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PubMed1988

Lipid binding properties of fatty acid binding protein isolated from mouse liver.

The present studies were designed (1) to examine the lipid composition of two protein fractions from mouse liver cytosol, and (2) to correlate this composition with the previously observed transfer and lipid binding properties of a protein named fatty acid binding protein. In the first part of this study, the ability of the cytosolic protein fractions to bind fatty acids was studied by gel filtration chromatography of cytosol in the presence of [1-14C] oleic acid. Two fractions with affinity for oleic acid were identified. The amount of phospholipids, cholesterol and cholesterol esters found in the low molecular weight protein fraction was significantly higher than that present in the protein fraction with high molecular weight. When mouse liver microsomes prelabeled with [1-14C] stearate were incubated with the protein fraction enriched in fatty acid binding protein, the protein removed radioactivity from the neutral rather than from the polar lipid fraction. Such an effect was drastically reduced when the protein was presaturated with oleic acid. The results suggest that fatty acid binding protein may bind cholesterol and its esters in addition to free fatty acids.

Animals↗

PubMed1984

Contribution of circulating maternal lipids to fetal tissues in the guinea pig.

Using a constant infusion of [1-14C] palmitic acid into 7 late pregnant guinea-pigs the contribution of circulating maternal lipid to the fetuses was assessed. Within the mother plasma radioactivity was found mainly in free fatty acid with only a small amount circulating as triacylglycerol. In contrast in the fetal plasma [14C] was distributed as one-quarter free fatty acid and three-quarters triacylglycerol, with total [14C] levels being four-fold higher than in the maternal plasma after 160 min of infusion. [14C] Triacylglycerol was found in the fetal liver within 10 min of commencing infusion and levels rose to 12 times the levels of [14C] triacylglycerol in the fetal plasma (per/g or ml) by 100-160 min. [14C] Distribution into the lipid moieties of fetal tissues was found to change with time. By 100-160 min the distribution was: liver, triacylglycerol 83%, phospholipid 7%, free fatty acid 4%; fat, triacylglycerol 80%; heart, triacylglycerol 38%, phospholipid 33%, free fatty acid 9%; kidney, triacylglycerol 15%, phospholipid 58%, free fatty acid 10%; muscle, triacylglycerol 70%, phospholipid 10%, free fatty acid 10%; lung, phospholipid 75%; brain, phospholipid 80%. It was concluded that free fatty acid transferred from the mother contributes substantially to fetal lipids in late gestation in this species.

Animals↗

PubMed1975

Adipose tissue metabolism in essential fatty acid deficienty. Effects of prostaglandin e1, epinephrine, and ACTH.

In an effort to better define some of the metabolic changes that accompany essential fatty acid deficiency (EFAD), we studied glucose metabolism in adipose tissue of EFAD and normal mice under basal conditions and in the presence of prostaglandin E1 (PGE1), epinephrine, and ACTH1-18. Isolated fat cells were incubated in Krebs-Ringer bicarbonate medium containing glucose 1(-14C) or 6(-14C), and the incorporation of radioactive carbon into CO2, total fat, fatty acids, and glyceride-glycerol was determined. It was found that EFAD increased glucose uptake over controls which could be attributed to increased oxidation to CO2 and fatty acid synthesis. The contribution of the pentose cycle to glucose oxidation was 50-80% higher in EFAD adipocytes as compared to controls. ACTH1-18 (0.1 mug/ml) suppressed this by 18 and 30% in the control and EFAD groups, respectively, while epinephrine decreased pentose cycle activity by 83 and 55% in the two groups, respectively. PGE1 alone had no significant effect, but in combination with epinephrine it abolished the inhibitory action of the catecholamine in both groups. It is suggested that, although EFA serve as prostaglandin precursors, the effects of EFAD on the metabolism of fat cells cannot be reversed by PGE1, in vitro. However, these fat cells retain their responsiveness to the action of both lipolytic (ACTH and epinephrine) and antilipolytic (PGE1) agents.

Adipose Tissue↗

PubMed1996

Triacylglycerol and cholesterol transport during absorption of glycerol trioleate vs. glycerol trielaidate.

