids. Nonhydroxy ceramide, nonhydroxy cerebroside and sphingomyelin containing longer-chain fatty acids had significant quantities of radioactivity with ³H/¹⁴C ratios similar to each other but lower than that of the injected material. The sphingolipids containing shorter-chain fatty acids were also significantly labeled; however, the ³H/¹⁴C ratios were much higher than that of the injected material. Hydroxy-ceramide and sulfatides contained very little radioactivity. However, hydroxy-cerebroside contained an amount of radioactivity comparable to that of the longer-chain nonhydroxy cerebroside with a similar ³H/¹⁴C ratio. It is proposed that the injected 3-ketoceramide was converted into ceramide, cerebroside, and sphingomyelin and that the fatty acids of these lipids were partly replaced by other fatty acids during the metabolic conversions.     

Incorporation of 14C-labelled polyunsaturated fatty acids by juvenile turbot, Scophthalmus maximus (L.) in vivo

The ability of juvenile turbot, Scophthalmus maximus (L.), to elongate and desaturate various polyunsaturated fatty acids (PUFA) was examined in relation to their lipid composition. Triacylglycerols were the most abundant lipid class present in the fish and phosphatidylcholine was the predominant phospholipid. In all lipid classes examined the levels of (n-3) PUFA exceeded that of (n-6) PUFA. 18C PUFA were minor components in comparison with 20:5(n-3) and 22:6(n-3). 20:4(n-6) was present in highest concentration in phosphatidylinositol in which it accounted for 16.9% of the fatty acids. When the fish were injected with either ¹⁴C-labelled 18:2(n-6), 18:3(n-3), 20:4(n-6), 20:5(n-3) or 22:6(n-3) the highest percentage recovery of radioactivity (69%) in body lipid was observed with 22:6(n-3). With all labelled substrates free fatty acids contained only a small proportion of the total recovered radioactivity whereas triacylglycerols were highly labelled. Phosphatidylcholine/sphingomyelin was the most highly labelled polar lipid fraction. With ¹⁴C-20:4(n-6) as injected substrate, 23.2% of the radioactivity recovered in total lipid was present in phosphatidylinositol in comparison with less than 6% with the other substrates. Only small proportions of radioactivity from ¹⁴C-18:2(n-6) and ¹⁴C-18:3(n-3) were recovered in the 20 and 22C fatty acids of triacylglycerols and total polar lipid. With ¹⁴C-20:5(n-3) as substrate, 27 and 33% of the total radioactivity recovered in the fatty acids of triacylglycerols and polar lipids respectively was present in 22C fatty acids. The corresponding values for ^l4C-20:4(n-6) as substrate were 19 and 18%. The results confirm the limited capacity of turbot to convert 18C PUFA to longer chain PUFA but demonstrate their ability to synthesize 22C PUFA from 20C PUFA. They also suggest a small but specific requirement for 20:4(n-6).     

Peroxisomal beta-oxidation of branched chain fatty acids in human skin fibroblasts.

Human skin fibroblasts in suspension are able to degrade [1-14C]-labeled alpha- and gamma-methyl branched chain fatty acids such as pristanic and homophytanic acid. Pristanic acid was converted to propionyl-CoA, whereas homophytanic acid was beta-oxidized to acetyl-CoA. Incubation of skin fibroblasts with [1-14C]-labeled fatty acids for longer periods produced radiolabeled carbon dioxide, presumably by further degradation of acetyl-CoA or propionyl-CoA generated by beta-oxidation. Under the same conditions similar products were produced from very long chain fatty acids, such as lignoceric acid. Inclusion of digitonin (> 10 micrograms/ml) in the incubations strongly inhibited carbon dioxide production but stimulated acetyl-CoA or propionyl-CoA production from fatty acids. ATP, Mg2+, coenzyme A, NAD+ and L-carnitine stimulated acetyl-CoA or propionyl-CoA production from [1-14C]-labeled fatty acids in skin fibroblast suspensions. Branched chain fatty acid beta-oxidation was reduced in peroxisome-deficient cells (Zellweger syndrome and infantile Refsum's disease) but they were beta-oxidized normally in cells from patients with X-linked adrenoleukodystrophy (ALD). Under the same conditions, lignoceric acid beta-oxidation was impaired in the above three peroxisomal disease states. These results provide evidence that branched chain fatty acid, as well as very long chain fatty acid, beta-oxidation occurs only in peroxisomes. As the defect in X-linked ALD is in a peroxisomal fatty acyl-CoA synthetase, which is believed to be specific for very long chain fatty acids, we postulate that different synthetases are involved in the activation of branched chain and very long chain fatty acids in peroxisomes.     

Characterization and comparison of lipids in different squid nervous tissues

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.     

