Effect of thyroid hormones on the metabolism of fatty acid components of hepatic plasma membrane lipids in rats of varying age

The metabolism of liver plasma membrane phospholipid acyl components of rats of various age was studied in vivo and in vitro. It was found that the level of incorporation of palmitic and linoleic acids into membranous phospholipids sharply increased in animals aged 1-3 months showing a decrease in 12- and 24 month-old animals. The incorporation of labelled fatty acids into phospholipids is inhibited in liver of adult animals and stimulated in liver of old rats by thyroid hormones.     

Age-associated changes in central nervous system glycerolipid composition and metabolism

In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.     

Incorporation of linoleic acid into membrane glycerophospholipids from rat brain submitted to ischemia and hypoxia

In the presence of ATP, Mg, and CoA-SH[1-¹⁴C]linoleic acid was incorporated into membrane phospholipids (P₂ fraction and synaptosomes) prepared from rat brain cortex. The relative order for linoleate incorporation was: phosphatidylcholine >phosphatidylethanolamine>phosphatidylinositol>ethanolamine plasmalogen >phosphatidylserine. The incorporation of labeled linoleate into P₂ fraction phospholipids was investigated in rats, aged 4, 16, and 90 days, after being subjected to ischemic and hypoxic conditions. With the exception of a small increase in the incorporation of the radioactivity into diacyl-GPC, little change in incorporation profile was observed with 4-day-old rats submitted to ischemic and hypoxic conditions. However, the incorporation of labeled linoleate into membrane phospholipids was decreased in 16-and 90-day-old rats after being subjected to ischemic and hypoxic conditions. Among the phospholipids, the decrease in incorporation of radioactivity was most prominent with ethanolamine plasmalogens and phosphatidylinositol although the radioactivity of phosphatidylcholine seemed to remain relatively constant. The decreased incorporation activity in these two age groups was noted along with concomitant increase in the FFA content, whereas an increase in FFA was not observed in the 4-day-old brain samples. Thus, the specific decrease in labeling of ethanolamine plasmalogens and phosphatidylinositol may be the result of increased enzymic degradation of these compounds after ischemic and hypoxic treatment. Furthermore, the decrease in incorporation of linoleate into membrane phospholipids may be due to an increase in the membrane, FFA pool which subsequently, gave a dilution of the labeled precursor.     

Insulin modifies aging-related inhibition of 1-stearoyl, 2-arachidonoylglycerol phosphorylation in rat synaptic terminals

The purpose of the present study was to analyze diacylglycerol kinase (DAGK) activity in synaptic terminals from cerebral cortex (CC) and hippocampus (Hp) from adult (3-4 month-old) and aged (26-28 month-old) rats. The effect of insulin through DAGK activity on synaptosomes from adult and aged rats was also analyzed under conditions favoring saturated or unsaturated phosphatidic acid (PA) formation, using exogenous di-palmitoil glycerol (DPG) or 1-stearoyl-2-arachidonoylglycerol (SAG) as substrates. Results showed that the enzymatic activity preferentially uses SAG as substrate, thus indicating the presence of ?-type DAGK. A significant decrease in DAGK activity transforming SAG into PA was also observed in both tissues from aged rats. Western blot detection of DAGK? showed that enzyme content undergoes no changes with aging. [3H] inositol incorporation into phosphoinosites was also analyzed to evaluate the role of DAGK? in their synthesis. Data obtained from 3H-inositol incorporation into phosphoinositides revealed that in synaptosomes from aged rats phosphatidylinositol (PI) synthesis is lower than in adult animals. Interestingly, in the presence of SAG, PI synthesis was restored to adult values. DAGK activity over SAG was more highly stimulated by insulin in CC and Hp synaptosomes of aged rats with respect to adult rats. On the other hand, insulin exerted a stimulatory effect on PI and phosphatidylinositol 4 phosphate (PI(4)P) synthesis in synaptosomal CC from aged rats. Taken together, our findings indicate that in aged rats insulin triggers a stimulatory mechanism that reverts the diminished synaptosomal ability to synthesize arachidonoyl phosphatidic acid (20:4 PA). The recovery of this PA species indicates that insulin positively regulates phosphoinositide synthesis.     