We used conscious, chronic lymph-fistula rats to compare intestinal lymphatic transport of glycerol trioleate (TO) vs. glycerol trielaidate (TE) and to determine the effect of TO vs. TE on absorption and transport of cholesterol. Rats were implanted with intestinal lymph fistulas and duodenal cannulas and then given intraduodenal infusions of lipid emulsions containing purified TO or TE (40 mumol/h) and cholesterol (7.8 mumol/h + 2 microCi [14C]cholesterol). Lymph samples were collected at 0, 2, 4, 5, 6, 7, and 8 h after the start of lipid infusion. Lymphatic output and luminal and gut wall recovery of radioactive lipid at 8 h were quantified. Triacylglycerol (TG) fatty acid isomers did not affect lymphatic output of TG; lymph TG fatty acid composition and output reflected infusate composition. Lymphatic output of cholesterol (mass and radioactivity) did not differ between groups; luminal and gut wall recovery of [14C]cholesterol was also similar between groups. Similar lymphatic transport of TG and cholesterol between triolein- and trielaidin-infused rats was maintained for up to 16 h after the cessation of an infused lipid load. These results indicate that TO and TE are transported into lymph similarly, and that cholesterol absorption and transport are similar irrespective of whether TO or TE is the TG source. The data suggest that trans fatty acid-induced hypercholesterolemia is not due to altered intestinal absorption and transport of cholesterol.

Animals↗

PubMed1980

In vivo incorporation of tritium from 3H2O into pulmonary lipids of meal-fed and starved rats.

In vivo fatty acid synthesis, as measured by tritium incorporation from 3H2O into fatty acids, was examined in the lungs of meal-fed and starved rats. In meal-fed animals, 74% of the radioactivity isolated from pulmonary lipids was found in the phospholipid fraction, with phospholipid fatty acids accounting for approximately 89% of total radioactivity in this fraction. Starving rats for 72 h markedly reduced in vivo 3H2O incorporation into pulmonary lipids. Intraperitoneal injection of (-)-hydroxycitrate prior to 3H2O administration also markedly reduced isotope incorporation into pulmonary lipid fractions of fed rats. These studies demonstrated net in vivo synthesis of fatty acids in pulmonary tissue of rats using a method that is not complicated by potential differences in metabolic pool sizes or peculiarities of specific carbon substrate sources. Synthesis of fatty acids in vivo was affected by the nutritional state of the animal and citrate appears to be a significant source of cytoplasmic acetyl-CoA for de novo pulmonary lipogenesis in the fed rat.

Animals↗

PubMed1976

Lithium induced alterations in rat ganglionic lipids.

Superior cervical ganglia from normal or lithium fed rats were incubated in vitro with [U-14C]pyruvic acid, in Krebs-Ringer solution with or without lithium ion. Acute lithium incubations were performed on ganglia from normal rats with 25 mEquiv./l of lithium ion, while chronic incubations were performed on ganglia from lithium fed rats with 0.5 mEquiv./l lithium in the bathing solution. Lipids were extracted, separated by thin layer chromatography and specific activities of individual lipids and their fatty acids determined. In control ganglia shinogolipids contained 60-70% of the lipid radioactivity, glycerophospholipids 20-30% and neutral lipids 10-25%. Phosphatidylcholine contained 65% of the glycerophospholipid label and 40% of the lipid phosphorus, while sphingomyelin contained 90% of the sphingolipid label and exhibited the highest specific activity over all. Cholesterol was the major neutral lipid. Niety-five per cent of the glycerophospholipid label and 4% of the sphingomyelin label was localized in the fatty acids. The lipid and fatty acid compositions of all ganglia were similar. However the lipid radioactivity in chronic ganglia was lower than in control, with the sphingolipids most affected. In 80 min stimulated chronic ganglia stearic and oleic acid, radioactivity was depressed with respect to both control fatty acids and to the [14C]palmitate of chronic tissues. In both chronic and acute lithium ganglia stimulated for 80 min, the specific activities of phosphatidylinositol were significantly lower than in control. In contrast to control, the labeling of sphingolipids in resting acute ganglia was higher than in stimulated tissues.

Animals↗

PubMed1984

Brominated vegetable oil myopathy: inhibition at multiple sites.

Skeletal muscle lipid storage was induced by feeding rats brominated vegetable oil (BVO). The defect was examined by measuring radioactive substrate oxidation, intermediates of fatty acid oxidation, and activities of oxidative enzymes. One- and U-[14C] palmitate and 1-[14C] pyruvate oxidation were reduced in muscle after four doses of BVO. Inhibition of U-[14C] palmitate oxidation occurred after two doses. Short chain acylcoenzyme A(CoA) derivatives accumulated in the muscle. Several enzymes of beta-oxidation were significantly reduced, with the greatest reduction in 3-ketoacyl-CoA thiolase. The inhibition probably affected multiple sites of CoA and CoA-derivative metabolism.

Animals↗

PubMed1986

The rate of uptake and efflux of phosphatidylcholine from human erythrocytes depends on the fatty acyl composition of the exchanging species.

