Masakazu Murata

Reciprocal responses to dietary diacylglycerol of hepatic enzymes of fatty acid synthesis and oxidation in the rat

The activities of hepatic enzymes of fatty acid synthesis and oxidation were compared in rats fed on diacylglycerol and triacylglycerol. In the first trial, rats were fed on diacylglycerol or triacylglycerol (rapeseed oil) for 14 d. The diacylglycerol preparation contained 65.2 g and 32.6 g fatty acids/100 g total fatty acids as 1,3-species and 1,2-species respectively. Fatty acid compositions of these dietary lipids were similar. Dietary acylglycerols were added to experimental diets to provide the same amounts of fatty acids (93.9 g/kg diet). Dietary diacylglycerol compared with triacylglycerol significantly reduced the concentrations of serum and liver triacylglycerol. The activities of enzymes of fatty acid synthesis (fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and malic enzyme (EC 1.1.1.40)) were significantly lower in rats fed on diacylglycerol than in those fed on triacylglycerol. In contrast, the rates of mitochondrial and peroxisomal oxidation of palmitoyl-CoA in liver homogenates were higher in rats fed on diacylglycerol than in those fed on triacylglycerol. In the second trial, varying amounts of dietary triacylglycerol were replaced by diacylglycerol while the dietary fatty acid content was maintained (93.9 g/kg diet). After 21 d of the feeding period the significant reductions in serum and liver triacylglycerol levels were confirmed in groups of rats fed on the diets in which diacylglycerol supplied more than 65.8 g fatty acids/kg diet (65.8 and 93.9 g/kg). Reductions in the activities of enzymes of fatty acid synthesis and increases in palmitoyl-CoA oxidation rates by both mitochondrial and peroxisomal pathways were also apparent when diacylglycerol replaced triacylglycerol in diets to supply more than 65.8 g fatty acid/kg. Increasing dietary levels of diacylglycerol also progressively increased the activities of enzymes involved in the beta-oxidation pathway (carnitine palmitoyltransferase (EC 2.3.1.21), acyl-CoA dehydrogenase (EC 1.3.99.3), acyl-CoA oxidase (EC 1.3.3.6), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), 2,4-dienoyl-CoA reductase (EC 1.3.1.34) and delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8)) in the liver. These results suggest that alteration of fatty acid metabolism in the liver is a factor responsible for the serum triacylglycerol-lowering effect of dietary diacylglycerol.

Effect of tetracosahexaenoic acid on the content and release of histamine, and eicosanoid production in MC/9 mouse mast cell

Abstract6,9,12,15,18,21-Tetracosahexaenoic acid (24∶6n−3) was isolated from a brittle star, Ophiura sarsi Lütken, at>95% purity to evaluate its physiological functions. The effects of 24∶6n−3 on the production of leukotriene (LT)-related compounds such as LTB4, LTC4 and 5-hydroxyeicosatetraenoic acid, and the accumulation and release of histamine in an MC/9 mouse mast cell line were studied. We found that 24∶6n−3 could inhibit the antigen-stimulated production of LT-related compounds as well as other n−3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (20∶5n−3) and docosahexaenoic acid (22∶6n−3), which are major n−3 PUFA in fish oils; 24∶6n−3 was also shown to reduce the histamine content in MC/9 cells at 25 μM (27% reduction from the control), and the effect was diminished with increase of the fatty acid concentration (up to 100 μM). These two n−3 PUFA, 20∶5n−3 and 22∶6n−3, also reduced the histamine content (16 and 20% reduction at 25 μM, respectively), whereas arachidonic acid (20∶4n−6) increased it (18% increase at 25 μM). Spontaneous- and antigen-induced release of histamine was not influenced with these PUFA (at 25 μM). Ionophore-stimulated release of histamine was suppressed by the PUFA (13,9,15, and 11% reduction with 20∶4n−6, 20∶5n−3, 22∶6n−3, and 24∶6n−3, respectively). The patterns of the effects of 24∶6n−3 on the synthesis of eicosanoids and histamine content were more similar to those of 22∶6n−3 than 20∶5n−3. From these results, 24∶6n−3 can be expected to have anti-inflammatory activity and antiallergic activities similar to those of 22∶6n−3.

