Phosphatidylethanolamine metabolism in rats fed a low methionine,choline-deficient diet

The metabolism of phosphatidylethanolamine (PE) was studied in male rats fed a low methionine diet for 7 days with or without supplemental choline. Groups of animals were injected with 2-¹⁴C-ethanolamine and killed at intervals thereafter up to 72 hr. Liver phospholipids were isolated, and PE and phosphatidylcholine (PC) were separated by argentation chromatography into diene (18∶2), tetraene (20∶4) and hexaene (22∶6) fractions. Fatty acid composition and the distribution of radioactivity and specific activity in the total phospholipids and in the fractions were determined. Choline deficiency did not affect total liver phospholipid, but it did increase the amount of PE and decreased that of PC. The major effect of the deficiency on phospholipid fatty acids was to decrease the proportion of PE arachidonate and to increase that of docosahexaenoate. Ethanolamine incorporation into liver PE of deficient rats was only slightly less than in the controls, but loss of the radioactivity from the PE was slower. Ethanolamine radioactivity appearing in the PC of deficient rats was about half that of the controls, even though specific activities of the PE precursors were similar to the control rats. Choline deficiency increased the biological half-lives of the total PE and its fractions. Although the proportion of PE tetraenoic fraction was reduced, the total amount of this liver PE fraction in deficient rats was not affected. However the amount of hexaenoic fraction was doubled, and it accounted for most of the increased quantity of liver PE seen in deficient animals. The results suggested that in choline deficiency PE synthesis was delayed but not appreciably suppressed, and that limited availability of methionine for methylating the PE fractions in their proper proportions affected the concentrations of the PE fractions and impaired their normal conversion to PC. Presented in part at the AOCS Meeting, Houston, May 1971.     

Sources of triacylglycerol accumulation in livers of rats fed a cholesterol-supplemented diet

The source of free fatty acids (FFA) and the pathways contributing to the accumulation of neutral fats in livers of rats fed a cholesterol-enriched diet were investigated in this report. Supplementation with 1% cholesterol in the diet for four weeks resulted in hepatomegaly in the rats. The contents of cholesterol and triacylglycerols (TG) per gram liver measured in rats fasted overnight increased by 48 mg (∼tenfold) and 66 mg (∼fourfold), respectively. The activities of glycerophosphate acyltransferase and diacylglycerol acyltransferase, the two key enzymes for TG synthesis in liver microsomes, were found to increase by 23 and 19%, respectively, in the cholesterol-fed rats. The secretion of plasma TG present predominantly in very low density lipoprotein was found to decrease by ∼30%. The incorporation of tritium from tritiated water in liver FFA increased by twofold in rats fed the cholesterol-supplemented diet, whereas the activity of CPT I in liver mitochondria decreased by 23%. The uptake of plasma FFAin vivo in livers of fasted rats maintained on the cholesterol-supplemented diet decreased by 60%. Our data thus indicate that the excess TG accumulated in livers of rats fed the cholesterol-enriched diet resulted from increased synthesis and decreased secretion of TG. To meet the demand of fatty acids for this purpose,de novo lipogenesis increased, whereas β-oxidation decreased. Although difference in the uptake of extrahepatic FFA may be discounted, a difference in the uptake of chylomicron remnants between the control and cholesterol-fed rats may not be ruled out.     

Development of hyperbetalipoproteinemia in pigs fed atherogenic diet

Hormel miniature pigs were studied over a period of 24 weeks to observe the changes in serum lipoprotein pattern, cholesteryl ester, free cholesterol, and triglyceride in the atherogenic-fed pigs. These pigs were compared to age-related control animals in our breeding herd. Pigs fed the atherogenic diet (20% tallow, 3% cholesterol, and a bile supplement) exhibited a heterogeneous response but showed mean increases in cholesteryl ester (571 mg/dl) and free cholesterol (226 mg/dl), a slight increase in triglyceride (58 mg/dl), and a severe hyperbetalipoproteinemia. Three animals with the highest cholesteryl ester (all above 600 mg/dl) had resolvable beta components in their 1.006 g/ml very low density lipoprotein fraction (type III), as well as huge increases in the Sf 12-20 low density lipoprotein fraction. The other four animals had substantial increases in Sf 0-20, and the three highest had much of their low density lipoprotein in the Sf 12-20, or "remnant" fraction. The test pigs all showed gross lesions in the aorta with an increase in cholesteryl ester and free cholesterol in the tissue as compared with control animals.     