The rate of uptake of radioactive phosphatidylcholine molecules of different fatty acid composition in intact erythrocytes as facilitated by a phosphatidylcholine-specific transfer protein has been studied. When trace amounts of radiolabeled phosphatidylcholine molecules are present in donor vesicles consisting of egg phosphatidylcholine and cholesterol, the transfer of the radiolabeled species depends strongly on their fatty acyl composition: dipalmitoylphosphatidylcholine is transferred at the lowest rate, 1-saturated-2-unsaturated species are transferred faster and the highest rate is observed for dioleoyl phosphatidylcholine. Transfer of the various phosphatidylcholine molecules was measured furthermore using donor systems in which the bulk phosphatidylcholine was varied in its fatty acyl composition. Also in this type of experiment, the transfer protein preferentially stimulated transfer of unsaturated phosphatidylcholine molecules, especially from an environment containing more saturated molecules. Finally, the efflux of labeled phosphatidylcholine from intact erythrocytes to plasma in the absence of the phosphatidylcholine-specific transfer protein was studied and it became clear that in this case the nature of the effused molecules itself, rather than the composition of the bulk lipids, determined the effuse rates. An important conclusion to be drawn from these experiments is that radiolabeled phosphatidylcholine molecules, when used as markers for phospholipid exchange or transfer, should resemble in their fatty acid composition the composition of the bulk lipid in order to provide reliable data on rates and extents of the process studied.

Androgen-Binding Protein↗

PubMed1981

Cholesterol and phospholipid biosynthesis in guinea pig megakaryocytes.

Megakaryocytes are the bone marrow cells which produce the blood platelets. Platelet lipids are thought to be important determinants of platelet activity in thrombosis and hemostasis. We have investigated lipid synthesis from [U-14C]-acetate in isolated guinea pig megakaryocytes with the goal of elucidating the genesis of platelet lipids. Cholesterol was the major product of megakaryocyte lipid synthesis from [U-14C]acetate. Incorporation of acetate (0.1 mM) into cholesterol per 10(6) megakaryocytes (1.1 mg protein) was 0.14 nmol in 0.5 h, 0.95 nmol in 1.5 h and 3.2 nmol in 4.0 h. Megakaryocytes also synthesized cholesterol from [U-14C]glucose. In contrast, platelet sterol synthesis was negligible. Both megakaryocytes and platelets synthesized phospholipids from [U-14C]acetate. Megakaryocytes incorporated 0.07 nmol acetate per 10(6) cells into phospholipid in 0.5 h, 0.38 nmol in 1.5 h and 1.8 nmol in 4.0 h. Platelets (10(9) cells, 1.3 mg protein) incorporated 0.15 nmol acetate into phospholipids in 1.5 h. Phosphatidylcholine accounted for 62% of the phospholipid radioactive label in megakaryocytes and 78% in platelets. Phospholipid radioactivity was associated with the fatty acids. We hypothesize that the megakaryocyte may synthesize a major portion of platelet cholesterol and that the phospholipid and fatty acid synthetic pathways available to the platelet are derived from the megakaryocytes.

Acetates↗

PubMed1984

Cholesterol synthesis and esterification in cultured intestinal mucosa. Evidence for compartmentation.

The current studies were undertaken to define the optimal conditions for measuring the absolute rates of cholesterol synthesis in cultured rabbit intestine and to assess whether the rate of sterol synthesis affects the esterification of locally formed or absorbed cholesterol. Using both [3H]water or [14C]octanoate (3 mM) as a precursor, sterol formation was linear during the 24 h culture, resulting in comparable estimates of the rate of synthesis equivalent to 129.5 and 118.7 nmol acetyl CoA incorporated per g per h, respectively. The presence of liposomal cholesterol or the hydroxymethylglutaryl-CoA reductase inhibitor mevinolin suppressed the rates of cholesterol synthesis by 24 and 92% of controls, respectively. Only 12% of total newly synthesized cholesterol was recovered in the medium and more than 97% was in the unesterified form, in both medium and biopsy. Even when the rate of sterol synthesis was stimulated over 90-fold by increasing concentrations of [14C]mevalonolactone, less than 8% of the label in total cholesterol was found in the sterol nucleus of the esterified cholesterol. Rather, the majority of the cholesterol ester-bound radioactivity was incorporated into the fatty acid moiety. On the other hand, there was only a limited decrease in the esterification of absorbed [3H]cholesterol both when the rate of sterol synthesis was increased with 10 mM mevalonolactone and when it was inhibited with mevinolin. The data suggest that locally synthesized and absorbed cholesterol is organized in distinct functional pools with different degrees of esterification in the mucosal epithelial cell.

