ENHANCEMENT OF BRAIN THIAMINE DIPHOSPHATASE ACTIVITY OF RATS BY INJECTION OF CHOLINERGIC DRUGS

Abstract— The effects of cholinergic drugs on thiamine diphosphatase (TDPase) in rat brain, liver and kidney were studied to clarify the role of the enzyme in the central nervous system.
Brain TDPase activity was markedly increased by intraperitoneal injection of a sub-lethal dose of physostigmine, ambenonium or pentetrazol. These drugs also increased the activity in the kidney, but not liver. Strychnine, atropine, and scopolamine did not affect the activity of brain TDPase, but decreased the enzyme activity of liver and kidney. Physostigmine also increased the activity of brain thiamine monophosphatase.
Brain TDPase activity reacheda maximum 30 minafterphysostigmine injection (1.0mg/kg). However, inhibition of brain acetylcholinesterase activity was greatest 45 min after physostigmine injection. The TDPase and AChE activities had both returned to normal values 60 min after the injection. The durations of these changes of TDPase and AChE activity corresponded to the duration of the tremor induced by physostigmine. The contents of total and phosphorylated thiamines in the brain but not in the liver or kidney were significantly reduced by physostigmine.
The relationship between ACh and activation of TDPase activity by cholinesterase inhibitors is discussed.     

Thyroid function and vitamin A deficiency.

Rats, when vitamin A deficient, had increased plasma T₃, T₄ and free thyroxine indexes. Pituitary TSH and hypothalamic TRH content were increased in vitamin A deficient animals compared to pair-fed controls. The plasma TSH response to TRH was normal in the vitamin A deficient rats. Basal prolactin, LH and FSH levels did not differ significantly in the two groups. Both groups had significant increases in LH and FSH after LRH. Vitamin A deficiency produces biochemical hyperthyroidism. Our data are consistent with an abnormality in thyroid hormone feedback on the hypothalamic pituitary axis.     

Characteristics of an acid active thiamine diphosphatase from beef brain

That thiamine has a role in nerve conduction as well as synaptic transmission is suggested by the following observations. (1) Thiamine phosphate esters are hydrolyzed and released from nerve membranes during nerve conduction. (2) Ultraviolet radiation of single nerve fibers at the wavelength specific for thiamine destroys the ability of that nerve to conduct an impulse. (3) Thiamine diphosphatase (TDPase) is present on synaptosomes. Previous articles have characterized an alkaline active TDPase in brain; this report characterizes a pH 5 active TDPase and compares its properties to the pH 9 enzyme. Both enzymes require a divalent cation for optimal activity. The pH 5 enzyme is more sensitive to ATP. Myelin fractions of brain have the highest specific activity for the acid TDPase, and the nerve ending particles the highest total activity. No PO₄ ^3– inhibition was observed. Kinetic constants of this enzyme activity are reported.     

Interaction of nutrition and infection: Effect of iron deficiency on resistance to Trypanosoma lewisi

• 1.1. Iron deficiency was studied in rats infected with Trypanosoma lewisi.
• 2.2. Iron-deficient and pair-fed control rats showed greater parasitemias than complete diet rats throughout the infection.
• 3.3. The average coefficients of variation in body length of T. lewisi cells indicated that the formation of the reproduction-inhibiting antibody (ablastin) occurred 9 days later in iron-deficient hosts compared to hosts fed complete diets. The duration of parasitemia in the deficient animals also indicated a delayed production of the terminal lytic antibodies.
• 4.4. Irrespective of diet, the infected animals consumed more food and gained more weight than un-infected controls.

