Effect of dietary fat on individual long-chain fatty acyl-CoA esters in rat liver and skeletal muscle

The effect of dietary fat on the long-chain acyl-CoA ester profile of liver and skeletal muscle was investigated by feeding weanling rats 12%-fat diets composed of high-linoleic safflower oil (73% 18∶2n−6), high-oleic safflower oil (70% 18∶1n−9) or olive oil (70% 18∶1n−9) for six and ten weeks. Approximately 50% of both hepatic and skeletal muscle acyl-CoA esters comprised linoleoyl-CoA or oleoyl-CoA with high-linoleic or oleic feeding, respectively. Total hepatic acyl-CoA ester concentration was 40% higher (p<0.05) in rats fed 12% fat compared with controls fed a 4%-fat diet. These data demonstrate that the long-chain acyl-CoA ester profile of liver and skeletal muscle reflects the dietary fatty acid profile.     

Effect of high fat/high erucic acid diet on phosphatidate synthesis and phosphatidate phosphatase in the subcellular fractions of rat heart and liver

Rats of weaning age were fed for a period of 1,3 or 6 weeks either a control diet (laboratory stock diet) or a semisynthetic diet containing 20% by weight of either mustard seed oil (1/3 of the total fatty acids were comprised of erucic acid) or corn oil (2/3 of the total fatty acids consisted of linoleic acid). Mitochondrial and microsomal fractions were isolated from the hearts and livers of these rats, and the rate of acylation ofsn-[U-¹⁴C] glycerol 3-phosphate (P) was examined using palmitoyl-CoA or erucoyl-CoA as the acyl donor. In addition, activities of phosphatidate phosphatase of the mitochondrial, microsomal and soluble fractions were assayed. Studies on the acylation of glycerol 3-P with palmitoyl-CoA demonstrated that feeding of the high fat/high erucic acid diet for 1,3 or 6 weeks significantly increased the rate of formation of monoacylglycerol 3-P by the cardiac subcellular fractions as compared to the control. The rate of formation of diacylglycerol 3-P also increased but to a lesser degree. Feeding the high fat/high linoleic acid diet tended to increase acylation of glycerol 3-P by cardiac subcellular fractions. However, neither high fat diet influenced acyltransferase activities of the hepatic subcellular fractions or phosphatase activities of the cardiac and hepatic fractions. Studies on the acylation of glycerol 3-P with erucoyl-CoA demonstrated that the rate of acylation was ca. 1/10 that measured using palmitoyl-CoA in all experiments; in particular, the formation of diacylglycerol 3-P was extremely slow, suggesting that erucoyl-CoA is an unsuitable substrate for the position-2 of the monoacylglycerol 3-P. The rate of acylation by the cardiac and hepatic subcellular fractions was not influenced by the feeding of the high-fat diets. The rate of glycerol 3-P acylation by both cardiac and hepatic mitochondrial fraction was ca. 2/3 of the rate of acylation by the respective microsomal fraction. In addition, the ratio of monoacyl-to diacylglycerol 3-P synthesized by the mitochondrial fraction was smaller than that by the microsomal fraction. These results suggest that acylation of glycerol 3-P by the mitochondrial cannot be attributed to the action of the contaminating microsomal enzymes.     

Distribution of the phosphatidate phosphohydrolase activity in the lamellar body and lysosomal fractions of lung tissue

Phosphatidate phosphohydrolase (PAPase) activity was measured in lamellar bodies purified from porcine lung tissue. After repeated freeze-thawing, only a negligible amount of PAPase activity was released into the soluble fractions, whereas there was release of 2 lysosomal marker enzyme activities, glucosaminidase and β-galactosidase into the soluble fraction. In addition, a lysosomal-enriched fraction was prepared from adult rat lung tissue by prior treatment of the rats with Triton WR 1339. Treatment with Triton WR 1339 resulted in the significant shift of the activities of the lysosomal marker enzymes, glucosaminidase and β-galactosidase, to less dense subcellular fractions. The highest specific activity of PAPase was found in a subcellular fraction which had a density that was intermediate between that of the mitochondrial and microsomal fractions and the distribution of PAPase activity was not affected by the prior treatment of the rats with Triton WR 1339.     

