Effect of sex and bezafibrate on incorporation of blood borne palmitate into lipids of rat liver nuclei

The aim of the present study was to investigate whether lipid metabolism in the nuclei is affected by changes in the metabolism of free fatty acids in the liver. The experiments were carried out on 3 groups of rats: 1 - control-male, 2 - female, and 3 - male, treated with bezafibrate (a peroxisome proliferator). The rats received 14C-palmitic acid intravenously. Thirty min later liver samples and blood from the abdominal aorta were taken. The liver nuclei were isolated in sucrose gradient. Lipids were extracted from the nuclei and the liver homogenate and subsequently separated into the following fractions: phospholipids, mono, di- and triacylglycerols, free fatty acids, cholesterol and cholesterol esters. The radioactivity of each fraction was counted. Furthermore, the content of free fatty acids and the fatty acid binding proteins was measured. It was found that radioactivity was present in each lipid fraction obtained from the liver homogenate and from the nuclei. In the female group, the total radioactivity of lipids in the liver homogenate was lower, whereas in the nuclei it was higher in comparison to the male group. The reduction in the radioactivity in the liver was mostly accounted for by decreased radioactivity in the fraction oftriacylglycerols and phospholipids. In the nuclei, the radioactivity of the fraction of phospholipids, free fatty acids and diacylglycerols was elevated. Bezafibrate did not affect the total radioactivity of lipids in the liver and reduced it in the nuclei. In the liver, the drug increased radioactivity mostly in the fraction of phospholipids and reduced it mainly in the fraction of triacylglycerols. In the nuclei, the radioactivity of each lipid fraction examined was reduced. The content of the fraction of free fatty acids in the liver and in the nuclei in the female and in the bezafibrate-treated groups did not differ from the respective value in the control group. The content of fatty acid binding proteins in the nuclei of the female and bezafibrate-treated groups increased in parallel to the elevation in their content in the cytosol. It is concluded that the female sex hormones and bezafibrate influence the transport of selected lipids into the nuclei. The effects seem to be a consequence of the action of these factors directly on the nucleus.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.     

In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats.

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.     

Influence of Seizures on Lipids of Homogenate and Neuronal and Glial Nuclei of Rat Neocortex

Lipid composition of homogenate and neuronal and glial nuclei of the brain cortex of Wistar rats was studied under normal conditions and after seizures induced by injection of picrotoxin. Seizures increased contents of lysophosphatidylcholine, sphingomyelin, and total phospholipids in the homogenate. In neuronal nuclei contents of total phospholipids, sphingomyelin, phosphatidylcholine, and phosphatidylserine decreased, and contents of free fatty acids and lysophosphatidylcholine increased. In glial nuclei content of total phospholipids decreased and content of free fatty acids increased. The role of changes in the lipid composition of the neocortex cells during seizures and the involvement of lipid messengers in signal mechanisms are discussed.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

Effect of triiodothyronine on the content of phospholipids in the rat liver nuclei.

The aim of the present study was to examine the effect of triiodothyronine (T3) on the content of phospholipids and on the incorporation of blood-borne palmitic acid into the phospholipid moieties in the nuclei of the rat liver. T3 was administered daily for 7 days, 10 microg x 100 g(-1). The control rats were treated with saline. Each rat received 14C-palmitic acid, intravenously suspended in serum. 30 min after administration of the label, samples of the liver were taken. The nuclei were isolated in sucrose gradient. Phospholipids were extracted from the nuclei fraction and from the liver homogenate. They were separated into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and cardiolipin. The content and radioactivity of each fraction was measured. It was found that treatment with T3 reduced the content of phosphatidylinositol and increased the content of cardiolipin in the nuclear fraction. In the liver homogenate, the content of phosphatidylinositol decreased and the content of phosphatidylethanolamine and cardiolipin increased after treatment with T3. The total content of phospholipids after treatment with T3 remained unchanged, both in the nuclear fraction and in the liver homogenate. T3 reduced the specific activity of phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and cardiolipin and had no effect on the specific activity of sphingomyelin and phosphatidylinositol both in the fraction of the nuclei and the liver homogenate. It is concluded that excess of triiodothyronine affects the content of phospholipids in the nuclei. The changes in the content of phospholipids in the nuclei largely reflect changes in their content in the liver.     