Molecular species of phosphoglycerides in liver microsomes of rats fed a fat-free diet

The influence of a fat-free diet on the molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) of rat liver microsomes was studied by using reversed-phase high-pressure liquid chromatography. In the three phosphoglyceride classes analyzed, the fat-free diet produced a large decrease in the 18:0/20:4n−6 species but less important changes were found in the 16:0/20:4n−6 species. In PC, the most abundant phosphoglyceride class of rat liver microsomes, the fall in the 18:0/20:4n−6 species was counterbalanced mainly by an enhancement in the 16:0/18:1n−9 species although it was not evident in PE. In PI, the decrease in the 18:0/20:4n−6 species was counterbalanced by an increase in the 18:0/20:3n−9 species. Fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene in liposomes of 16:0/18:1n−9-, 18:0/18:1n−9-, 16:0/20:4n−6-, and 18:0/20:4n−6-PC indicated that the change in the saturated fatty acid in the sn-1 position accompanying the replacement of 20:4n−6 by 18:1n−9 could be very important for a homeoviscous compensation, maintaining the membrane physical properties without large alterations in spite of the essential fatty acid deficiency due to the fat-free diet.     

Peroxisomes in mice fed a diet supplemented with low doses of fish oil

The influence of low dietary doses (0.1 and 0.8% w/w) of a commercial fish oil preparation on peroxisomes in normal mice was studied and compared to the known strong inductive effects of high (10%) fish oil diets. Low fish oil doses were chosen to supply the mice with a concentration of docosahexaenoic acid, which was beneficial to patients with a peroxisomal disease. Peroxisomes were evaluated by cytochemical, morphometric, and enzymological techniques. The 0.1% fish oil diet had no effect on peroxisomes in liver, heart, and kidney even after prolonged treatment. The 0.8% diet did not change the peroxisomal number nor the catalase (EC 1.11.1.6) activity in the liver. Hepatic peroxisomal β-oxidation, however, was increased by 50% after 14 d. This was accompanied by reduced peroxisomal size. The 0.8% diet also caused a small increase (+25%) in myocardial catalase activity. No effect was observed in kidneys. Our results indicate that in mice a low (<0.8%) dietary fish oil dose has no or only a slight effect on hepatic peroxisomal β-oxidation. This may be of particular interest to patients with a peroxisomal fatty acid β-oxidation defect and who display a severe deficiency of docosahexaenoic acid—diets supplemented with low fish oil doses will improve the docosahexaenoic acid level without adding a strong load to the disturbed fatty acid metabolism.     

Effect of cocaine intake on the development of fatty liver in rats fed a low-protein diet

It has been shown that cocaine given in the diet is able to reduce fat accumulation in the liver of protein-malnourished rats (Arch. Latinoamer. Nutr. 19: 69-79, 1969). This study was, therefore, designed to approach the probable action of the drug upon the process (increased triglycerides synthesis and normal/decreased capacity for exporting triglycerides from the liver into the blood) which leads to an increased fat accumulation in the liver under this physiological condition. To accomplish this purpose, the total and fractioned lipids in the liver and total lipids as well as lipoproteins in serum were determined in female Wistar rats (120-130 g) fed either a 5% corn protein diet or a 20% casein diet, with and without cocaine (15 mg HCl cocaine/10 g of diet) for 18 days. The results, aside from confirming the reduction (p less than 0.001) of fat accumulation in the liver of rats fed on the 5% corn protein diet plus cocaine, revealed that this drug also reduced triglycerides concentration (significantly, (p less than 0.001, when results were calculated by difference, and slightly reduced them when results were determined) in this tissue. Nevertheless, it increases both total lipids (p less than 0.05) and triglycerides-rich pre-beta lipoprotein (p less than 0.10) levels in the serum of these animals. Otherwise, these lipidic parameters were not modified by cocaine in rats on the 20% casein diet, except for the total cholesterol level in liver and the cholesterol-rich beta lipoprotein level in serum. Respectively, these were slightly and significantly (p less than 0.001) reduced by the drug. These evidences and their statistical significance suggest that cocaine given chronically with the 5% corn-protein diet for 18 days, reduces at least partially (other biochemical event in the liver could have also accounted for its effect at this level) the liver fat accumulation, by increasing the triglycerides output from the liver into the blood. Elsewhere, cocaine appears to be able to induce some metabolic alterations in the hepatic cholesterol of well-nourished rats.     