Difficulties in the assay of phosphatidate phosphohydrolase activity. Influence of ionic strength,detergent, and selection of substrate

In the present paper, problems in connection with assay of the activity of magnesium-dependent rat liver phosphatidate phosphohydrolase (PAP) are discussed. PAP activity is usually measured by following the production of diacylglycerol or inorganic phosphate from the substrate phosphatidate. These two methods may give widely different results due to a number of factors that may affect the assay. One such factor is the composition of the substrate. Higher apparent enzyme activity was observed with dioleoyl-phosphatidate than with dipalmitoyl-phosphatidate. This substrate-dependent difference in apparent PAP activity was 2-2.5-fold in the absence and 10-fold in the presence of Triton X-100, respectively. Triton X-100 reduced the activity as measured with the dipalmitoyl-phosphatidate substrate. In contrast, the activity of PAP as measured with dioleoyl-phosphatidate was stimulated by Triton X-100 The stimulatory effect of Triton was reduced or abolished when the ionic strength in the assay mixture was increased. Assays based on³²P-labeled substrate are rapid and sensitive. It is shown here that³³P can be used as an alternative. This radionuclide has a longer half-life and also emits particles with lower energy, thus posing less potential health hazards for the user.     

One-step synthesis of radioactive acyl-CoA and acylcarnitines using rat liver mitochondrial outer membrane as enzyme source

Rat liver mitochondrial outer membrane enriched preparations have proven to be a convenient enzyme source for synthesizing coenzyme A (CoA) and carnitine esters of radioactive fatty acids. These membranes are simple to isolate and they retain acyl-CoA ligase and carnitine palmitoyltransferase activities well upon storage. Enzyme purification is not required. A novel aspect of the present procedure is that the same enzymatic incubation step allows both the acyl-CoA and the acylcarnitine esters to be obtained simulataneously when carnitine is present, but produces acyl-CoA ester only when carnitine is not included. Under the conditions described, the conversion of [1-¹⁴C]octanoic acid to the respective esters was about 95%; the corresponding figure for [1-¹⁴C]palmitic acids was over 70%. The procedure seems suitable for synthesizing the labeled CoA and carnitine esters from a variety of radioactive fatty acids. A preliminary account of this work has been published (ref. 1).     

Age-dependent accumulation of phosphatidylcholine hydroperoxide in the brain and liver of the rat

Age-related changes in phosphatidylcholine hydroperoxide (PCOOH) content as an index for oxidative membrane lipid damage were determined by high-performance liquid chromatography using chemiluminescence detection. Brain and liver PCOOH content increased significantly in male and female rats with age. The brain PCOOH content of male 18-month-old rats was 4.4 times that of 1-month-old rats, and that of female 18-month-old rats was 3.5 times that of 1-month-old females. The liver PCOOH content of the male 18-month-old rats was 9.3 times that of the 1-month-old; and of the famale 18-month-old rats was 4.7 times that of the 1-month-old. PCOOH levels in heart and lung did not show age dependency. In both brain and liver (but not in heart and lung), the phosphatidylcholine content significantly decreased upon aging. The results indicate that oxidative deterioration, such as phospholipid hydroperoxidation, is prevalent in the membrane lipids of brain and liver of the rat due to aging.     

Animal endogenous triglycerides: III. Swine,rat and chicken liver: Comparison with adipose tissue

The endogenous triglycerides of swine, rat and chicken livers were fractionated by silver ion thin layer chromatography and the resulting fractions were analyzed for their fatty acid composition and distribution. Whereas the endogenous triglycerides of swine adipose tissue differ markedly from those of rat and chicken adipose tissue in the location of the major fatty acids, the liver triglycerides of the three species are quite similar. They also resemble rat and chicken adipose triglycerides.     

Relationship between desaturation and chain elongation of palmityl-CoA in rat liver microsomes

The relationship between desaturation and chain elongation of palmityl-CoA was studied in rat liver microsomes. Enzyme activities for desaturation and chain elongation were stimulated by re-feeding starved rats. The activities of desaturation and chain elongation in re-fed rats were four- and twofold greater than those in normal rats, respectively. When a sonicated dispersion of lecithin was added to the incubation medium containing both reactions, chain elongation activity, especially formation of stearic acid, was stimulated and desaturation activity was apparently depressed.     