Octadecatrienoic acids as the substrates for the key enzymes in glycerolipid biosynthesis and fatty acid oxidation in rat liver

The activities of key enzymes in glycerolipid biosynthesis and fatty acid oxidation were compared using CoA esters of naturally occurring positional isomers of octadecatrienoic acids (18∶3) as the substrates. The trienoic acids employed were 9,12,15–18∶3 (α-18∶3), 6,9,12–18∶3 (γ-18∶3), and 5,9,12–18∶3 (pinolenic acid which is a fatty acid contained in pine seed oil, po-18∶3). The activities of microsomal glycerol 3-phosphate acyltransferase obtained with various 18∶3 were only slightly lower than or comparable with those obtained with palmitic (16∶0), oleic (18∶1), and linoleic (18∶2) acids. Mitochondrial glycerol 3-phosphate acyltransferase was exclusively specific for saturated fatty acyl-CoA. The activities of microsomal diacylglycerol acyltransferase measured with various polyunsaturated fatty acyl-CoAs were significantly lower than those obtained with 16∶0- and 18∶1-CoAs. Among the polyunsaturated fatty acids, γ-18∶3 gave the distinctly low activity. The Vmax values of the mitochondrial carnitine palmitoyltransferase I were significantly higher with α-18∶3 and po-18∶3 but not γ-18∶3, than with 16∶0 and 18∶2, while the apparent Km values were the same irrespective of the types of acyl-CoA used except for the distinctly low value obtained with γ-18∶3. The response to an inhibitor of the acyltransferase reaction, malonyl-CoA, was appreciably exaggerated with 18∶2, α-18∶3, and po-18∶3 more than with 16∶0 and 18∶1. However, the response with γ-18∶3 was the same as with 16∶0. Thus, some of glycerolipid biosynthesis and fatty acid oxidation enzymes could discriminate not only the differences in the degree of unsaturation of fatty acids but also the positional distribution of double bond among the naturally occurring 18∶3 acids.

THE HIGH CONTENT OF MONOENE FATTY ACIDS IN THE LIPIDS OF SOME MIDWATER FISHES : FAMILY MYCTOPHIDAE

The total lipids of eleven species of Myctophids caught at depths between 20 and 700 m in the northern Pacific Ocean were analyzed using silicic acid column chromatography (lipid classes) and capillary gas chromatography (fatty acid and fatty alcohol composition). The major components in the lipid classes were triacylglycerols or wax esters; triacylglycerols were the dominant acyl neutral lipids (68.1–96.1%) in eight species, and wax esters were found as the dominant lipid (85.5–87.9%) in three species. The major fatty acids and alcohols contained in the was esters of the three fishes were 18:1n–9, 20:1n–9, 20:1n–11, and 22:1n–11 for fatty acids, and 16:0, 18:1, 20:1, and 22:1 for fatty alcohols. Fatty acids in the triacylglycerols ranging from C14 to C22 were predominantly of even chain length. The major components were 16:0, 16:1n–7, 18:1n–9, 20:1n–11, 22:1n–11, 20:5n–3 (icosapentaenoic acid), and 22:6n–3 (docosahexaenoic acid). In both the triacylglycerols and the wax esters, the major fatty components were monoenoic acids and alcohols. It is suggested from the lipid chemistry of the Myctophids that they may prey on the same organisms as the certain pelagic fishes such as saury and herring, because the large quantities of monoenoic fatty acids are similar to those of saury, herring, and sprats whose lipids originate from their prey organisms such as zooplanktons which are rich in monoenoic wax esters.

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, delta 4-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane-isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15-3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09-1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic-colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.