The effect of exercise on the skeletal muscle phospholipidome of rats fed a high-fat diet

The aim of this study was to examine the effect of endurance training on skeletal muscle phospholipid molecular species from high-fat fed rats. Twelve female Sprague-Dawley rats were fed a high-fat diet (78.1% energy). The rats were randomly divided into two groups, a sedentary control group and a trained group (125 min of treadmill running at 8 m/min, 4 days/wk for 4 weeks). Forty-eight hours after their last training bout phospholipids were extracted from the red and white vastus lateralis and analyzed by electrospray-ionization mass spectrometry. Exercise training was associated with significant alterations in the relative abundance of a number of phospholipid molecular species. These changes were more prominent in red vastus lateralis than white vastus lateralis. The largest observed change was an increase of ~30% in the abundance of 1-palmitoyl-2-linoleoyl phosphatidylcholine ions in oxidative fibers. Reductions in the relative abundance of a number of phospholipids containing long-chain n-3 polyunsaturated fatty acids were also observed. These data suggest a possible reduction in phospholipid remodeling in the trained animals. This results in a decrease in the phospholipid n-3 to n-6 ratio that may in turn influence endurance capacity.     

Hypertension in rats does not potentiate hypercholesterolemia and aortic cholesterol deposition induced by a hypercholesterolemic diet

The effect of a hypercholesterolemic diet (HCD) on hyperlipemia and atherogenesis was investigated using normotensive Wistar/Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP), with systolic blood pressures increasing in that order. Feeding an HCD diet containing cholesterol, cholate and suet induced hypercholesterolemia in all the strains examined as compared with a normal diet. The plasma cholesterol levels were significantly higher in WKY than in SHR and SHRSP fed the HCD diet. The HCD diet also induced hepatic fat deposition, particularly deposition of cholesteryl esters, a slight increase in aortic cholesterol deposition, and elevation of both monoenoic/saturated fatty acid ratios and linoleate/arachidonate ratios in tissue lipids. The changes induced in the three strains by the HCD diet were not positively correlated with blood pressures. The HCD diet affected hepatic acyl-CoA:cholesterol acyltransferase and plasma lecithin:cholesterol acyltransferase activities differently in WKY and SHR which, in addition to the induction of Δ9 desaturase, may partly account for the difference in the diet-induced changes in the fatty acid compositions of plasma cholesteryl esters. The results indicate that hypertensionper se does not stimulate the development of hypercholesterolemia and arterial cholesterol deposition induced by an HCD diet, suggesting that other factors are involved.     

Liver phospholipids of rats fed a choline-deficient diet supplemented with choline or methionine

The low amount of arachidonic acid in the total phospholipids in the liver of rats fed a standard type of choline-deficient diet was corrected by either choline or methionine, which also increased food intake. Choline increased the content of this fatty acid in the phosphatidyl ethanolamine but not in the phosphatidyl choline. Methionine increased both the amount of phosphatidyl choline and its content of arachidonic acid.     

Activity of fatty acyl CoA-lysophospholipid acyltransferases in liver microsomes of rats fed a choline-deficient diet

Feeding a choline-deficient diet to rats has no effect on the activity of fatty acyl CoA-lysophospholipid acyltransferases, even though liver microsomes are severely depleted of lecithins. Thus lecithins appear to have no functional role in the activity of these transferases. It can be excluded that the latter enzymes are involved in the production of the changes in the fatty acid composition of liver phospholipids occurring in choline-deficient rats. These changes result most likely from alterations in the biosynthesis of liver lecithins and cephalins.     

Cholesterol synthesis and degradation in normal rats fed a cholesterol-free diet with excess cystine

Feeding a diet with excess cystine to rats resulted in hypercholesterolemia. To understand the mechanism of the hypercholesterolemia’ cholesterol synthesis and degradation’ bile acid content of bile’ and fecal steroids were determined. The in vivo incorporation of tritiated water into hepatic cholesterol’ and activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in rats fed a high-cystine diet were significantly higher than those in rats fed a control diet. The activity of hepatic cholesterol 7α-hydroxylase was similar between two groups. Little effect of cystine supplementation was found on fecal sterol excretion although there were some changes in biliary excretion of cholic acid derivatives. These results indicate that hypercholesterolemia caused by feeding of a high-cystine diet may be due to the stimulation of hepatic cholesterol synthesis.     