Stereospecific analysis of hepatoma,host liver,and normal rat liver triglycerides from animals on chow and fat free diets

Triglycerides from normal liver, host liver, and hepatoma of rats maintained on chow and fat-free diets were subjected to stereospecific analysis. Normal and host liver triglycerides from animals on the same diet did not exhibit significant differences. Fat-free diet reduced polyunsaturated fatty acids in normal and host liver triglycerides, but had no effect upon hepatoma triglycerides. Each position of hepatoma and liver triglyceride glycerol exhibited a characteristic fatty acid composition. Palmitate concentrations were reduced dramatically and stearate levels were increased significantly at the 1 position of hepatoma triglycerides, relative to the corresponding position of liver triglycerides which were affected little by diet or tumor. Except for higher percentages of C-20 and higher fatty acids, common to all three positions, the composition of hepatoma triglycerides at the 2 position appeared normal. The 3 position of hepatoma triglycerides contained significantly higher percentages of stearate than liver. Data obtained previously for Ehrlich ascites cell triglycerides were in good agreement with this hepatoma. Data from these two neoplasms suggest that the metabolic system that regulates or controls the fatty acid composition at the 1 and 3 positions of normal tissue triglycerides does not function normally in neoplasms.     

Interrelationship between the dietary regulation of fatty acid synthesis and the fatty acyl-CoA desaturases

In this paper we present further evidence for the close control of fatty acid synthetase and stearoyl-CoA desaturase. Furthermore, we have established that whereas dietary palmitic acid may influence the activity of this desaturase but not of fatty acid synthetase, dietary linoleic acid appears to control both these enzymes. Finally, we have studied the influence of dietary fat and carbohydrate on the activities of the delta6 and delta5 desaturases. The former is only slightly affected by these dietary components. The delta5 desaturase activity is stimulated as the dietary fat content rises but is unaffected by dietary carbohydrate. The control of these enzymes is therefore independent of the control of fatty acid synthetase and stearoyl-CoA desaturase. From the data presented, the magnitude of the controlling effect of polyunsaturated fatty acids on fatty acid synthetase and stearoyl-CoA desaturase activity is determined and its relevance to lipogenesis in man based on daily intake of carbohydrate and linoleic acid is discussed.     

Effects of high fat diets on the activity of palmitoyl-CoA hydrolase in rat liver

Palmitoyl-CoA hydrolase [EC 3.1.2.2.] activity in rat liver was found to be enhanced by high fat diets. Partially hydrogenated marine oil and high-erucic acid rapeseed oil diets produced a greater increase than a diet containing soybean oil. With diets containing from 5 to 30% (w/w) of partially hydrogenated marine oil the increase in palmitoyl-CoA hydrolase activity was similar to the increase observed in peroxisomal β-oxidation activity (correlation coefficient r=0.94). A positive correlation (r=0.86) also was observed between the activity of palmitoyl-CoA hydrolase and previously determined levels of long-chain acyl-CoA. The results presented may suggest a common “induction” mechanism for palmitoyl-CoA hydrolase and peroxisomal β-oxidation enzymes, possibly exerted through an increased cellular level of long-chain acyl-CoA.     

Effects of perturbations in hepatic free and esterified cholesterol pools on bile acid synthesis,cholesterol 7α-hydroxylase,HMG-CoA reductase,acyl-CoA:Cholesterol acyltransferase and cytosolic cholesteryl ester hydrolase