Animals↗

PubMed1995

Role of glycosyl-phosphatidylinositol hydrolysis as a mitogenic signal for epidermal growth factor.

We have investigated the role of the hydrolysis of glycosyl-phosphatidylinositol (GPI) as one of the signalling pathways elicited after interaction of epidermal growth factor (EGF) with its specific plasma membrane receptor (EGFR). Endogenous GPI was characterized in both NIH 3T3 mouse fibroblast cells and in EGFR-transfected NIH 3T3 cells (designated EGFR T17). GPI molecules isolated from both cell lines were identical and they incorporated radioactivity from both sugar and fatty acid substrates. Incubation of EGFR T17 cells with EGF, produced a rapid and transient hydrolysis of GPI. Maximum hydrolysis occurred after a 1-min incubation with 50 nM EGF. No such effects of EGF were observed in the parental cell line. Both inositol phosphoglycan (IPG)- and EGF-induced cell proliferation was inhibited in the presence of an IPG-antibody to different extents. The relationship between GPI hydrolysis and the activity of the EGFR was studied using the tyrosine kinase inhibitors tyrphostin (RG50864) and genistein. These agents were able to significantly inhibit EGF-mediated cell proliferation, EGF-dependent hydrolysis of GPI and EGF-regulated autophosphorylation of the EGFR. It is concluded that GPI hydrolysis is one of the earliest intracellular events generated in response to EGF.

3T3 Cells↗

PubMed1966

Incorporation of acetate-1-14C into lipids by the perfused liver of normal, X-irradiated, or partially hepatectomized rats.

In order to study lipid metabolism in the liver without interference due to transport from and to the liver the isolated livers of normal, X-irradiated, and partially hepatectomized rats were perfused with acetate-1-(14)C and the distribution of radioactivity in total lipids, total fatty acids, individual lipids, and fatty acids of individual lipids was determined. In X-irradiated animals, an increased incorporation of acetate into many lipids, particularly into cholesterol, was observed. Lipids in the liver of the partially hepatectomized rats exhibited a marked increase in triglyceride content together with a decreased rate of incorporation into all but the phospholipid fractions. It is concluded that the increase usually observed in lipid pontent of the regenerating liver is due to the changes in transcort rather than to changes in synthesis. The changes observed in irradiated liver could be the result of alterations in the metabolism of precursors common to most lipids.

Acetates↗

PubMed1983

The uptake of dietary polyprenols and their modification to active dolichols by the rat liver.

The uptake of dietary polyprenols was studied by administering, through a gastric tube, labeled alpha-saturated and alpha-unsaturated polyprenols, with 11 and 19 isoprene residues. The lipids appeared in all organs but mostly in the liver after 16 h where those with 11 isoprenes were in much higher concentration than the prenols with 19 isoprene residues; the distribution in the liver was studied in detail. About 45% of the polyprenols taken up were esterified with fatty acids. A part of the radioactivity (6-30%) appeared in the supernatant but mostly in water-soluble form. Among subcellular fractions, the highest uptake was found in the outer mitochondrial membranes. After 16 h, both 11- and 19-residue alpha-unsaturated injected prenols were present to a large extent as alpha-saturated compounds in liver homogenates and subcellular fractions. About 10-15% of the lipids were phosphorylated. The results suggest that a part of the dolichol phosphate pool, participating in glycosylation reactions, may derive from dietary unsaturated polyprenols which after uptake can be reduced and phosphorylated.

Animals↗

PubMed1993

Site-specific/stable radioiodination of 1,2-Pal-3-IPPA: an agent for the potential clinical evaluation of pancreatic insufficiency by urine analysis.

To measure pancreatic lipase activity, we synthesized a triglyceride containing a radioiodinated fatty acid. The urinary excretion of radioactivity was measured in five rats following administration of the agent by feeding tube. We attached 15-phenylpentadecanoic acid (PPA) to position-3 of 1,2-dipalmitoyl-rac-glycerol (1,2-Pal) to form 1,2-Pal-3-PPA. The 1,2-Pal-3-IPPA (expected lipase substrate) was prepared by the thallation-iodide displacement method. In a dual-label study, the 125I-1,2-Pal-3-IPPA triglyceride was administered with the 131I-IPPA free acid to rats (n = 5) by oral gavage. Urine and feces were collected daily and the tissue distribution of both tracers was evaluated over a five-day period. A significant portion of the administered activity was excreted in 24 hr in the urine (125I, 30.31% + 4.32%; 131I, 35.0% + 7.29%), which cochromatographed with hippuric acid by thin layer chromatography. Release of the acidic components from the conjugated excretory products by acid hydrolysis of the urine provided the radioactive acidic metabolites. Analysis of the Folch extracts of fat samples demonstrated that the radioactive components cochromatographed in the triglyceride region. This agent appears useful for the evaluation of various gastrointestinal diseases.