SIMULTANEOUS INCORPORATION OF ORALLY ADMINISTERED [9, 10-3H2]OLEIC AND [1-14C]LINOLEIC ACIDS INTO LIPIDS OF THE ADULT RAT BRAIN

—The incorporation of an orally administered mixture of [9,10-³H₂joleic acid and [1-¹⁴C]linoleic acid into the brain and spinal cord lipids was maximal after 24 h compared with 4 h for extraneural tissue. In the latter, both acids were utilized equally well for triglyceride biosynthesis, but linoleate entered phosphatidylcholine more rapidly than oleate. Oleic acid was preferentially incorporated into newly synthesized cholesterol esters although 4 h after dosing most cholesterol esters present in serum were formed preferentially from linoleate presumably by the action of lecithin-cholesterol acyl transferase. In neural tissue, a considerable amount of [1-¹⁴C]linoleate was metabolized to higher polyunsaturated fatty acids, whereas in the case of oleate, 90 per cent of the tritium activity remained in monoenic acids at all time periods studied. Both acids were initially incorporated most rapidly into the lecithin fraction of brain and spinal cord, but after 7 days diacyl phosphatidylethanolamine had the highest specific activity. These data are consistent with the view that the uptake of labelled fatty acids by the brain takes place principally as free acids but that some uptake of esterified forms, probably largely as phosphatidylcholine, also occurs. The low linoleate content of the brain and probably also of cerebrospinal fluid cannot be explained on the basis of a selective restriction on the uptake of this lipid from plasma.     

The role of holotrichs in the metabolism of dietary linoleic acid in the rumen

The uptake and metabolism of linoleic acid by rumen holotrichs (mainly Isotricha prostoma and I. intestinalis) has been examined in in vitro infusion experiments. Maximum absorption and metabolism of [1-14C]linoleate by 2 . 10(6) Isotricha suspended in 100 ml buffer was obtained using an infusion rate of 1.6 mg linoleate/h. After 90 min, 84% of the added substrate was recovered within the cells, mainly as free fatty acid or phospholipid. There was a rapid incorporation of radioactivity into phospholipid, mainly phosphatidylcholine, at the commencement of linoleate infusion but no further incorporation after about 40 min. The presence of bacteria during incubations, in approximately the same Isotricha/bacteria ratio as found in the rumen, reduced the uptake of linoleate and the accumulation of free fatty acid by holotrichs but the incorporation into phospholipid remained similar to that obtained in the absence of bacteria. Very little biohydrogenation of linoleic acid occurred in incubations with holotrichs alone. Bacterial suspensions converted linoleic acid to mainly trans monoene and a small amount of stearic acid, but in incubations containing both bacteria and holotrichs, both stearic acid and trans monoene were major products. Using the latter mixed culture, about 20% of the added [1-14C]linoleic acid was present in holotrich phospholipid of which 62% remained as octadecadienoic acid. The Isotricha population was 3 . 10(3)--2 . 10(4)/ml rumen fluid and it contributed about 23% of the linoleic acid in the rumen of a cow on a hay diet.     

Effects of drought on [1-14C]-oleic and [1-14C]-linoleic acid desaturation in cotton leaves

The effects of water stress on [1-¹⁴C]-oleic and [1-¹⁴C]-linoleic acid desaturations were studied in leaves of two varieties of cotton ( Gossypium hirsutum L.), one drought-sensitive (Reba) and the other more resistant (Mocosinho). After 24 h incorporation, [1-¹⁴C]-oleate led to the appearance of linoleate in phospholipids and, additionally, of linolenate in galactolipids. [1-¹⁴C]-Linoleate was desaturated to linolenate only in galactolipid fractions. Water stress markedly inhibited the incorporation of the precursors into the leaf lipids. The two desaturation steps were affected, particularly the transformation of linoleate to linolenate in monogalactosyldiacylglycerol in the drought-sensitive variety of cotton. The metabolic implications of the inhibition of the biosynthesis of C₁₈-polyunsaturated fatty acids are discussed.     

Effect of propionyl-L-carnitine treatment on membrane phospholipid fatty acid turnover in diabetic rat erythrocytes

In this work we have examined the effect of the oral administration of propionyl-L-carnitine (PLC) on the membrane phospholipid fatty acid turnover of erythrocytes from streptozotocin-induced diabetic rats. A statistically significant reduction in radioactive palmitate, oleate, and linoleate, but not arachidonate, incorporation into membrane phosphatidylcholine (PC) of diabetic rat erythrocytes with respect to control animals was found. Changes in radioactive fatty acid incorporation were also found in diabetic red cell phosphatidylethanolamine (PE), though they were not statistically significant. Oral propionyl-L-carnitine (PLC) treatment of diabetic rats partially restored the ability of intact red cells to reacylate membrane PC with palmitate and oleate, and reacylation with linoleate was fully restored. The analysis of the membrane phospholipid fatty acid composition revealed a consistent increase of linoleate levels in diabetic rat red cells, and a modest decrease of palmitate, oleate and arachidonate. The phospholipid fatty acid composition of diabetic red blood cells was not affected by the PLC treatment. Lysophosphatidylcholine acyl-CoA transferase (LAT) specific activity measured with either palmitoyl-CoA or oleyl-CoA was significantly reduced in diabetic erythrocyte membranes in comparison to controls. In addition LAT kinetic parameters of diabetic erythrocytes were altered. The reduced LAT activity could be partially corrected by PLC treatment of diabetic rats. Our data suggest that the impaired erythrocyte membrane physiological expression induced by the diabetic disease may be attenuated by the beneficial activity of PLC on the red cell membrane phospholipid fatty acid turnover.Abbreviations LAT lysophosphatidylcholine acyl-CoA transferase - PC phosphatidylcholine - PE phosphatidylethanolamine - PLC propionyl-L-carnitine - STZ streptozotocin     