Effects of expansion of the hepatic free cholesterol pool on bile acid and cholesterol metabolism and homeostasis were examined in rats fed cholesterol in high-fat diets or treated with oleyl-p-(n-decyl)-benzenesulfonate (ODS) or progesterone. Cholesterol feeding for 10–16 days, which increased free (33%) and esterified (6-fold) cholesterol, had no effect on cholate synthesis, total bile acid synthesis, or cholate turnover, whereas these activities were increased 60–80% by ODS and progesterone, which produced only small increases (19%) in free cholesterol. Cholesterol feeding reduced β-hydroxy-β-methylglutaryl (HMG)-CoA reductase (72%) and cholesteryl ester hydrolase (48%) and increased acyl-CoA:cholesterol acyltransferase (184%), whereas ODS and progesterone reversed these compensatory responses in cholesterol-fed rats. Cholesterol 7α-hydroxylase was changed no more than 22% by any treatment. A bolus of ODS elevated biliary cholesterol output 41% and shifted biliary bile acid synthesis and composition toward 12-deoxy bile acids. These effects were not seen in ODS-fed or progesterone-treated rats, in which cholesteryl ester stores were depleted. It is concluded that effects of free cholesterol on bile acid synthesis and biliary cholesterol are probably mediated by specific precursor or regulatory pools which can be independently regulated and which represent a relatively small fraction of hepatic free cholesterol.     

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase,triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 ×g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the three activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.     

Effect of hyperpnea on enzymes of the CDPcholine path for phosphatidylcholine synthesis in rat lung

We have examined the activity of three enzymes in pulmonary surfactant phosphatidylcholine synthesis following the hyperpnea induced by having rats either inspire 5% CO₂/13%O₂/82% N₂ for 24 hr or swim in thermoneutral water for 30 min. Both stimuli markedly increase frequency and tidal volume of breathing and promote the release of surfactant. Lungs were perfused to remove blood, lavaged, and then homogenized in 1 mM Hepes, 0.15M KCl at pH 7.0. The homogenate was centrifuged at 9,000 g (av) for 10 min to sediment the mitochondria and lamellar bodies and at 100,000 g (av) for 60 min to obtain the microsomal and cytosol fractions. Incubations were carried out under determined optimal conditions and zero order kinetics. Choline kinase (CK), cholinephosphate cytidylytransferase (CP-cyT) and choline phosphotransferase (CPT) were assayed by the incorporation of [methyl-¹⁴C] choline chloride into phosphocholine, [methyl-¹⁴C]phosphocholine into CDPcholine, and [¹⁴C]CDPcholine into phosphatidylcholine, respectively. The incubation products were separated by thin-layer chromatography. Whereas both forms of hyperpnea increased the activity of CP-cyT in the microsomal fraction, they had no effect on the activity of either cytosolic CP-cyT and CK, or microsomal CPT. A similar increase in tidal volume in an isolated perfused rat lung had no effect. We conclude that,in vivo, hyperpnea increases the activity of CP-cyT, the rate-limiting enzyme in phosphatidylcholine synthesis. Whether this is due to an increase in the amount of enzyme, or of a cofactor, is unknown.     

Application of thin-layer chromatography to the quantitative estimation of tissue triglycerides II. Influence of methyl parathion on the composition of liver triglycerides in the rat

Methyl parathion fed at 10 ppm in a high protein low fat diet inhibited 46.9% of the total liver carboxylesterase activity. The total fatty acid composition of liver triglycerides was not significantly altered. However, the methyl parathion-fed rats showed a higher percentage of saturated acids in the 2-position of the glyceride molecule. Triglyceride analysis employing the multiple TLC-GLC technique (5) also showed a slightly higher percentage of saturated glycerides and those containing 1 and 2 double bonds than those in the control group. Triglyceride patterns in both groups were in general agreement with those calculated from the fatty acid distribution as suggested by Vander Wal (11). Presented at the AOCS meeting in Houston, Texas, April, 1965.     

The incorporation of14C-glycerol into different species of diglycerides and triglycerides in rat liver slices

The relative rates of de novo synthesis of species of diglycerides and triglycerides from¹⁴C-glycerol were examined in rat liver slices. Diglycerides containing one or two double bonds per molecule and triglycerides containing four or more double bonds per molecule represented 70% and 60% respectively of the newly synthesized diglycerides and triglycerides. The newly synthesized triglycerides were more unsaturated than the endogenous triglycerides. Our results suggest that a nonrandom synthesis of species of diglycerides occurred followed by an almost random utilization of the various diglyceride species for the biosynthesis of triglycerides.     