Reduction of essential fatty acid deficiency in rats fed a low iron fat free diet

Young male rats were fed ad libitum for 8 weeks a low iron fat-free (FF-Fe) diet or a fat-free diet supplemented with iron (FF+Fe). The relative levels of 16∶1 to 16∶0 and 18∶1 to 18∶0 in the total fatty acids of liver and other tissues (plasma, erythrocytes and intestinal mucosa) were considerably decreased because of a lack of dietary iron. In rats fed the FF-Fe diet, the levels of essential fatty acids (18∶2ω6+20∶4ω6) in tissues were 2-to 3-fold greater than in the corresponding tissues of rats fed the FF+Fe diet. Eicosatrienoic acid (20∶3ω9) levels in tissue lipids from rats fed the FF+Fe diet were high (8–16%), whereas they were low (2–5%) in the case of animals fed the FF-Fe diet. The proportion of 20∶4 in total fatty acids of tissues was 2-to 3-fold greater in rats fed the FF-Fe diet than when they were fed the FF+Fe diet. Therefore, the relative levels of 20∶3ω9/20∶4ω6 varied from 1-2.9 in tissue lipids of rats fed the FF+Fe diet, while it varied only from 0.2–0.3 in animals fed the FF-Fe diet. These results suggest that a lack of dietary iron may reduce the synthesis of 16∶1, 18∶1, 20∶3 and 20∶4 and the metabolism of 20∶4.     

Levels of polyunsaturated fatty acids in tissues from zinc-deficient rats fed a linseed oil diet

The effect of zinc deficiency on the levels of n−6 and n−3 polyunsaturated fatty acids (PUFA) in lipids from tissues of rats fed a diet containing linseed oil was investigated. Rats were fed either a control diet (25 mg Zn/kg) or a zinc-deficient diet (0.8 mg Zn/kg) for 10 d. To avoid energy and nutrient deficiency, 11.6 g of diet per day was administered by gastric tube. At the end of the experiment, rats fed the zinc-deficient diet had drastically reduced plasma zinc concentration and alkaline phosphatase activity consistent with severe zinc deficiency in these rats. Zinc-deficient rats had higher levels of n−3 PUFA, in particular eicosapentaenoic acid (EPA), and lower levels of n−6 PUFA, in particular linoleic acid, in liver and plasma phosphatidylcholine (PC) and in erythrocyte membrane total lipids than did control rats. By contrast, the levels of n−3 PUFA in PC from testes and heart, and in phosphatidylethanolamine (PE) from liver, testes and heart, were only slightly different between zinc-deficient and control rats. The study suggests that desaturation of α-linolenic acid is not inhibited by zinc deficiency, and that in zinc-deficient rats, n−3 PUFA preferentially incorporated into phospholipids at the expense of n−6 PUFA, especially EPA into PC. The study also shows that the effect of zinc deficiency on PUFA levels is different for PC and PE in rat tissues.     

Changes in lipids in liver and serum of rats fed a histidine-excess diet or cholesterol-supplemented diets

The influence of dietary excess (5%) L-histidine on serum and liver lipids was examined in rats. Feeding a histidine-excess diet for 3, 6, 14 or 30 days caused growth retardation, hepatomegaly and decreased liver lipids throughout the period of the experiment. Hypercholesterolemia was observed after feeding a histidine-excess diet for 6 days; then serum cholesterol continuously increased for 30 days. Serum triglyceride on day 30 in rats fed the histidine-excess diet showed a significant decrease compared to rats fed the basal diet. Serum phospholipids of rats fed the histidine-excess diet for 7 or 14 days showed a significant increase compared to rats fed the basal diet. When rats were fed a basal, histidine-excess or cholesterol-supplemented diet (0.5% and 1.0% cholesterol) for 6 days, the distribution of serum high density (HDL), low density (LDL) and very low density lipoprotein cholesterol in rats fed the histidine-excess diet was similar to that of rats fed the basal diet, whereas LDL-cholesterol increased and HDL-cholesterol decreased in rats fed the cholesterol-supplemented diet.     

Stimulation of hepatic lipogenesis by eicosa-5,8,11,14-tetraynoic acid in mice fed a high linoleate diet

Liver slices, from mice fasted for one day and then refed for three days either a 15% corn oil diet or a 15% corn oil diet containing eicosa-5,8,11,14-tetraynoic acid (TYA), were incubated with [1-¹⁴C] acetate or [³H]H₂O to determine lipogenic capacity. Dietary TYA produced a twofold stimulation in fatty acid and cholesterol synthesis. TYA also caused an increase in the relative proportion of linoleate (C_18∶2) and a decrease in that of arachidonate (C_20∶4) in liver. Thus, (a) despite high levels of C_18∶2, hepatic lipogenesis can be increased, and (b) even short term feeding of TYA can alter the hepatic fatty acid composition presumably by inhibition of arachidonate synthesis from linoleate.     