Influence of Age on the Fatty Acid Composition of Breast and Thigh Muscles of Male Turkeys

Large White male turkeys were sacrificed at 4-week intervals from 4 to 28 weeks of age to study the fatty acid distribution in lipid of breast and thigh muscles. A total of 70 turkeys were sampled for this experiment.

Fatty acid distribution varied with advancing maturity and between muscle types. The most abundant fatty acids in the tissues were those with carbon chain lengths of 15:0, 16:0, 18:0, 18:1, 18:2, 20:4 and 24:0. Thigh muscle contained significantly more linoleate (18:2) than did breast. Larger proportions of pentadecanoic (15:0), arachidonic (20:4) and lignoceric (24:0), however, appeared in breast. Indications of minor fatty acids appeared on the chromatograms, but their low concentrations made their estimation and identification difficult.     

Distribution and fatty acid composition of phosphoglycerides in normal human brain

A thin-layer chromatographic procedure for the isolation of tissue phospholipids and their subsequent analysis is described. The method has been applied to the determination of the fatty acids of phosphoglycerides in human brain from the early fetal stage to old age. The study shows changes in the distribution and fatty acid composition of each phosphoglyceride in normal brain, although they are quite small after early childhood. A lipid-specific fatty acid pattern for each of the four major phosphoglycerides was found. Besides this, the pronounced differences between fatty acids of the lipids from the cerebral cortex and from the adjacent white matter justify speaking of a tissue-specific fatty acid pattern for brain phosphoglycerides. The phospholipids of cerebral white matter contained more monoenoic acid but much less polyunsaturated fatty acid than those of cerebral cortex. The brain phosphoglycerides also showed an age-dependent fatty acid pattern. With increasing age the concentration of the fatty acids of the linoleate family diminished while that of the linolenate family increased. Brain inositol phosphoglycerides, the fatty acid composition of which has not been studied systematically before, were characterized by a large concentration of arachidonate which was nearly as high for white as for gray matter and showed only small changes with age.     

Alteration of fatty acid composition of LM cells by lipid supplementation and temperature.

Alteration of the fatty acid composition of monolayer cultures of LM cells grown in chemically defined medium was achieved by supplementation with fatty acids complexed to bovine serum albumin. Phospholipids containing up to 40% linoleate were found in cells grown in medium containing 20 mu g of linoleate/ml. Incorporation of linoleate into phospholipids reached a plateau after 12-24 hr, and cells remained viable for at least 3-4 days. Although linoleic, linolenic, and arachidonic acids were incorporated into LM cells equally well, only the latter was elongated by these cells under these experimental conditions. Nonadecanoic acid was incorporated to a lesser extent than the polyunsaturated fatty acids. Phosphatidylcholine and phosphatidylethanolamine of LM cells had different fatty acid compositions; phosphatidylethanolamine contained more longer chain and unsaturated fatty acids. Cells were also grown in the absence of choline and presence of choline analogs such as N,N-dimethylethanolamine, N-methylethanolamine, 3-amino-1-propanol, and 1-2-amino-1-butanol. The analog phospholipids in these cells had fatty acid compositions which were intermediate between those of phosphatidylethanolamine and phosphatidylcholine of control cells grown in the presence of choline. Linoleate was found in both phosphatidylcholine and phosphatidylethanolamine of cells supplemented with linoleate. The sphingolipid fraction of these cells, however, did not contain significant amounts of linoleate. When linoleate was present in the phospholipids, compensatory decreases in the oleate and palmitoleate content of phospholipids were observed. Lowering of the growth temperature to 28 degrees produced an increase in unsaturate fatty acid content of the phospholipids. When linoleate was supplied to cells grown at 28 degrees, there was no further increase in the unsaturated fatty acid composition of the phospholipids. Using both fatty acid supplementation and lowered growth temperature, LM cell membranes can be produced which have phospholipids with vastly different fatty acid compositions.     