Age-related changes of lipid fractions and total fatty acids in the serum of female and male rats aged from 37 to 1200 days

1. Total lipids and the lipid fractions cholesterol ester, triacylglycerols, free cholesterol, free fatty acids and phospholipids as well as the fatty acid patterns of total lipids were measured in the serum of female and male rats (Wistar SPF, strain Hannover) aged 37-1213 days. 2. All parameters were monitored every 49th day. Five female and five male animals were used in each experiment. 3. All lipid fractions showed a significant positive linear regression vs age in both sexes. There were multiple significant differences in lipid fractions between the sexes. 4. Among the fatty acids only docosahexaenoic acid shows a significant correlation with age in both sexes.     

Lipid and fatty acid composition of the fat body during the female reproductive cycle of Labidura riparia (Insecta Dermaptera)

During the reproductive cycle of the female Labidura riparia, cytological observations show cyclical modifications of lipid droplets in the periovarian adipocyte. Fat body lipids and their constitutive fatty acids are analyzed. The lipids are predominantly triacylglycerols, which increase after adult ecdysis during vitellogenic and non-vitellogenic periods. Small amounts of diacylglycerols and phospholipids are found. Diacylglycerols increase during vitellogenesis and decrease during the non-vitellogenic period. Cytological modifications of lipid droplets are probably related to diacylglycerol fluctuations. Gas-liquid chromatography of fatty acid methyl esters shows oleic acid to be the predominant fatty acid in total lipids and triacylglycerols; unsaturated acids are approximately twice as abundant as saturated acids all along the reproductive cycle. Fatty acid composition of diacylglycerols and phospholipids differs from triacylglycerols and total lipids composition. Palmitic, stearic, oleic and linoleic acids represent the major fatty acids; their relative amounts vary during the different periods of the reproductive cycle. The correlations between fat body lipid changes and ovarian development were discussed and compared with observations made on other insect species. Accepted: 23 April 1997     

Fatty acid composition and incorporation of arachidonic acid into phospholipids of hemocytes from the tobacco hornworm Manduca sexta

The fatty acid compositions were determined for total lipids, triacylglycerols, phospholipids and four phospholipid fractions, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/phosphatidylinositol (PS/PI) and cardiolipin (CA) obtained from hemocytes and cell-free serum from second day, fifth instar larvae of the tobacco hornworm Manduca sexta and the standard Manduca rearing medium. The hemocyte fatty acid profiles were considerably different from the profiles of the medium the insects were reared on and from the profiles of the cell-free serum. Hemocyte neutral lipids had lower proportions of polyunsaturated fatty acids than phospholipids. The fatty acid profiles of PC, PE, PS/PI and CA differ from each other and from the total lipid profiles, indicating selective fatty acid incorporation into hemocyte phospholipid species. Studies with radioactive arachidonic acid similarly indicated selective incorporation of polyunsaturated fatty acids into hemocyte lipids. Under our in vitro conditions, >40% of the total radioactivity was incorporated into hemocyte lipids. About 93% of the incorporated radioactivity was found in phospholipids. Within phospholipids. most of the radioactivity was associated with PC (46%), and less with PE (28%) and PS/PI (21%). Very little radioactivity was recovered in CA (0.9%).     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

Studies on (Na+ + K+)-activated ATPase. XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na+ + K+)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na+ + K+)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na+ + K+)-ATPase activity. These results indicate that cholesterol is not essential for (Na+ + K+)-ATPase activity.     

Isolation and composition of the carotenoid-containing oil droplets from cone photoreceptors.

