As a model system for investigating the mechanism of the hepatic NAD-lowering effect of leucine in rats, aerobically grown Saccharomyces carlsbergensis was used in this paper.
Tryptophan supplementation of the medium doubled total niacin pro-duction by S. carlsbergensis. This elevation in total niacin was mainly due to increases in niacin (14 times) and niacinamide nucleotides (2 times). Among nucleotides, the NAD level doubled whereas NADH, NADP and NADPH levels dropped significantly. Simultaneous supplementation of the medium with leucine suppressed the elevation in total and free niacin levels.
In the presence of tryptophan, approximately 50% of the total niacin was secreted in the medium in the form of free niacin, while in the presence of both tryptophan and leucine most of the total niacin remained in the cell. The specific activity of quinolinate phosphoribosyltransferase [EC 2.4.2.19] was not affected by supplementation of the medium with tryptophan and/or leucine. In contrast, the specific activity of nicoti-namide deamidase [EC 3.5.1.19] increased fivefold in the presence of tryptophan. Simultaneous supplementation of the medium with leucine suppressed the increase in nicotinamide deamidase.
Cellular incorporation of tryptophan was not affected by leucine simul-taneously added as a supplement to the medium. Leucine did not have any inhibitory effect on total niacin synthesis from 3-hydroxyanthranilate.
From the results, a possible mechanism for the inhibitory effect of leucine on the tryptophan-NAD pathway was discussed.

View full abstract

L-Kynurenine 3-hydroxylase [EC 1.14.1.2] was partially purified from the mitochondrial outer membrane fraction of Saccharo-myces carlsbergensis by Sephadex G-200 gel chromatography, and the effects of leucine and its related compounds on the enzyme were investi-gated.
α-Keto acid derivatives of the three branched chain amino acids were found inhibitory to the partially purified kynurenine 3-hydroxylase, but branched chain amino acids were without effect. α-Ketoisocaproate (KIC), a keto acid analogue of L-leucine, inhibited kynurenine 3-hy-droxylase noncompetitively with apparent K; values of 4.2 and 8.3 mM for kynurenine and NADPH respectively. a-Ketoglutarate and pyruvate were mixed-type inhibitors of the enzyme.
KIC production by S. carlsbergensis grown in medium containing no leucine was negligible, while that in leucine-supplemented medium in-creased in proportion to the amount of L-leucine incorporated into cells. From the results, it was proposed that KIC produced from leucine lowered synthesis of NAD from tryptophan by inhibiting L-kynurenine 3-hydroxylase, a possible rate-limiting enzyme in the tryptophan-NAD pathway in Saccharomyces carlsbergensis.

View full abstract

In the preceding paper, we found that branched chain α-keto acids, α-ketoglutarate and pyruvate were inhibitory to kynurenine 3-hydroxylase [EC 1.14.1.2] from Saccharomyces (Shin, M, et al. (1982): J. Nutr. Sci. Vitaminol., 28, 191-201). As kynurenine 3-hydroxylase is reported to be a rate-limiting enzyme in the tryptophan-NAD pathway in rats, branched chain amino acids, branched chain a-keto acids and several other keto acids were tested for their effects on kynurenine 3-hydroxylase activity in the mitochondrial outer membrane fraction prepared from rat liver. In contrast with the yeast enzyme, the rat liver enzyme was resistant to α-keto acid-inhibition and more than 70% of the enzyme activity was maintained even in the presence of 20mM of each α-keto acid. The present result implies that the mechanism of the hepatic NAD-lowering effect of leucine in rats might be completely different from that operative in Saccharomyces.

View full abstract

The effect of high-protein pyridoxine-deficient diet on the localization of lysosomes and acid phosphatase activity in the rat liver was studied using light and electron microscopy. Numerous lysosomes con-taining lipid droplets appeared to arise directly from GERI (Golgi apparatus, endoplasmic reticulum and lysosomes) near bile canaliculi and thereafter large crystal clefts were frequently found in these lysosomes. The increased appearance of lysosomes in hepatocytes was compatible with increased lipid droplets and represented an indication of breakdown of stored lipids. Acid phosphatase activity was localized almost exclusively in lysosomes with or without lipid droplets. We postulated that one of the causes of accumulation of lipid in hepatocytes, including that of tri-glyceride and cholesteryl ester, might be associated with a relative deficiency of intralysosomal digestion in these conditions.