Dietary modifications of the biliary bile acid glycine: taurine ratio and activity of hepatic bile acid-CoA:amino acid N -acyltransferase ( EC 2.3.1) in the rat

Effects of dietary manipulations on the biliary bile acid glycine:taurine (G:T) ratio and the activity of hepatic bile acid-CoA:amino acid N-acyltransferase (EC 2.3.1) in the post-mitochondrial fraction of liver homogenates were examined in the rat. The G:T ratio in rats fed on the diet containing 100 g pectin/kg (2.18) was markedly higher than that in the animals fed on the diet containing 100 g cellulose/kg (0.09). The diets containing either 10 g cholesterol/kg or 5 g sodium cholate/kg, especially the latter, also increased the G:T ratio (0.77 and 2.33 respectively) compared with a control diet free of these steroids (0.34). When the saturating concentrations of taurine (20 mM) and glycine (100 mM) were the substrates, dietary pectin relative to cellulose significantly increased the activity of both taurine- and glycine-dependent bile acid-CoA:amino acid N-acyltransferase, but neither dietary bile acid nor cholesterol influenced it. In spite of the marked difference in the G:T ratio among the rats given various types of experimental diet, the bile acid-CoA:amino acid N-acyltransferase reaction produced taurine-but little glycone-conjugated bile acid when both taurine and glycine coexisted at physiological concentration ranges in the assay media. Dietary manipulations modified the hepatic taurine concentrations and the changes were inversely correlated with those in the G:T ratio. However, hepatic concentration of taurine (1.67-4.82 mumol/g) in rats given various types of experimental diet was comparable with or even higher than the reported Michaelis constant (Km) value of N-acyltransferase for this compound (0.8-2.5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Depressions by Dietary Phospholipids of Soybean and Egg Yolk Origins of Hepatic Triacylglycerol and Fatty Acid Synthesis in Fasted-Refed Rats

Effects of various phospholipid preparations from soybean and egg yolk origins on various indices for lipid biosynthesis in the liver were compared in fasted-refed rats. Phospholipid preparations employed were soybean phospholipid mainly consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidic acid (soybean PL), egg phospholipid mainly consisting of phosphatidylcholine and phosphatidylethanolamine (egg PL) and purified phosphatidylcholines from soybean (soybean PC) and egg yolk (egg PC) origins. The diet containing 3% fatty acids as soybean and egg PLs compared to the those containing the same amount of fatty acids as the fat blend simulating fatty acid composition of soybean PL and no fatty acids profoundly decreased hepatic concentration of triacylglycerol and liver microsomal concentration of diacylglycerol. Soybean and egg PCs compared to the fat blend also decreased the concentrations of these lipid components in the liver but with the much less extents. Soybean and egg PLs compared to the fat blend also profoundly decreased the activities of enzymes in fatty acid synthesis and of microsomal glycerol 3-phosphate acyltransferase in the liver. These phospholipid preparations compared to the fat blend also significantly decreased the activity of microsomal diacylglycerol acyltransferase when it was measured with endogenous microsomal diacylglycerol substrate. The purified PCs from soybean and egg yolk origins compared to the fat blend also significantly decreased some of these parameters in lipid biosynthesis in the liver but with the much less extents.(ABSTRACT TRUNCATED AT 250 WORDS)

Soybean Protein-Dependent Changes in Triacylglycerol Synthesis and Concentration of Diacylglycerol in the Liver Microsomes of Fasted-Refed Rats