     

The isoelectric fractionation of hen''s-egg ovotransferrin

1. ATP sulphurylase was assayed in various organs from vitamin A-deficient and pair-fed control rats at different stages of deficiency. Activity decreased slightly in the liver and markedly in the adrenal gland. Striking differences in liver activity were observed between pair-fed control and ad libitum-fed animals. This observation suggested that diet (apart from vitamin A) strongly influenced the activity of ATP sulphurylase. 2. Total starvation caused a severe decrease in activity in liver within 48hr. This was due to a lack of protein intake. 3. By feeding groups of vitamin A-deficient and pair-fed control rats on a diet containing 80% protein, the specific activity of the liver ATP sulphurylase was maintained in the pair-fed control group at the normal level of an ad libitum-fed rat, whereas it decreased by 25% (statistically significant at P<0.01) in the deficient rat. On a 20%-protein diet, there were no significant differences between vitamin A-deficient and pair-fed control rats. These relationships held also for enzyme activity expressed per g. of liver, per total liver and per g. of DNA. There were no differences in liver protein or DNA concentration between vitamin A-deficient and control rats on either protein intake. 4. Control rats on a 20%-protein diet had liver specific enzyme activities about one-half of those in control rats on an 80%-protein diet, as well as lower liver protein concentrations. 5. It is concluded that, when the effect of protein deprivation on ATP sulphurylase is separated from the effect of vitamin A deficiency, a lowering of the enzyme activity caused by the vitamin deficiency is demonstrable.     

Zinc and insulin metabolism

A review of experimental studies of the effect of zinc nutrition on insulin metabolism is presented. In addition to a short introduction to the synthesis, secretion, and action of insulin, the effects of zinc deficiency—specifically on glucose tolerance, insulin secretion, insulin synthesis and storage, and on total insulin-like activity—are dealt with. The concentrations of zinc and chromium in serum, pancreas, and liver are compared to those of zinc-deficient animals and pair-fed controls. In contrast to pair-fed controls, zinc-deficient rats had unaltered proinsulin contents after glucose stimulation, but they showed a diminished glucose tolerance, lowered serum insulin content, and an elevated total insulin-like activity. The serum zinc concentration of the deficient animals was greatly reduced and did not change during glucose stimulation, whereas it rose in the case of the pair-fed controls. The serum chromium concentration increased in both groups in response to glucose stimulation. In the pancreas of the deficient animals, the zinc concentration was reduced 60% and it increased during the glucose tolerance test. In the liver there were no significant differences. The chromium concentrations were elevated in both the pancreas and liver of the zinc-deficient rats by 60 and 100%, respectively, and were not influenced by glucose injection. These studies show clearly that nutritional zinc deficiency influences insulin metabolism and action.     

The inhibition of rat brain ATP: citrate oxaloacetate-lyase by L-glutamate

Abstract—

• 1 Among 16 amino acids tested only D- and L-glutamate were found to be specific inhibitors of citrate lyase from adult rat brain. Glutamate also inhibited citrate lyase from the liver of starved animals while it was without effect on lyase from those refed with carbohydrate. L-Glutamate did not inhibit the citrate lyase from brains of newborn rats.
• 2 The inhibitory effect of L-glutamate was increased when the time of preincubation was prolonged, but only in the presence of both ATP and MgCl₂. This time-dependent inhibition could be reversed by addition of high concentrations of ATP.
• 3 L-Glutamate was without effect on K_m and V_max values for MgCl₂. Excess of Mg²⁺ ions was indispensable for glutamate inhibition.
• 4 L-Glutamate was shown to be competitive with respect to the ATP inhibitor of the lyase, with a K_i of 0.3mM.
• 5 The mechanism of L-glutamate inhibition may be due to the reaction of a glutamate-Mg complex with the phosphorylated intermediary form of the lyase, resulting in the formation of a lyase-glutamate complex.
• 6 The physiological significance of the inhibitory action of glutamate is discussed.

     

EFFECT OF THYROID HORMONE ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: CONVERSION OF GLUCOSE-CARBON INTO AMINO ACIDS

• 1 The rapid and extensive conversion of glucose-carbon into amino acids is an index of the final coordination of the mechanisms underlying energy metabolism in the adult brain. This phenomenon develops in the rat during a short period extending from 10 to about 19 days after birth. The underlying factors have been analysed.

• 2 The development of the pattern of distribution of glucose-carbon characteristic of the adult brain was markedly influenced by the thyroid state of the animals. The age-curve for the conversion of glucose-carbon into brain amino acids was displaced to the left after treatment with thyroid hormone (T₃) in infancy thus indicating an accelerated maturation. Conversely, neonatal thyroidectomy resulted in a significant retardation in the conversion of glucose-carbon into amino acids.

