The cerebro-hepato-renal (Zellweger) syndrome. Impaired de novo biosynthesis of plasmalogens in cultured skin fibroblasts

In tissues of patients with the cerebro-hepato-renal (Zellweger) syndrome the plasmalogen content is very low. In order to study the biosynthesis of plasmalogens, skin fibroblasts of Zellweger patients, controls and heterozygotes, and amniotic fluid cells of controls were cultured in a medium supplemented with [1-14 C]hexadecanol or 1-O-[9,10-3H2]octadecylglycerol. The incorporation of 14C-label into the alkenyl moiety of plasmalogens was strongly reduced in Zellweger patients as compared to controls. The low concentration of 14C-labeled plasmalogens was not compensated for by an elevated levels of 14C-labeled alkyl phospholipids. Hexadecanol was partly oxidized to fatty acid in all cell lines and the incorporation of 14C-labeled fatty acid into phospholipids was comparable for patients and controls. [3H]Alkylglycerol was incorporated into plasmalogens with the same efficiency in Zellweger patients as in controls. These results indicate that only the reaction(s) involved in the introduction of the ether bond in the process of plasmalogen synthesis are deficient in Zellweger patients. The results also suggest that the hexadecanol incorporation patterns can be used for the (prenatal) diagnosis of the Zellweger syndrome.     

Plasmalogen biosynthesis in peroxisomal disorders: fatty alcohol versus alkylglycerol precursors

In recent years a growing number of inherited diseases have been recognized to originate from an impairment in one or more peroxisomal functions. Since it is well established that the first two steps in the biosynthesis of plasmalogens proceed in peroxisomes, we studied the biosynthesis of plasmalogens in cultured skin fibroblasts from patients with different peroxisomal and related disorders. When de novo plasmalogen biosynthesis was studied by growing the cells in the presence of [14C]hexadecanol, impaired plasmalogen biosynthesis was found in rhizomelic chondrodysplasia punctata, cerebrohepatorenal (Zellweger) syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. In all these cases, alkyl-acyl phospholipids, the precursors of plasmalogens, did not accumulate and 1-O-[9,10-3H2]octadecylglycerol was converted into plasmalogens with equal efficiency as in controls. This indicated that impaired de novo plasmalogen biosynthesis as measured by [14C]hexadecanol incorporation was due to a deficient formation of the glycero-ether bond. Using this procedure, normal de novo plasmalogen biosynthesis was found in X-linked adrenoleukodystrophy, adrenomyeloneuropathy, X-linked chondrodysplasia punctata, adult Refsum disease, as well as in heterozygotes for Zellweger syndrome and infantile Refsum disease. The data have indicated that the average extent of the deficiency in glycero-ether bond formation is different in Zellweger syndrome, chondrodysplasia punctata, neonatal adrenoleukodystrophy, and infantile Refsum disease.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.     

Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts

The objective of this study was to determine the contribution of myocardial triglycerides to overall ATP production in isolated working rat hearts. Endogenous lipid pools were initially prelabeled (pulsed) by perfusing hearts for 60 min with Krebs-Henseleit buffer containing 1.2 mM [1-14C]palmitate. During a subsequent 60-min period (chase), hearts were perfused with either no fat, low fat (0.4 mM [9,10-3H] palmitate), or high fat (1.2 mM [9,10-3H]palmitate). All buffers contained 11 mM glucose. During the "chase," 14CO2 production (a measure of endogenous fatty acid oxidation) and 3H2O production (a measure of exogenous fatty acid oxidation) were determined. Oxidative rates of endogenous fatty acids during the chase were 279 +/- 50, 88 +/- 14, and 88 +/- 8 nmol of [14C]palmitate oxidized per g dry weight.min in the no fat, low fat, and high fat groups, respectively, compared to exogenous palmitate oxidation rates of 0, 361 +/- 68, and 633 +/- 60 nmol of [3H]palmitate/g dry weight.min, in the no fat, low fat, and high fat groups, respectively. Endogenous [14C]palmitate oxidation rates were matched by loss of [14C]palmitate from endogenous myocardial triglycerides. Overall triglyceride content decreased during the no fat and low fat chase perfusion but did not change during the high fat chase. Loss of triglyceride [14C]palmitate during the high fat chase was matched by incorporation of exogenous [3H]palmitate in triglycerides. In a second series of perfusions, three groups of hearts were perfused under similar conditions, except that unlabeled palmitate was used during the "pulse" and that 11 mM [2-3H/U-14C]glucose and unlabeled palmitate was present during the chase. During the chase, both glycolysis (3H2O production) and glucose oxidation (14CO2 production) rates were measured. Rates of glucose oxidation were inversely related to the fatty acid concentration in the perfusate (1257 +/- 158, 366 +/- 40, and 124 +/- 26 nmol of glucose oxidized per min.g dry weight in the no fat, low fat, and high fat groups, respectively), while rates of glycolysis were not significantly different between these groups. Calculation of overall ATP production from both oxidative and glycolytic sources determined that even in the presence of high concentrations of fatty acids, myocardial triglyceride turnover can provide over 11% of steady state ATP production in the aerobically perfused heart. In the absence of fatty acids, myocardial triglyceride fatty acids can become the major energy substrate of the heart.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of the isolated perfused rat lung in studies on lung lipid metabolism

A procedure for the use of the isolated perfused rat lung in studies on metabolic regulation has been developed. The procedure, reasonably uncomplicated, yet physiological, maintains the lung so that edema is not observed. The phospholipid content remains normal, and incorporation of [1-(14)C]-palmitate, [2-(14)C]acetate, and [U-(14)C]glucose is linear with time for a minimum of 2 hr. The incorporation of [1-(14)C]-palmitate and [2-(14)C]acetate into the total lung phospholipid fraction and into the phosphatidylcholine and phospatidylethanolamine fractions has been studied. Increasing the concentration of palmitate in the medium from 0.14 to 0.51 mm increased by 60% the incorporation of [1-(14)C]palmitate into the total lung phospholipid fraction at 2 hr. When the palmitate concentration of the medium was 0.14 mm, addition of 0.11 and 0.79 mm oleate to the medium decreased [1-(14)C]palmitate incorporation into the total lung phospholipid fraction at 2 hr by 37 and 49%, respectively. The results suggest that the incorporation of exogenous fatty acids, present in the medium perfusing the lung, into lung phospholipids may depend upon the fatty acid composition of the medium. Known specific acyltransferase activities may be responsible for the ordered incorporation of available fatty acids into lung phospholipids.     

Role of carnitine and carnitine palmitoyltransferase as integral components of the pathway for membrane phospholipid fatty acid turnover in intact human erythrocytes.