Relationship between dietary supply of long-chain fatty acids and membrane composition of long- and very long chain essential fatty acids in developing rat photoreceptors

The present study was designed to determine if dietary supply of long-chain fatty acid (LCFA, C20∶4n-6, and/or C22∶6n-3), reflecting levels that might be incorporated into infant formulas, influences the fatty acid composition of the visual cell membrane. The rod outer segment (ROS) of the retina was analyzed from rats fed diets varying in the ratio of 18∶2n-6 to 18∶3n-3 with or without 20∶4n-6 [arachidonic acid (AA)] and 22∶6n-3 (docosahexaenoic acid) from birth to six weeks of age. The level of very long chain fatty acids (VLCFA, C₂₄−C₃₆) was identified using gas chromatography and gas chromatography-mass spectrometry. In the ROS, the highest relative percent of AA was attained in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of animals fed 1% AA diet, whereas feeding 0.7% docosahexaenoic acid (DHA) diet significantly increased the DHA level in PC, phosphatidylserine, and phosphatidylinositol compared to feeding diets containing AA. VLCFA of n-6 and n-3 up to C₃₆ were found in PC, with the most abundant fatty acids being C₃₂ and C₃₄. In PC, phosphatidylserine and PE, the n-6 tetraenoic VLCFA level was highly increased in animals fed 1% AA compared to other dietary groups. This study suggests that dietary fat containing small amounts of AA or DHA is an important factor influencing membrane fatty acid composition of the visual cell during development. Based on a presentation at the AOCS Annual Meeting & Expo in San Antonio, Texas, May 7–11, 1995.     

Effects of dietary cholesterol and triglycerides on lipid concentrations in liver, plasma, and bile

Dietary cholesterol (CHL) and triglycerides (TG) can influence plasma, hepatic, and biliary lipid composition, but effects on lipids in these three compartments during the early stages of CHL gallstone formation have not been studied in parallel. We fed prairie dogs diets containing one of four tes oils (safflower, coconut, olive, or menhaden) at either 5 or 40% of calories, in the presence of 0 or 0.34% CHL, for 3 wk. In the absence of dietary CHL, increases in dietary TG produced 50–200% increases in the concentrations of biliary CHL and hepatic cholesteryl ester (CE), while the concentrations of hepatic free CHL (FC) as well as plasma FC and CE remained relatively unchanged. Increasing dietary CHL to 0.34% resulted in increases in hepatic FC of approximately 50% for all four fats regardless of whether they were supplied at 5 or 40% of calories. CHL supplementation caused more pronounced increases in biliary CHL (200–400%), hepatic CE (50–200%), plasma FC (up to 100%), and plasma CE (up to 150%), and these increases were exacerbated by concurrent supplementation of dietary fat and CHL (biliary CHL: 300–700%; hepatic CE: 100–250%; plasma FC: up to 165%; plasma CE: 100–350%). These results indicate that enhanced secretion of biliary CHL and, to a lesser extent, increased synthesis of hepatic CE, may be primary mechanisms for maintaining the hepatic FC pool. Furthermore, dietary CHL and high levels of fat intake are independent risk factors for increasing biliary CHL concentrations, and adverse effects on lipid concentrations in plasma and bile tend to be exacerbated by ingestion of diets rich in both fat and CHL.     

Relationships between cholesterogenesis,microsomal sterols and HMG-CoA reductase in the perfused rat liver

The relationships between cholesterogenesis and the activity of HMG-CoA reductase of microsomes prepared with or without sodium fluoride, and between changes of cholesterogenesis and microsomal sterols were studied in the isolated rat liver perfused with or without oleic acid in the presence of AY-9944. AY-9944 inhibits the conversion of 7-dehydrocholesterol, measured colorimetrically as “fast-acting” sterols, to cholesterol, measured colorimetrically as “slow-acting” sterols. The level of “fast-acting” sterols is used to estimate cholesterogenesis and changes in microsomal sterols. It was observed that the activity of HMG-CoA reductase of microsomes prepared with or without fluoride reflects the relative changes in cholesterogenesis of the perfused livers. In addition, the amount of “fast-acting” and “slow-acting” sterols in microsomes correlates with increases in the activity of HMG-CoA reductase and cholesterogenesis.