Yasuo Wakabayashi

Anti-obesity and anti-diabetic effects of CL 316, 243, a highly specific β3-adrenoceptor agonist, in yellow KK mice

The anti-obesity and anti-diabetic effects of CL 316,243, a highly specific beta 3-adrenoceptor agonist (beta 1: beta 2: beta 3 = 0:1:100,000), were evaluated in obese diabetic yellow KK mice and C57Bl control mice. The study compound was fed through a gastric tube at a rate of 0.1 mg/kg/day for 2 weeks. The following parameters were compared in the treated and control animals given distilled water: brown adipose tissue thermogenesis, resting metabolic rate, insulin receptors in adipocytes, and blood glucose and serum insulin levels during a glucose overloading test. CL 316,243 significantly increased brown adipose tissue thermogenesis and resting metabolic rate in both yellow KK mice and C57Bl controls. The amount of white adipose tissue decreased, although food intake was not affected. The effects contributed to the mitigation of obesity in yellow KK mice. CL 316,243 also increased the concentration of insulin receptors and decreased the levels of serum insulin and blood glucose during the glucose overloading test in yellow KK mice. These observations suggest that CL 316,243 possesses anti-obesity and anti-diabetic effects and consequently may be useful for treating obesity as well as non-insulin-dependent diabetes mellitus in obese persons, without causing excessive side effects.

Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate: I. Pyrroline-5-carboxylate synthase

The in vivo synthesis of arginine from glutamate in mammals requires seven enzymes to cooperate. Pyrroline-5-carboxylate synthase (PCS) is the first enzyme required. In order to establish the interorgan dependency of arginine synthesis, we quantitated PCS activity in as many as 32 rat tissues and found that the activity was concentrated only in the upper small intestine. Minor activity was found in pancreas, thymus, lymph node, and some other tissues: this was confirmed by the dependency on specific substrates, the loss of activity in the presence of an inhibitor, and identifying the reduced product as proline. No difference in activity was found between male and female rats on a milligram protein basis. The strict tissue localization of PCS and the localization of other enzymes of arginine synthesis previously reported clearly indicate that the upper small intestine is an indispensable tissue for the arginine synthesis from glutamate. Many of the tissues examined showed an activity to form an unknown product from glutamate. When assayed by the previously reported radiometric assay procedure using an AG1-X8 column (acetate), the product was not separated from PC and caused false-positive activities of PCS. An improved procedure was developed to overcome this technical difficulty. The new procedure enabled us to detect even 20 pmol PC without contamination by the adjoining unknown product. A preliminary characterization of the unknown product was achieved.

Presence of d-aspartate oxidase in rat liver and mouse tissues

Rat liver D-aspartate oxidase activity, which had been reported to be undetectable, was found to be well detectable in dialyzed liver homogenate. The requirements of the enzyme for activity and its sensitivity to inhibitors were identical with the known properties of the enzyme from other sources. We also demonstrated for the first time the presence of the enzyme activity in mouse tissues and some other rat tissues using dialyzed tissue homogenates.

Thiamine transport mutants of Saccharomyces cerevisiae.

Abstract Two mutants, PT-R1 and PT-R2, which are resistant to the inhibitory action of pyrithiamine, were isolated by nitrosoguanidine treatment from Saccharomyces cerevisiae . They were found to be, respectively, partially and almost totally defective in the thiamine-specific transport system. The mechanism of resistance of the mutants to pyrithiamine is discussed.

Administration of D-aspartate increases D-aspartate oxidase activity in mouse liver

Oral administration of D-aspartate to mice for 2 weeks by addition of the amino acid to drinking water produced a nearly 4-fold increase in liver D-aspartate oxidase (EC 1.4.3.1) activity, whereas no increase was induced by L-aspartate administered in the same way. Administration of D-aspartate also produced a small significant increase in the kidney enzyme activity, but L-aspartate administration increased the activity as well. The enzyme activity in the brain and muscle was not affected by administration of either D- or L-aspartate. Intraperitoneal administration of D-aspartate increased the enzyme activity only in the liver, and other compounds tested, including D-glutamate and D-alanine, could not replace D-aspartate. The results indicate a specific relationship between D-aspartate and D-aspartate oxidase and suggest that the amino acid is, in fact, a physiological substrate of the enzyme.

Enzymological evidence for the indispensability of small intestine in the synthesis of arginine from glutamate: II. N-acetylglutamate synthase☆

We describe here a concise assay procedure for N-acetylglutamate (AGA) synthase (AGAS) and its application to an extensive study of tissue distribution of AGAS activity. Crude mitochondria from several tissues were incubated in a pair of assay mixtures with [14C]glutamate in the absence and presence of acetyl-CoA at 15 degrees C for 10 min. Anionic components including [14C]AGA were first isolated from glutamate by a cation exchanger column. In order to remove anionic contaminants such as succinate, the AGA was converted to glutamate enzymatically by aminoacylase, and then the glutamate was isolated by cation exchange chromatography and counted. Recoveries were corrected individually. The difference between the pair incubations was taken as the activity. An extensive survey of AGAS activity in rats showed that, although the liver expressed the highest activity, the small intestine, testis, lung and submaxillary gland also exhibited considerable activity. Sexual differences in activity were not found in the liver and small intestine. We also detected activity in the human small intestine for the first time. Optimization of incubation temperature and time and the presence of arginine in an assay mixture was essential and we demonstrated that the AGAS reaction with crude mitochondria as an enzyme source was unstable without arginine and at higher temperatures. This procedure appears suitable for studying the physiological and nutritional role of AGAS in non-hepatic tissues. In the accompanying paper we applied this procedure to study the ontogeny of AGAS in developing rat tissues.