The deacylation and reacylation process of phospholipids is the major pathway of turnover and repair in erythrocyte membranes. In this paper, we have investigated the role of carnitine palmitoyltransferase in erythrocyte membrane phospholipid fatty acid turnover. The role of acyl-L-carnitine as a reservoir of activated acyl groups, the buffer function of carnitine, and the importance of the acyl-CoA/free CoA ratio in the reacylation process of erythrocyte membrane phospholipids have also been addressed. In intact erythrocytes, the incorporation of [1-14C]palmitic acid into acyl-L-carnitine, phosphatidylcholine, and phosphatidylethanolamine was linear with time for at least 3 h. The greatest proportion of the radioactivity was found in acyl-L-carnitine. Competition experiments using [1-14C]palmitic and [9,10-3H]oleic acid demonstrated that [9,10-3H]oleic acid was incorporated preferentially into the phospholipids and less into acyl-L-carnitine. When an erythrocyte suspension was incubated with [1-14C]palmitoyl-L-carnitine, radiolabeled palmitate was recovered in the phospholipid fraction, and the carnitine palmitoyltransferase inhibitor, 2-tetradecylglycidic acid, completely abolished the incorporation. ATP depletion decreased incorporation of [1-14C]palmitic and/or [9,10-3H]oleic acid into acyl-L-carnitine, but the incorporation into phosphatidylcholine and phosphatidylethanolamine was unaffected. In contrast, ATP depletion enhanced the incorporation into phosphatidylcholine and phosphatidylethanolamine of the radiolabeled fatty acid from [1-14C]palmitoyl-L-carnitine. These data are suggestive of the existence of an acyl-L-carnitine pool, in equilibrium with the acyl-CoA pool, which serves as a reservoir of activated acyl groups. The carnitine palmitoyltransferase inhibition by 2-tetradecylglycidic acid or palmitoyl-D-carnitine caused a significant reduction of radiolabeled fatty acid incorporation into membrane phospholipids, only when intact erythrocytes were incubated with [9,10-3H]oleic acid. These latter data may be explained by the differences in rates and substrates specificities between acyl-CoA synthetase and the reacylating enzymes for palmitate and oleate, which support the importance of carnitine palmitoyltransferase in modulating the optimal acyl-CoA/free CoA ratio for the physiological expression of the membrane phospholipids fatty acid turnover.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Phosphatidyglycerol in rat lung. II. Comparison of occurrence, composition, and metabolism in surfactant and residual lung fractions.

A comparison of the occurrence, fatty acid composition, and metabolism of phosphatidyglycerol and phosphatidylcholine in the surfactant and residual fraction of rat lung has been carried out. The surfactant and residual fractions were separated by discontinuous sucrose density gradient centrifugation. The surfactant fraction was found to contain 69 percent phosphatidylcholine and 7 percent phosphatidylglycerol. The residual fraction contained 46 percent phosphatidylcholine and 3 percent phosphatidylglycerol. Phosphatidylcholine and phosphatidylglycerol were found to contain 85 and 79 percent palmitate in the surfactant fraction and 67 and 68 percent in the residual fraction, respectively. Isolated rat lungs were perfused with medium containing [U-14C]glucose, [9,10-3H]palmitate, and [1-14C]acetate and the incorporation into palmitate isolated from the alpha and beta position of phosphatidylcholine and phosphatidylglycerol was determined. Each radioactive substrate was found to be incorporated into palmitate of phosphatidylcholine equally at the alpha and beta position of the surfactant fraction. In the residual fraction the specific activity of the beta position palmitate was found to be twice that of the alpha position. The incorporation of [9,10-3H]palmitate and [1-14C]acetate into palmitate at the alpha and beta positions of phosphatidylglycerol was similar in both the surfactant and residual fractions. In each case palmitate at the alpha position had approximately twice the specific activity of that at the beta position. The incorporation of [U-14C]glucose into phosphatidylglycerol of the surfactant fraction was, however, greater in palmitate at the beta position than at the alpha. The results show that phosphatidylglycerol is associated with the lung surfactant fraction and suggest that palmitate esterified to the alpha and beta positions of phosphatidylglycerol and phosphatidylcholine occurs at different rates and is dependent upon the precursor source of palmitate.     

Polyunsaturated fatty acid incorporation into plasmalogens in plasma membrane of glioma cells is preceded temporally by acylation in microsomes.