View full abstract

Levels of thiamin and its derivatives in brain, heart and liver were determined in control, thiamin-deficient, pyrithiamin-treated and oxythiamin-treated rats at various times after the start of treatment. With 10μg of thiamin/100g/day subcutaneously, rats rapidly lost thiamin from the tissues until growth maintenance levels were reached. Oxythiamin had a minimal further effect on tissue thiamin while pyrithiamin caused further marked reduction. With higher levels of thiamin intake, tissue thiamin levels increased with intakes of up to 150-200μg/ 100g/day after which they did not show any further significant increase. Levels of pyrithiamin above 50μg/ 100g/day for 20 days had only minimal further effect upon tissue thiamin levels over that with 50μg.

View full abstract

In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol-inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehy-dratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5'-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn²⁺, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn²⁺, such displacement did not take place. In contrast, enzyme-bound adeninyl-butylcobalamin and adenosylethylcobalamin were essentially not displace-able by the free coenzyme even in the presence of ATP and Mn²⁺. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn²⁺. These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Coβ ligands was markedly lowered in the presence of ATP and Mn²⁺, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol-inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg²⁺, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated vation (10, 21). This inactivation is believed to result from the irreversible cleavage of the activated carbon-cobalt bond of the coenzyme by reaction with oxygen. Figure 4 shows that the oxygen-inactivated glycerol dehydratase was also re-activated in situ in the presence of ATP, Mn²⁺ and adenosylcobalamin, although the rate of reactivation was rather slow. This slow rate may be attributed at least in part to damage to the apoprotein itself, to extraordinarily tight binding of the compound derived from the coenzyme, or to denaturation of the reactivating system during the oxygen-inactivation procedure.
It is known that diol dehydratase is gradually inactivated during dehydration of l, 2-ethanediol (1, 3). Diol dehydratase also undergoes gradual inactivation during catalysis when arabino-adenosylcobalamin is used as a coenzyme instead of adenosylcobalamin (17). The inactivated enzymes thus obtained were also re-activated in situ by ATP, Mn2+ and adenosylcobalamin, although the data are not shown here.

View full abstract

A study on the relation of plasma- and RBC-tocopherol to plasma lipids and adiposity led to the following findings. Plasma tocopherol increased in parallel with increased plasma total lipid and beta-lipoprotein levels, while RBC tocopherol levels had little cor-relation with those of plasma lipids or β-lipoproteins. The RBC-to-plasma tocopherol ratio decreased with increased plasma lipids and -lipo-proteins. Though the relations of HDL-cholesterol, total lipids and f3-lipoproteins to RBC tocopherol levels resembled each other, those of HDL-cholesterol to plasma tocopherol levels and RBC-to-plasma to-copherol ratios differed greatly from the relationship in total lipids and β-lipoproteins, especially the RBC-to-plasma ratios which had no definite pattern. Plasma tocopherol was unrelated to obesity grade, while the RBC tocopherol and RBC-to-plasma tocopherol ratios decreased with in-creased obesity.

View full abstract

To study the mechanism which cobalamin is taken up by the mitochondria, ⁵⁷Co-cyanocobalamin-binder complex and freshly pre-pared mitochondria were prepared from the enterocytes of rats. Subsequently, the binder complex was incubated together with mitochon-dria in a calcium-containing medium in vitro. Uptake of cobalamin was determined by measuring the radioactivities bound to the mitochondria. Consequently, lysosomal and microsomal binders enhanced cobalamin uptake into the mitochondria, but intrinsic factor did not.
It was found that the uptake into the mitochondria was inhibited by previous treatment with calcium-chelating agents. The uptake was com-pletely restored after addition of calcium ions to the mitochondria.

View full abstract

A glycoprotein isolated from Kintoki beans (Phaseolus vulgaris cultivar Kintoki) agglutinated human erythrocytes of all types and erythrocytes of rat, rabbit, sheep, and mouse. The lectin activity was not affected by 1 hr heating at 60°C, but decreased slightly on heating for the same period at 70-80°C and markedly at 90-100°C. The activity was inhibited by galactose, lactose, N-acetyl galactosamine and fetuin. The inhibition was, however, weak, as often found for nonspecific lectins. the activity did not change when tyrosine residues or small parts of amino groups were modified, but decreased considerably when histidine residues or carboxyl groups were modified. This lectin was found to be relatively resistant to trypsin, and, particularly, to pepsin. All mice died within 48hr when 200μg lectin per gram body weight was injected intraperitoneally and 14μg intravenously. The toxic activity changed in parallel with the lectin activity upon various treatments of the glycoprotein. In addition, blood analyses of injected mice suggested that the toxicity might be developed by the action of the lectin on blood cells.