A procedure for isolating the carotenoid-containing oil droplets of cone retinal photoreceptors of Gallus domesticus is described. The oil droplets, composed almost entirely of neutral lipids and carotenoids, have been separated into ten chromatographic components. Similar separations have been carried out on the total retinal neutral lipids for comparison. The neutral lipids represented 26.1% of the total retinal lipid. Cholesterol, cholesterol ester, mono-, di- and triacylglycerols represented 92.6% of the total neutral lipid. Each of these and other minor neutral lipid components were also present in the lipids extracted from the isolated oil droplets in correspondingly similar concentrations. However, the concentrations of carotenoids were greatly enriched in the neutral lipids of the oil droplets. Each of the major fatty acyl-containing neutral lipids from the chromatography of oil droplet lipids is greatly enriched in polyunsaturated fatty acids when compared with the corresponding component from the total neutral lipid chromatography. In the acylglycerols and free fatty acid fraction from the oil droplets, linoleic and arachidonic acid together represented 52-83% of the total polyunsaturated fatty acids present. The remainder was generally distributed about equally among six other acids. Except for the diacylglycerol fraction, linoleic acid was usually the most enriched acid in a specific oil droplet fraction when compared with any other polyunsaturated fatty acids. A similar pattern of polyunsaturated fatty acid enrichment observed in the fatty acids of the outer segment phospholipids relative to the corresponding total phospholipid fractions of this cone rich retina (Johnston, D. and Hudson, R.A. (1974) Biochim. Biophys. Acta 369, 269) suggest possible metabolic relationships between the oil droplet neutral lipids and the outer segment membrane phospholipids of the cone photoreceptors. A mechanism for the accumulation of the carotenoids in the oil droplets is also discussed.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Lipogenic enzyme induction and incorporation of exogenous fatty acid into liver and liver nuclei

The incorporation of exogenous fatty acid into lipids of liver and liver nuclei of rats fed diets with or without fat was compared. When [3H]palmitic acid was injected into rats, more radioactivity was incorporated into triacylglycerols and phospholipids of liver and liver nuclei from rats fed the fat-free diet than from those fed the fat diet. The results were supported further by an autoradiographic study. On the other hand, the enzyme induction and quantity of malic enzyme mRNA were decreased by fat feeding. Other lipogenic enzymes were also coordinately decreased. Thus, it may be possible that exogenous fatty acid is involved in nuclear regulation in addition to cytosolic regulation of lipogenic enzyme induction.     

Age-related changes of lipid fractions and total fatty acids in liver lipids and heart lipids of female and male rats aged 37-1200 days (liver) and 331-1200 days (heart)

1. Total lipids and the lipid fractions cholesterol ester, triacylglycerol, free cholesterol, free fatty acids and phospholipids, as well as the fatty acid patterns of total lipids, were measured in liver homogenates of female and male rats (Wistar SPF, strain Hannover) aged 37-1213 days. 2. The same parameters were measured in the apex of the heart in female and male rats aged 331-1213 days. 3. All parameters were monitored every 49th day. Five female and five male animals were used in each experiment. 4. The lipid fractions in liver showed a positive linear regression vs age, whereas all lipids in rat heart showed a negative regression vs age in both sexes. 5. The significance of regression vs age of fatty acids was much less than that in the lipid fractions of liver and heart of these animals.     

The role of exogenous lipids in lycopene synthesis in the mucoraceous fungus Blakeslea trispora

The addition of plant oils to the growth medium stimulated growth and lipid synthesis in the fungus Blakeslea trispora. However, only oils with high content of linoleic and especially linolenic acid enhanced lycopene formation. The increase in lycopene formation was accompanied by accumulation in the neutral lipid fraction of the fatty acids prevailing in plant oils. In contrast, the influence of exogenous lipids on the fatty acid composition of bulk fungal phospholipids was insignificant. Nonetheless, a marked increase in the content of membrane lipids and of their phosphatidylethanolamine content was revealed. Presumably, the main mechanism of stimulation of lycopene formation by the oils involves an increase in the solubility of lycopene in the triacylglycerols of the lipid bodies, which is due to an increase in the desaturation degree of their fatty acids. The predominance of linoleic and especially of linolenic fatty acid in plant oils is regarded as a criterion for selecting the oil species for the purpose of intensifying lycopene synthesis.     