Anti-obesity and anti-diabetic effects of mazindol in yellow KK mice: its activating effect on brown adipose tissue thermogenesis.

1. The anti‐obesity and anti‐diabetic effects of mazindol were evaluated in obese diabetic yellow KK mice and C57B1 control mice.

Slow- and tight-binding inhibition of aspartate aminotransferase by l-hydrazinosuccinate

The inhibition of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) by L-hydrazinosuccinate has been studied. The velocity of the enzyme reaction decreased with time when the reaction was initiated by the addition of enzyme to a mixture of the assay components and L-hydrazinosuccinate, while it increased slowly from a low level when a preincubated mixture of the enzyme and the inhibitor was added to the reaction mixture to initiate the reaction. Nearly 50% decrease in the initial reaction velocity was produced by a prolonged preincubation of the enzyme with the inhibitor, both at low concentrations of about 2 nM. These findings indicate that the inhibition is of the slow- and tight-binding type. The time-course of the reaction of the enzyme and the inhibitor, examined by the change in activity, was not in accord with single-step mechanisms, but rather appeared to follow biphasic kinetics. The inhibition could be fully reversed only in the presence of L-cysteine sulfinate or large excess of L-aspartate to convert the regenerated enzyme to its pyridoxamine form. The time-course of the reversal followed pseudo-first-order kinetics. Quantitative analysis of the experimental data has shown that the results are consistent with a mechanism of enzyme-inhibitor interaction which involves a reaction of two consecutive, reversible steps. The overall inhibition constant for L-hydrazinosuccinate was calculated to be approx. 0.2 nM.

Dopamine and serotonin uptake inhibitors on the release of dopamine and serotonin in the nucleus accumbens of young and aged rats

Nucleus accumbens (ACC) of young (4 months old) and aged (24 months old) Wistar rats were perfused with dopamine (DA) uptake blocker, cocaine, or the serotonin (5-HT) selective reuptake inhibitor, fluoxetine, through the microdialysis probe membrane, used to assess the dopamine transporter (DAT) or serotonin transporter (SERT) modulation. The basal extracellular DA release in the ACC was significantly lower in aged rats than young rats. Analysis of DA and 5-HT concentrations in the ACC with increased positive GFAP revealed that DA and DOPAC levels of aged rats were decreased to 55 and 60% of those in young rats, respectively. After co-perfusion with cocaine, both DA and 5-HT releases in the ACC were increased in the young and aged groups. However, the magnitude of the increased DA release was lower in aged rats than young rats. Co-perfusion with fluoxetine showed lower magnitude of the increased DA release in aged rats. It appears that the DAT and SERT system responds initially to ACC cell loss with age, and that especially ACC DAT in the aged rat is more degenerative compared with the young rats. These findings suggest that the serotonergic system with SERT in the remaining ACC neurons show an early adaptive response and resistance to the normal aging and maintain the multiple regulatory system in the ACC despite neural loss since the dopaminergic neurons in the aged animals are vulnerable to aging.

Development of pyrroline-5-carboxylate synthase and N-acetylglutamate synthase and their changes in lactation and aging

Using newly developed assay procedures, we studied the development of pyrroline-5-carboxylate synthase (PCS) and N-acetylglutamate synthase (AGAS) activity in rat tissues. PCS in the small intestine of fetuses was 1/5 that of adults and reached an adult level as early as postnatal Day 1. The highest peak was observed at Day 14, and then activity decreased to the adult level. However, PCS in the brain was highest at birth and quickly inactivated in a few days. AGAS in the fetus small intestine was 1/3 that of adults and became higher than the adult level by 40% at Day 1 but was reduced to 1/2 that of adults at Day 3. Subsequently activity increased gradually to the adult level at Day 24. On the contrary, AGAS in the fetus liver was only 1/20 that of adults, and activity increased slowly up to 10 weeks and more. Pregnancy and lactation reduced liver AGAS markedly up to Day 8 and intestinal PCS considerably up to Day 14 after parturition. PCS in the small intestine of senescent rats was almost halved compared to young controls on a whole tissue basis. AGAS in the small intestine was also halved on a gram wet weight basis. Nonetheless the liver AGAS of 430-day-old rats was higher than that of the controls, although that of 630-day rats was lower. The results indicate that the arginine synthesizing enzymes in the small intestine are highly activated in suckling and weaning, and raise a question whether arginine remains fully dispensable in pregnancy, lactation, and senescence.