The incorporation of oleic and linoleic acids and their desaturation products into the glycerolipids of maize leaves

[1-¹⁴C]Oleic and [1-¹⁴C]linoleic acids were rapidly desaturated when incubated with maize leaves from 8-day-old plants and the labeled fatty acids, and their desaturation products, were rapidly incorporated into glycerolipids. Oleic acid was desaturated to linoleate at the rate of 0.7 nmol/100 mg tissue/h and further desaturated to linolenate at about one-third this rate. The rates of linolenate formation were similar when either oleic acid or linoleic acid was the substrate although there was a 2-h lag period when oleic acid was substrate. When radioactive oleic, linoleic, and linolenic acids were substrates, phosphatidylcholine was the most extensively labeled glycerolipid followed by monogalactosyldiacylglycerol. The relative rates of incorporation of label into individual glycerolipids are consistent with a movement of labeled fatty acids from phosphatidylcholine to monogalactosyldiacylglycerol and then to diagalactosyldiacylglycerol. The rates of labeling of phosphatidylcholine oleate and of phosphatidylcholine linoleate are consistent with a precursor-product relationship in that there was a delayed accumulation of phosphatidylcholine linoleate relative to that of phosphatidylcholine oleate and phosphatidylcholine linoleate continued to accumulate while phosphatidylcholine oleate declined. Linoleate formed from oleate was widely distributed in glycerolipids but neither phosphatidylcholine linolenate nor linolenate-containing diacylglycerol was detected at short and intermediate incubation times when either oleic or linoleic acid was substrate. The kinetics of incorporation of linoleate and linolenate into monogalactosyldiacylglycerol suggest a transfer of linoleate from phosphatidylcholine. The initial rate of accumulation of labeled linolenate in monogalactosyldiacylglycerol was very similar to the rate of desaturation of linoleate and it is suggested that desaturation of linoleate occurs while associated with monogalactosyl-diacylglycerol.     

Metabolism of hexacosatetraenoic acid (C26:4,n-6) in immature rat brain.

Rat brain was recently found to contain polyenoic very-long-chain fatty acids (VLCFA) belonging to the n-3 and n-6 series with four, five and six double bonds and even-carbon chain lengths from 24 to 38 [Robinson, Johnson & Poulos (1990) Biochem. J. 265, 763-767]. In the present paper, the metabolism in vivo of hexacosatetraenoic acid (C26:4,n-6) was studied in neonatal rat brain. Rats were injected intracerebrally with [1-14C]C26:4,n-6 and the labelled metabolites were examined after 4 h. Radioactivity was detected mainly in non-esterified fatty acids, with smaller amounts in other neutral lipids and phospholipids. Radiolabelled fatty acid products included C28-36 tetraenoic and C26-28 pentaenoic VLCFA formed by elongation and desaturation of the substrate, and C14-24 saturated, C16-24 monoenoic, C18-24 dienoic, C18-22 trienoic and C20-24 tetraenoic fatty acids formed from released [1-14C]acetate either by synthesis de novo or by elongation of endogenous fatty acids. The data suggest that polyenoic VLCFA are synthesized in brain from shorter-chain precursor fatty acids and undergo beta-oxidation.     

Fatty-acid desaturation and microsomal lipid fatty-acid composition in experimental hyperthyroidism.

We have studied the influence of experimental hyperthyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty-acid composition. Tri-iodothyronine treatment (25 micrograms/100 g body weight) daily for 3 weeks caused no significant changes in delta 9 (stearate) desaturation but a 24% decrease in delta 6 (linoleate) desaturation. Much larger doses of tri-iodothyronine increased delta 9 desaturation. Liver microsomal fatty-acid composition in hyperthyroidism is altered with significantly increased proportions of stearate and arachidonate and decreased proportions of palmitate, palmitoleate, linoleate (C18:2) and eicosa-8,11,14-trienoate (C20:3). These changes, other than the decreases proportion of C20:3 fatty acid, which may be due to the diminished delta 6 desaturase activity, cannot be attributed to changes in fatty-acid desaturation. Most of these changes were also found to be due not simply to the decreased weight gain or the increased food intake of the hyperthyroid animals. Only the decreased C18:2 fatty-acid proportions could be mimicked by restricting food intake of control animals and none of the changes were prevented by restricting food intake of hyperthyroid animals. Thus most of the changes in microsomal lipid fatty-acid composition are likely to be due to a thyroid hormone effect on peripheral lipid mobilization or lipid degradation.     