Animals↗

PubMed1975

[Effect of alcohol on the metabolic conversion of 14C erucic acid in the liver of rats receiving rapeseed or peanut oil].

The effects of addition of ethanol to diets containing rapeseed or ground nut oil on the metabolic conversions of 14 14C erucic and 9-10 3H oleic acid were studied in the rat liver. 1. Whatever the diet more 14C than 3H radioactivity was recovered in liver lipids 2 and 19 hours after injection of labelled fatty acids. Ethanol has little effect on this incorporation. 2. Only small amounts of 3H oleic acid were converted. 3. In all cases, the metabolic conversion of erucic acid was identical: the main part of 14C was not recovered as erucic acid but was present in other monounsaturated fatty acids n-9: oleic acid (18 : 1), which was the most labelled acid, 16 : 1, 20 : 1 and nervonic acid (24 : 1). 4. The amount of erucic acid converted to shorter chain fatty acids was unchanged by addition of ethanol but the alcohol increased the proportion of 14C radioactivity recovered as nervonic acid. This latter effect was opposite to the effect of rapeseed oil diet, which consisted in a decrease in the conversion of erucic to nervonic acid. 5. A high amount of 14C radioactivity was recovered in the F.F.A. fraction of the liver as an unknown compound (13 and 80% of 14C radioactivity respectively after 2 and 19 h.) Its identification is presently under investigation.

Animals↗

PubMed1988

Radioiodinated p-phenylene bridged fatty acids as new myocardial imaging agents: syntheses and biodistribution in rats.

Due to the inhibition of beta-oxidation, radiolabeled long-chain fatty acids with substituents in the alkyl chain often can enhance the retention of myocardial radioactivity. In this connection the synthesis and biodistribution of 12 new radioiodinated omega-(p-iodophenyl)fatty acids is described featuring a p-phenylene group as an inhibiting group. Phenyl fatty acid esters were acylated (Friedel-Crafts) with omega-(p-halophenyl)fatty acid chlorides, reduced and hydrolyzed (Wolff-Kishner) to form the omega-(p-halophenyl)-p-phenylene-fatty acid (PHIPA) derivatives. The peak heart uptake and the loss of radioactivity from the myocardium of fasted Sprague-Dawley rats depended on the alkyl-chain length and p-phenylene position. Thus, compared to radioiodinated 15-(p-iodophenyl)pentadecanoic acid (IPPA) some of the 131I labeled PHIPA derivatives with 12, 13 or 14 methylene groups proved to show an about equal initial heart uptake (about 5% inj. dose/g at 2.5 min). This was especially true for those PHIPA derivatives with a p-phenylene moiety close to the carboxylic acid group. The retention of radioactivity in the heart was also significantly prolonged when the p-phenylene group was in this position. Among the PHIPA derivatives investigated, 13-(p[131I]iodophenyl)-3-(p-phenylene)tridecanoic acid (PHIPA 3-10) and 14-(p-[131I] iodophenyl)-4-(p-phenylene)tetradecanoic acid (PHIPA 4-10) showed the most promising characteristics for potential use as new myocardial imaging agents. Both agents showed an initial heart/blood activity ratio of about 10 which increased to 15 after 10-30 min. This high value was maintained for at least 4 h.

Animals↗

PubMed1991

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein.

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.

Carrier Proteins↗

PubMed1976

Plasma transport forms of ingested fatty alcohols in the rat.

Previous studies have shown that ingested fatty alcohols are absorbed as fatty acids and fatty acid esters, particularly triglycerides. The present study was carried out to determine whether fatty alcohols are also transported as 0-alkyl glyceryl ethers, alk-1-enyl glyceryl ethers, and as wax esters. Oxidation of fatty alcohols to other lipids was assessed by using a mixture of [1-3H] hexadecanol and [1-14C] hexadecanol of predetermined ratio. The results indicate that the absorption of fatty alcohol, and of its transport forms, parallels the absorption of labeled fatty acids. Six to 25% of plasma radioactivity was present as 1-0-alkyl diacylglyceryl ethers with a smaller proportion of ether lipids in the phospholipid fraction. In addition, 4-13% of the ingested hexadecanol appeared in the plasma as a material having the chromatographic properties of wax ester. Fatty alcohols were not detected in the plasma as alk-1-enyl lipids.

Animals↗