View full abstract

The effect of soybean phospholipid (SP) on the chemical composition of lymph chylomicrons and excretion of steroids into bile and feces was examined. Rats were meal-fed (9 am-10 am) or ad libitum-fed on diets containing phospholipid and neutral lipid (soybean oil or corn oil) for 2 weeks in order to assess the amount of chylomicrons secreted from the intestine during the absorption or post-absorption of dietary lipids. In meal-fed rats, dietary SP decreased the concentration of surface com-ponents (phospholipid, cholesterol and apoprotein A-I) of large chylomi-crons and increased the core components (triglyceride) of small chylomi-crons. In ad libitum-fed rats given SP, the concentration of triglyceride in small chylomicrons appeared to increase. Dietary SP increased the relative proportion of linoleic acid and decreased arachidonic acid at the 2-position of chylomicron-phospholipids. The calculated diameter of large and small chylomicron particles in meal-fed rats given SP increased and the particle number per ml lymph decreased compared to those values in rats given neutral lipid. The particle size of the small chylomicron in ad libitum-fed rats given SP also increased. Apoprotein A-I in lymph d>1.006 g/ml decreased in the SP-fed rats.
The permeability of glucose-6-phosphate to microsomal membrane increased in the SP-fed rats. The excretion of neutral, but not acidic steroids into the bile and feces was increased in SP-fed rats. The results suggest that dietary SP inhibits the supply of the surface components of chylomicrons to the chylomicron precursors by interfering with absorption of the lipids in the upper part of the intestine and altering the subsequent metabolism of these lipids and apoproteins in the intestinal cells.

View full abstract

The effect of soybean phospholipid (SP), given at the rate of 100g/kg diet for 2-3 weeks, on the chemical composition of serum lipoprotein in rats was examined. Soybean oil (SO) containing almost comparable amounts of linoleic acid was used as a control diet. SP significantly decreased the concentration of serum cholesterol and apo-lipoprotein A-I (apo A-I) and increased apolipoprotein B (apo B). There was no significant change in the concentration of serum apolipoprotein E (apo E) and the other lipids. The reduction of cholesterol levels in all the lipoprotein fractions was observed. The reduction of the serum apo A-I was brought about by the addition of SP at the level of 1 % while that of the cholesterol at the level of 4%. The subunit composition of apo A-I, but not apolipoprotein C, in high density lipoprotein (HDL) was also altered by SP. The high molecular weight of apo B was responsible for the increment of apoproteins in the low density lipoprotein (LDL). The triglyceride, but not the cholesterol ester, also increased in LDL. There was no significan4 difference between the groups in the fatty acid compositions of serum triglyceride and phospholipid. In the liver, SP significantly increased the concentration of cholesterol ester and decreased triglyceride. These results are discussed in the context of the role of the lipoprotein and apoprotein in the regulation of serum cholesterol concentration.

View full abstract

The present study was undertaken to determine whether the timing of sucrose meal feeding relative to periods of physical activity affects plasma triacylglycerol (TG) levels in rats. Animals were daily meal-fed on a basal diet and a 35% sucrose diet for 10 weeks. Meal times were at 08.00-09.00 hr and 21.00-22.00 hr. Voluntary running in wheels was allowed between 22.00-08.00 hr, but was restricted from 08.00 to 22.00 hr. The sucrose diet was given at the morning meal time to one group (M-S eaters) and at the evening meal time to another group (E-S eaters). The timing of the sucrose meal did not have any influence on consumption of either of the two diets, physical activity, weight gain, or the weight of several organs and tissues. Plasma TG, however, was significantly higher in the M-S eaters than in the E-S eaters. Lipoprotein lipase activity of several tissues was not affected by the timing of the sucrose meal. The Triton-induced increase in fasting plasma TG was significantly higher after the sucrose meal than after the basal meal regardless of the timing of the sucrose diet. The TG accumulation rate during the physically inactive period was significantly greater in the M-S eaters than in the E-S eaters, while during the physically active period it was equal in both groups. These results suggest that the effect of sucrose feeding on plasma TG may be conditioned by the timing of sucrose feeding and rats' physical activity.

View full abstract