Age-related characteristics of the metabolism of fatty acid components of nuclear phospholipids

The metabolism of liver nuclear phospholipid acyl components of rats of various age was studied in vitro. It was found that the activity of phospholipases A1 and A2 in the nuclei sharply increased in animals aged 1-3 months, showing a decrease in 12- and 24-month-old animals. The incorporation of labeled arachidonic acid into nuclear phospholipids remained practically unchanged thereby. The age-specific fluctuations in the activity of nuclear phospholipids A1 and A2 may be one of possible reasons of changes in the fatty acid composition of nuclear lipids during ontogenesis.     

Turnover of palmitic and arachidonic acids in the phospholipids from different brain areas of adult and aged rats

[³H]Palmitic acid and [¹⁴C]arachidonic acid were injected together into the cerebral ventricle of 4-month and 24-month-old rats. At different time intervals from the injection, the distribution of these fatty acids in the lipids from different brain areas was examined. The fatty acids were rapidly incorporated into the lipids through different mechanisms. The time-specific activity relationship indicate that the utilization of the fatty acid differs according to the different areas and aging decreases the utilization of both the fatty acids. The decline of arachidonic acid incorporation into phospholipids is particularly evident, indicating that aging affects mainly the utilization of polyunsaturated fatty acids.     

Acetate-1-14C incorporation into polyunsaturated fatty acids of phospholipids of developing chick brain

The incorporation of acetate-1-(14)C into the polyunsaturated fatty acids of glycerophosphatides of chick embryonic brain has been studied. After the injection of acetate-1-(14)C into the yolk sac, differences were found in the degree of labeling of the major fatty acids of the omega3 and omega6 series. Arachidonic acid (20:4omega6) showed a high degree of radioactivity while docosahexaenoic acid (22:6omega3) was poorly labeled, at a period of brain development when both fatty acids were being actively deposited. Evidence is presented to indicate that the low activity in docosahexaenoic acid is not explicable on the basis of either a low or high rate of turnover of this polyenoic acid. Similar results were obtained whether the rapid early or slower late stage of brain development was examined. It is suggested that the elongation of omega3 and omega6 series acids may be under the control of different regulatory mechanisms.     

Brain arachidonic acid incorporation is decreased in heart fatty acid binding protein gene-ablated mice

Heart fatty acid binding protein (H-FABP) is expressed in neurons, but its role in brain fatty acid incorporation and metabolism is poorly defined. We examined the effect of H-FABP gene ablation on brain incorporation of arachidonic ([1-(14)C]20:4n-6) or palmitic ([1-(14)C]16:0) acid in vivo. Analysis of brain mRNA confirmed gene ablation and demonstrated no compensatory changes in the levels of other FABP mRNA in the gene-ablated mice. In brains from H-FABP gene-ablated mice, the incorporation coefficient for [1-(14)C]20:4n-6 was reduced 24%, while that for [1-(14)C]16:0 was unaffected. Within the organic and aqueous fractions, significantly more [1-(14)C]20:4n-6 was distributed into the aqueous fraction, suggesting a disruption in the metabolic targeting of 20:4n-6 in these mice. There was less incorporation of [1-(14)C]20:4n-6 into total phospholipids and a marked reduction (51%) in the level of incorporation into the choline glycerophospholipids (ChoGpl). Because FABP can influence steady-state lipid mass, brain individual lipid masses were measured. The brain total phospholipid mass was reduced 17% by gene ablation, ascribed to a 27% and 32% reduction in the masses of ChoGpl and sphingomyelin, respectively. Plasmalogen subclass masses were also reduced, suggesting that H-FABP may augment brain plasmalogen synthesis. In gene-ablated mice, the phosphatidylinositol 20:4n-6 level was reduced 25%, while the proportion of total n-6 fatty acids was reduced in the major phospholipid classes. Thus, these results demonstrate for the first time that H-FABP expression influences brain 20:4n-6 uptake and trafficking as well as steady-state brain lipid levels.     

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.     

Aging influence on delta-6-desaturase activity and fatty acid composition of rat liver microsomes

The activity of delta-6-desaturase (D6D) in liver microsomes and fatty acid composition of microsomal lipids of rats of different ages were studied. The D6D activity was similar in suckling rats and in weaning rats. However, the enzyme showed a significantly decreased activity in oldest animals, and a linear correlation was found between the D6D activity and the animal age. The fatty acid composition data on total lipids of liver microsomes were consistent with the age-dependent changes in fatty acid desaturase activity. The major changes occurred in the linoleate and arachidonate fractions; the 20:4/18:2 ratio in liver microsomes decreased together with D6D activity during aging. The loss of D6D activity may be a key factor in aging through altering lipid membrane composition.