Studies on furan fatty acids of salmon roe phospholipids

Mature salmon roe lipids were found to consist of triacylglycerols (63%), phospholipids (30%), sterols (4.2%), steryl esters (0.7%), and other minor components. In the steryl esters and phospholipids, furan fatty acids were detected instead of the triacylglycerols of the testes lipids in male fish. The representative 12,15-epoxy-13,14-dimethyleicosa-12,14-dienoic acid (F6) amounted to 3.8% and 0.6% of the total fatty acids in each fraction, respectively. However, the absolute amount of the acid in the phospholipid was much more than that contained in the steryl esters. The characteristic distribution of the furan acids found in the phospholipids was common to the steryl esters in the liver. Large amounts of furan acids were contained in phosphatidylcholine (PC) rather than in phosphatidylethanolamine. For positional analysis of furan fatty acids in PC, furan-containing species in the molecule were concentrated fourteenfold by using selective hydrogenation and repeated silica gel column chromatography. A series of furan fatty acids in PC was found to be exclusively linked to the sn-1 position. The amount of the acids in the roe exclusively linked to the sn-1 position. The amount of the acids in the roe phospholipids was comparable with that in the testes triacylglycerols. The physiological roles of furan fatty acids are discussed.     

Aliphatic Chains of Esterified Lipids in Isolated Eyespots of Euglena gracilis var. bacillaris

Isolated eyespot granules of Euglena gracilis Klebs var. bacillaris Pringsheim contained approximately 6% lipids (based on protein). Separation of the lipid extracts by thin layer chromatography revealed four major fractions: wax esters, triacylglycerols, free fatty acids, and phospholipids. Methanolysis of each fraction yielded between 27 and 29 different fatty acids ranging from 12:0 to 22:6. Acetates of the fatty alcohols of the wax fraction consisted of 11:0 to 18:0 carbon chains, with 14:0 being the major component; unsaturated alcohols were not detected.     

The enzymic degradation of lipids resulting from physical disruption of cucumber (Cucumis sativus) fruit

Homogenization of fresh tissue from cucumber fruits results in a loss of endogenous lipid catalysed by acyl hydrolase enzymes. Deacylation of lipids is not accompanied by accumulation of free fatty acids. The levels of both saturated (mainly palmitic) and polyunsaturated (linoleic and linolenic) fatty acids in the lipids are reduced. Losses of the major acyl lipid constituents of cucumber (triacylglycerols and phospholipids) are mainly responsible for the observed hydrolysis. Triacylglycerol acyl hydrolase (lipase), phospholipase D and polar lipid acyl hydrolase enzyme activities were demonstrated. It is suggested that hydrolytic attack on endogenous lipids is the initial event on disruption of cucumber tissue, in the formation of lipid degradation products, amongst which are the volatile carbonyl compounds responsible for the characteristic flavour of cucumber.     

Lipids of the Green Alga BotryococcusCultured in a Batch Mode

The lipid fraction of the green alga Botryococcuscultured in a batch mode was found to contain polar lipids (more than 50% of the total lipids), di- and triacylglycerols, sterols and their esters, free fatty acids, and hydrocarbons. In aging culture, the content of polar lipids somewhat decreased and that of triacylglycerols increased by more than four times. The content of hydrocarbons in the algal biomass did not exceed 0.9% and depended little on the culture age. Intracellular lipids contained saturated and unsaturated (mono-, di-, and trienoic) fatty acids. The maximum content of C_{16 : 3}and -C_{18 : 3}fatty acids (up to 35% of the total fatty acids) was detected in the phase of active growth. The extracellular and intracellular lipids of the alga differed in the proportion of particular lipids and in the fatty acid pattern.