Effects of soybean protein, casein and whole egg protein on various indices for lipid biosynthesis in the liver were compared in fasted-refed rats. Soybean protein compared to casein and whole egg protein significantly reduced activities of glucose 6-phosphate dehydrogenase and fatty acid synthetase. The protein source also slightly reduced the activity of the malic enzyme. Soybean protein compared to other proteins not only reduced the microsomal triacylglycerol but also phosphatidylcholine syntheses when the activities were measured with endogenous diacylglycerol substrate. The protein-dependent changes disappeared, if artificial dispersion of dioleoylglycerol was employed as a substrate. The concentrations of microsomal diacylglycerol and triacylglycerol in whole liver in rats fed soybean protein were lower than those fed other proteins. When the diets containing soybean protein and casein were supplemented with DL-methionine (0.5 and 0.3%, respectively) to meet the nutritional requirement of the animals, soybean protein-dependent reductions in these indices for lipid biosynthesis were still detectable but considerably attenuated. Thus, it is plausible that a soybean protein-dependent decrease in fatty acid synthesis reduced the availability of microsomal diacylglycerol substrate for triacylglycerol synthesis and in turn modified hepatic triacylglycerol concentration. The dietary availability of sulfur amino acids may, at least in part, be responsible for the consequence observed in the present study.

Microsomal Triacylglycerol Synthesis and Diacylglycerol Concentration in the Liver of Rats Fed with Soybean and Egg Yolk Phospholipids

Diets containing 10% soybean oil, soybean phospholipid and egg yolk phospholipid decreased rat liver microsomal diacylglycerol acyltransferase activity when measured with an endogenous diacylglycerol substrate relative to that in rats fed with a low-fat diet (0.5% soybean oil). Although no significant difference was detected in the enzyme activity among the rats fed with the diets containing 10% lipids, the extent of the decrease was more prominent with two kinds of phospholipids compared to soybean oil. When the enzyme activity was measured with an exogenous dioleoylglycerol substrate dispersed in ethanol, it was also significantly depressed by the diets containing 10% phospholipids and soybean oil, but to a much lesser extent. Dietary phospholipids reduced the microsomal concentration of diacylglycerol to a value less than one-third that in the animals fed with a low-fat diet. Soybean oil also decreased this parameter, but to a lesser extent. In addition, soybean and egg yolk phospholipids compared to soybean oil were more effective in reducing the triacylglycerol concentration in the liver. It seemed that dietary phospholipid compared to soybean oil exerted a more potent triacylglycerol lowering effect by modifying the concentration of microsomal diacylglycerol available for triacylglycerol synthesis in the liver.

The acyl-acceptor specificity of microsomal diacylglycerol acyltransferase as a possible determinant in regulating hepatic triacylglycerol synthesis in rats fed a polyunsaturated fat diet

Abstract Rat liver microsomal diacylglycerol acyltransferase activity was determined using molecular species of 1,2-diacylglycerol substrates differing in the degree of unsaturation. When C 16:0 -CoA was used as an acyl-donor, molecular species of diacylglycerol substrates dispersed in ethanol modified the V max without influencing K m values of the enzyme reaction. The enzyme activities measured with diC 18:2 and diC 18:3 glycerols were lower than that obtained with diC 18:1 glycerol. The latter value was the same as that obtained with 1-C 16:0 , 2-C 18:1 glycerol. On the other hand, 1-C 16:0 , 2-CP 18:2 glycerol gave the lowest activity among the various diacylglycerol substrates examined. The similar results were obtained when C 18:2 -CoA was served as an acyl-donor. To test the possibility that observed differences in dependencies on molecular species of diacylglycerols of diacylglycerol acyltransferase in vitro may be a factor in regulating hepatic triacylglycerol synthesis in vivo, rats were fed diets containing 20% fats with different degrees of unsaturation for 3 days after 2 days of fasting. Safflower oil compared with palm and olive oils increased the proportions of polyunsaturated fatty acids in liver microsomal diacylglycerol. Safflower oil compared with the other oils significantly decreased the microsomal diacylglycerol acyltransferase activity measured with endogenous diacylglycerols as the acyl-acceptors and C 16:0 -CoA as an acyl-donor irrespective of the fact that microsomal concentration of diacylglycerol was the same among the rats fed these 20% fat diets. Thus, it is plausible that the acyl-acceptor specificity of microsomal diacylglycerol acyltransferase is a determinant in regulating triacylglycerol synthesis in rat liver.