Incorporation into phospholipid classes and metabolism via desaturation and elongation of various 14C-labelled (n-3) and (n-6) polyunsaturated fatty acids in trout astrocytes in primary culture

The incorporation and metabolism of [1-14C]18:3(n-3), [1-14C]20:5(n-3), [1-14C]18:2(n-6), and [1-14C]20:4(n-6) were studied in primary cultures of trout brain astrocytes. There were no significant differences between the amounts of individual fatty acids incorporated into total lipid at 22 degrees C, with greater than 90% of all the fatty acids being incorporated into polar lipid classes. The distributions of 18:2(n-6), 18:3(n-3), and 20:5(n-3) in individual phospholipid classes at 22 degrees C were very similar, with 57-63 and 18-24% being incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Approximately equal amounts of 20:4(n-6), approximately 30% of the total, were incorporated into each of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. The metabolism of the (n-3) fatty acids to longer-chain and more unsaturated species was significantly greater than that of (n-6) acids, but delta 4-desaturase activity was very low. A culture temperature of 10 degrees C increased the incorporation of all the fatty acids into total lipid and that of C20 fatty acids into polar lipid. At 10 degrees C, the incorporation of C20 fatty acids into phosphatidylethanolamine and phosphatidylinositol was increased, and the incorporation into phosphatidylcholine and phosphatidylserine was decreased. The distribution of C18 fatty acids was unchanged at the lower temperature, as was the desaturation and elongation of all the polyunsaturated fatty acids incorporated.     

FATP1 channels exogenous FA into 1,2,3-triacyl-sn-glycerol and down-regulates sphingomyelin and cholesterol metabolism in growing 293 cells

Biosynthesis of lipids was investigated in growing 293 cells stably expressing fatty acid (FA) transport protein 1 (FATP1), a bifunctional polypeptide with FA transport as well as fatty acyl-CoA synthetase activity. In short-term (30 s) incubations, FA uptake was increased in FATP1 expressing cells (C8 cells) compared with the vector (as determined by BODIPY 3823 staining and radioactive FA uptake). In long-term (4 h) incubations, incorporation of [(14)C]acetate, [3H]oleic acid, or [(14)C]lignoceric acid into 1,2,3-triacyl-sn-glycerol (TG) was elevated in C8 cells compared with vector, whereas incorporation of radiolabel into glycerophospholipids was unaltered. The increase in TG biosynthesis correlated with an increase in 1,2-diacyl-sn-glycerol acyltransferase activity in C8 cells compared with vector. In contrast, incorporation of [(14)C]acetate into sphingomyelin (SM) and cholesterol, and [3H]oleic acid or [(14)C]lignoceric acid into SM was reduced due to a reduction in de novo biosynthesis of these lipids in C8 cells compared with vector. The results indicate that exogenously supplied FAs, and their subsequently produced acyl-CoAs, are preferentially channeled by an FATP1 linked mechanism into the TG biosynthetic pathway and that such internalized lipids down-regulate de novo SM and cholesterol metabolism in actively growing 293 cells.     

Lipid composition of human serum lipoproteins

1. The lipid compositions of the low-density lipoproteins, the high-density lipoproteins and the ultracentrifugal residue of human serum are presented, with emphasis on certain lipoprotein classes and lipid components not previously described. 2. Except for the lipoproteins with the lowest and highest densities, there is a trend for stepwise successive increase or, respectively, decrease in the relative amounts of the main constituents of lipoproteins. 3. High-density lipoprotein-2 and high-density lipoprotein-3 have different amounts of certain lipids; high-density lipoprotein-2 has relatively more free cholesterol and sphingomyelin; high-density lipoprotein-3 has more free fatty acids, diglycerides and ceramide monohexosides. 4. All the lipoproteins contain hydrocarbons of the alkane series. The greatest amount, which averages 4.4% of total lipid extracted, is in the ultracentrifugal residue; n-alkanes comprise 18-50% of the hydrocarbons. 5. All the lipoproteins contain ceramide monohexosides. The highest relative contents of these glycolipids are in high-density lipoprotein-3 and in the ultracentrifugal residue. 6. The ultracentrifugal residue contains 55% of the total quantity of free fatty acids present in serum. The remaining free fatty acids are distributed among the other lipoprotein classes. 7. The choline-containing phospholipids (phosphatidylcholine, lysophosphatidylcholine and sphingomyelin) comprise about 90% of the phospholipids in all the lipoprotein classes except the low-density lipoprotein-2, which contains about 80% of these phospholipids. 8. The presence of a large amount of lysophosphatidylcholine in the ultracentrifugal residue and the successive decrease of sphingomyelin from the low-density lipoprotein-1 to the ultracentrifugal residue was confirmed. 9. The low-density lipoprotein-2 and the ultracentrifugal residue are characterized by relatively high contents of the lower glycerides.     

Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in chick duodenal mucosal cell. Relationship to its mechanism of action

Pretreatment of the D-deficient chick with 1,25-dihydroxyvitamin D3 increases de novo synthesis of phosphatidylcholine by a stimulation of CDP-choline: sn-1,2-diacylglycerol choline-phosphotransferase reaction. The time course of change in the incorporation of [3H]choline and [14C]ethanolamine into the brush border lipid fraction after 1,25-dihydroxyvitamin D3 treatment correlates closely with the time course of change in calcium uptake into the brush border membrane vesicles. Prior treatment with cycloheximide does not block this increase in phosphatidylcholine synthesis. In addition, 1,25-dihydroxyvitamin D3 administration increases the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction of the brush border to a great extent but does not increase the incorporation of [3H]palmitic acid into the phosphatidylcholine fraction. The incorporation of these 3H labeled fatty acids into diacylglycerol is not changed by 1,25-dihydroxyvitamin D3. These data indicate that 1,25-dihydroxyvitamin D3 enhances the synthesis of phosphatidylcholine independent of new protein synthesis, and also increases the incorporation of unsaturated fatty acids into phosphatidylcholine. From these results we suggest that changes in phospholipid metabolism in the enterocyte are the mechanisms by which 1,25-dihydroxyvitamin D3 acts to enhance calcium entry across the brush border membrane.