The effect of clofibrate on heart and plasma lipids in rats fed a diet containing rapeseed oil

The effect of clofibrate on heart and plasma lipids in rats fed a diet containing 30% of the calories as peanut oil (PO) or rapeseed oil (RSO) (42.7% erucic acid and 0.5% eicosenoic acid) was studied. A decrease of erucic acid content to one-third and concomitant increase in the content of 18∶1, 16∶1 and 16∶0 fatty acids in plasma triacylglycerols were observed after administration of clofibrate to rats fed the RSO-diet. It is suggested that these changes reflect the increased capacity of the liver to chainshorten very long chain length fatty acids. The extent of lipidosis in the heart of rats fed the RSO-diet was decreased by 50% by clofibrate. However, the concentration of erucic acid in heart triacylglycerols decreased much less (30%) than the concentration of all other fatty acids (50–65%). It is concluded that the clofibrate administration increased the oxidative capacity of the heart mitochondria and that the heart cell does not have an efficient system to handle very long chain length monounsaturated fatty acids as does the liver.     

Changes in fatty acid composition of phospholipids from liver microsomes and nuclei in rats fed a choline-free diet

Male F-344 rats were fed a choline-free (CF) diet, and changes in phospholipid content, phospholipid fatty acids and phospholipase A₂ activity in liver nuclei and microsomes were examined during the first 72 hr. Both nuclei and microsomes showed a decrease in phosphatidylcholine (PC) content. Microsomes showed an increase in PC arachidonate while nuclei showed a decrease. Also, microsomes showed increased activity of phospholipase A₂ (PLA₂) while nuclei did not. These observations are consistent with the hypothesis that the absence of diene conjugates in liver microsomes in the rats on the CF diet may reflect the increased rate of removal of peroxidized fatty acids by phospholipase A₂.     

Effect of dietary fat upon aflatoxicosis in rats fed torula yeast containing diet

This study was designed to study the possible interrelationships between Torula yeast, vitamin E, and the dietary fat source on aflatoxin-induced tumors. Rats were fed Torula yeast-containing basal diets which included 1.7 ppm aflatoxin B₁ with either lard, corn oil or no fat, and with or without vitamin E supplements for 3 months. Thereafter, the respective diets without aflatoxin were fed for ca. 9 months. Animals receiving the vitamin E-deficient diets had a high mortality. Although the vitamin E-deficient, aflatoxin-treated rats had lower wt gains than did the vitamin E-deficient controls, they lived twice as long. In addition, regardless of the dietary fat source, the kidneys and adrenals of these vitamin E-deficient, aflatoxin-supplemented rats were found to be significantly heavier than the controls, and plasma cholesterol levels were elevated. Increased amounts of liver lipid were observed in response to aflatoxin in both corn oil-fed and fat-deficient rats. No such differences were observed in the responses of the vitamin E-supplemented groups to aflatoxin. On the corn oil diet, aflatoxin administration resulted in an increased deposition of polyunsaturated fatty acids in cholesteryl ester and phospholipid fractions in livers of vitamin E-deficient rats and the phospholipid fraction of vitamin E-sufficient rats. The vitamin E-deficient rats exhibited necrosis of the liver, which was alleviated when aflatoxin was included in the diet, and calcification of the kidneys, which was potentiated by the dietary aflatoxin. No tumors were observed in these animals. In animals maintained on vitamin E-sufficient diets for 1 year, growth was depressed as a result of aflatoxin administration with the greatest depression occurring in the group fed corn oil. Spleen wt were decreased in all groups given aflatoxin. However, there were no changes in either plasma or liver cholesterol or total liver lipids which could be attributed to aflatoxin administration. When aflatoxin was fed with lard, the cholesteryl ester, triglyceride, and free fatty acid fractions of plasma had decreased amounts of the C20:4 acid. In the cholesteryl ester fraction only, this change was accompanied by increased levels of C16:0, C18:0, and C18:1 acids. In the liver phospholipids, there were increased levels of mono- and polyunsaturated fatty acids and decreases in the saturated fatty acids. All of the animals receiving aflatoxin exhibited severe necrosis and tumor formation in the kidneys; the animals fed lard had the highest level of involvement and those in the fat-free group the least. Liver pathology was the least marked among the rats fed the fat-free diet. Since aflatoxin-induced tumors are rich in lipids, the fat-free diet may be protective to the animal.