Plasmalogens (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) are major phospholipids in many tissues and cells, particularly of neural origin. Using cultured C6 glioma cells and subcellular fractions isolated on Percoll gradients we investigated selectivity for esterification of several polyunsaturated fatty acids (PUFA) in the sn-2 position of plasmalogens compared to [1-14C]hexadecanol, representative of de novo synthesis of the ether-linked sn-1 position. In whole cells at a final concentration of 105 microM PUFA, 2-4 nmol plasmalogen/mg protein was labeled in 4 h and 10-14 nmol in 24 h, representing 8-15% and 35-50%, respectively, of initial plasmalogen mass. Incorporation of label from hexadecanol was lower than PUFA incorporation (20:5(n-3) greater than 20:4(n-6) greater than 18:3(n-3) much greater than 18:2(n-6)) suggesting deacylation-reacylation at the sn-2 position. Plasmalogens accounted for 50% of total cell ethanolamine phospholipids and 75% in plasma membrane. Using a novel, improved method for extraction of subcellular fractions containing Percoll, plasma membrane also was enriched in plasmalogen relative to microsomes (107.4 +/- 5.2 vs. 40.0 +/- 2.9 nmol/mg protein). Selectivity for esterification at the sn-2 position of plasmalogens with respect to chain length and unsaturation of the fatty acyl chain was similar in both subcellular fractions and reflected that of whole cells. Labeling of plasma membrane with PUFA and fatty alcohol lagged behind that of microsomes. Chase experiments in cells prelabeled with [1-14C]18:3(n-3) for 2 h showed no significant reduction of label in plasmalogen of any subcellular fraction although accumulation of label in the microsomal fraction was slowed initially. Reduction of plasmalogen label (40-50%) did occur in microsomes and plasma membrane when cells prelabeled for 24 h were switched to chase medium with or without chase fatty acid. Our data suggest that esterification of PUFA to plasmalogen may occur at the endoplasmic reticulum with subsequent translocation to plasma membrane resulting in accumulation of relatively stable pools of plasmalogen that are not readily accessible for deacylation-reacylation exchange with newly appearing PUFA. Alternatively, deacylation-reacylation may occur in a more stable phospholipid pool within the plasma membrane but would involve a slower process than at the endoplasmic reticulum.     

Differential turnover of polyunsaturated fatty acids in plasmalogen and diacyl glycerophospholipids of isolated cardiac myocytes

To investigate the relative turnover of esterified polyunsaturated fatty acids in diacylglycerophospholipids and plasmalogens in isolated cardiac myocytes, we characterized the phospholipid composition and distribution of radiolabel in different phospholipid classes and in individual molecular species of diradyl choline (CGP) and ethanolamine (EGP) glycerophospholipids after incubation of isolated cardiac myocytes with [3H]arachidonate or [14C]linoleate. Plasmalogens in CGP (55%) and EGP (42%) quantitatively accounted for the total plasmalogen content (39%) of cardiac myocyte phospholipids. Plasmalogens comprised 86% and 51% of total arachidonylated CGP and EGP mass, respectively, and [3H]arachidonate was primarily incorporated into plasmalogens in both CGP (65%) and EGP (61%) classes. The specificity activity of [3H]arachidonylated diacyl-CGP was approximately 2- to 5-fold greater than that of [3H]arachidonylated choline plasmalogen, whereas comparable specific activities were found in the [3H]arachidonate-labeled ethanolamine plasmalogen and diacyl-EGP pools. Of the total linoleate-containing CGP and EGP mass, 54% and 57%, respectively, was esterified to plasmalogen molecular species. However, [14C]linoleate was almost exclusively incorporated into diacyl-CGP (96%) and diacyl-EGP (86%). The specific activities of [14C]linoleate-labeled diacyl-CGP and diacyl-EGP were 5- to 20-fold greater than that of the [14C]linoleate-labeled plasmalogen pools. The differential incorporation of polyunsaturated fatty acids in plasmalogens and diacylglycerophospholipids demonstrates that the metabolism of the sn-2 fatty acyl moiety in these phospholipid subclasses is differentially regulated, possibly fulfilling separate and distinct physiologic roles.     

Synthesis of alkyl-ether glycerophospholipids in rat glomerular mesangial cells: evidence for alkyldihydroxyacetone phosphate synthase activity

We studied the ability of rat glomerular mesangial cells and their microsomal fractions to incorporate 1-[14C]hexadecanol to glycerophospholipids via an O-alkyl ether linkage and assessed the presence and activity of the required enzyme: alkyl-dihydroxy acetone phosphate synthase. Suspensions of cultured mesangial cells incorporated 1-[14C]hexadecanol to the phosphatidyl ethanolamine and phosphatidyl choline lipid pools, via a bond resistant to acid and base hydrolysis. When cell homogenates or microsomal fractions were incubated with palmitoyl-DHAP and 1-[14C]hexadecanol, alkyl-DHAP and 1-O-alkyl glycerol were formed (alkyl:hexadecyl). The activity of the enzyme responsible for the O-alkyl product formation was calculated to be 2.5 +/- 0.3 and 544 +/- 50 pmoles/min/mg protein for mesangial cell homogenates and mesangial cell microsomes, respectively. These observations provide evidence that mesangial cells may elaborate either linked lipid precursors de novo for the biosynthesis of O-alkyl glycerophospholipids.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Biosynthesis of triacylglycerols in the intestines of rainbow trout (Salmo gairdnerii) fed marine zooplankton rich in wax esters

Intestinal preparations from rainbow trout fed a diet rich in wax esters incorporated [1(-14)C]hexadecanoic acid and [1(-14)C]hexadecanol into triacylglycerols at the same rate. The ratio of the number of H atoms from C1 of hexadecanol to the number of molecules of hexadecanol incorporated into triacylglycerols was 1.6 : 3.0. [U-14C]Glucose was incorporated much faster into the glycerol moiety of triacylglycerols than was [U-14C]aspartic acid. We conclude that the oxidation of absorbed fatty alcohol to fatty acid and its subsequent incorporation into triacylglycerols is closely linked with the reductive formation of triacylglycerol-glycerol from glucose. The ability of trout intestines to metabolise fatty alcohol to triacylglycerols was the same in fish fed wax esters as in those fed triacylglycerols.     

Generation of Arachidonic Acid and Diacylglycerol Second Messengers from Polyphosphoinositides in Ischemic Fetal Brain

Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.     

Sequence of prolactin effects on phospholipid synthesis in mouse mammary gland explants

In cultured mouse mammary gland explants derived from 12-14 day pregnant mice, the effect of prolactin (PRL) on the rate of incorporation of several precursors into neutral lipids and phospholipids was determined. Employing [14C]-acetate as a substrate, PRL stimulates its incorporation into a) neutral lipids by 4-6 hours, b) phosphatidyl choline (PC) and phosphatidyl inositol-phosphatidyl serine (PI-PS) by 1-2 hours, and c) phosphatidyl ethanolamine (PE) by 2-4 hours. Using [3H]-glycerol as a substrate, the temporal response to PRL for its incorporation into the neutral lipids was the same as that for [14C]-acetate, however, PRL did not enhance the rate of [3H]-glycerol incorporation into the phospholipids at any time through 16 hours. PRL similarly had no effect on the rates of [3H]-choline, [3H]-serine, [3H]-ethanolamine, or [32P]O4 incorporation into the phospholipids at hormone exposure periods of 8 hours or more. And finally, PRL had no effect on the rates of [3H]-arachidonate or [14C]-linoleate incorporation into neutral lipids or phospholipids at culture periods up to 18 hours. These data suggest that the early effect of PRL on [14C]-acetate incorporation into the phospholipids is due to either the insertion of newly synthesized fatty acids and/or the extension of fatty acids contained in the phospholipids.     

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

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

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).     

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.     

Differences in glycerolipid synthesis and insulin regulation in rat hepatocytes and adipocytes

Glycerolipid synthesis was studied by determining radioactive incorporation from either [1-14C] acetate or [U-14C] palmitate. Glycerolipid synthesis in adipocytes, mainly from exogenous palmitate, was preferentially directed to the formation of triacylglycerols, whereas in hepatocytes triacylglycerols and phospholipids were synthesized at similar rates. Insulin stimulated glycerolipid synthesis from acetate in both types of cells, being triacylglycerols more significantly increased than phospholipids. The most relevant difference was the finding that in adipocytes insulin strongly stimulated the formation of diglycerides, apparently from phosphatidate, whereas in hepatocytes insulin only slightly increased diglyceride levels. A possible role of diacylglycerol in insulin action in adipocytes, but not in hepatocytes, is also discussed.