• 3 The specific radioactivity of glutamate increased five-fold in the brain of normal rats from the 10th to the 19th day of age. The values (as a percentage of those for littermate controls) were 220 in the case of the 10 day-old thyroid treated rats and about 30 for the 19 day-old thyroid deficient animals. At the age of 10 days neither treatment affected the concentration of glutamate which was also only slightly less than the control values in the brain of 19 day-old thyroid deficient animals (–17 per cent).

• 4 Specific pool(s) of glutamate associated with the formation of GABA can be demonstrated in the brain of 19 day-old rats after administration of [U-¹⁴C]glucose as a result of anoxia post mortem. These pools did not develop in the brain of 10 day-old animals. Neonatal thyroidectomy retarded the development of these glutamate pools.

• 5 Evidence is summarized which indicates that the development of the rapid conversion of glucose-carbon into amino acids reflects the enlargement, during maturation, of the metabolic compartments which are associated with neuronal processes.

     

Effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail

• 1.1. The effects of niacin deficiency on the relative turnover rates of proteins in various tissues of Japanese quail were investigated.
• 2.2. The level of liver NAD was not affected by niacin deficiency whereas the level of pectoral muscle NAD was markedly reduced.
• 3.3. In all dietary treatments the liver had the highest turnover rates of proteins, heart and brain had intermediate rates, and pectoral muscle had the lowest rates.
• 4.4. Relative turnover rates of proteins in all tissues (particularly pectoral muscle) of the niacin deficient group were significantly higher than those of pair-fed control group, although there were no significant differences in turnover rate between pair-fed control and control groups.
• 5.5. The high turnover rate of proteins in niacin deficiency was primarily attributed to enhanced degradation rate of proteins rather than enhanced synthesis rate of proteins.
• 6.6. Optical density scanning (or densitometric) of water-soluble pectoral muscle proteins separated by isoelectric focusing revealed several additional minor protein bands between major protein bands in the niacin deficient group which were more pronounced in the acidic region of the gel.
• 7.7. These results suggest that proteins with a low pI value in pectoral muscle of the niacin deficient animal are highly sensitive to protein degradation.

     

The effects of pyridoxine deficiency and of caloric restriction on lipids in the developing rat brain

Abstract— The effects of dietary deficiency of pyridoxine upon the contents of lipids in the brain were determined at several times after birth for three groups of rats. The mothers of the nursing pups were fed one of the following dietary regimes: pyridoxine-deficient diet ad lib., pyridoxine-supplemented diet ad lib., or pyridoxine-supplemented diet in restricted amounts. At 7 and 14 days of postnatal age there were no significant differences between supplemented and deficient animals for any of the cerebral lipids studied. At 21 days the content of sphingomyelin in the brains of deficient animals was significantly lower than that in brains from the supplemented or calorically restricted animals in terms of percentage of total lipid and phospholipid phosphorus or tissue weight. On a per brain basis the content of sphingomyelin in the brains of calorically-restricted rats was significantly lower than in the brains of rats fed the supplemented diet ad lib. The contents of cerebrosides but not of sulphatides or ceramides were also significantly lower in brains of the deficient group than in brains from the other two groups. The contents of pyridoxine in brains and in livers of the deficient animals were considerably lower than the contents found in the same organs of the other dietary groups. The results suggest that one reason for the abnormal development of the brains of rats on a pyridoxine-deficient diet during the early postnatal period may be due to decreased quantities of sphingolipids.     

Zinc-stimulated microtubule assembly and evidence for zinc binding to tubulin

• 1.1. Microtubule reassembly was studied in supernatant fluids from rat brain. Tubulin in extracts from zinc-deficient animals showed an impaired ability to repolymerize compared to extracts from controls; 10 μM zinc stimulated reassembly of tubulin in extracts from zinc-deficient animals.
• 2.2. Low zinc concentrations (250–900 μM zinc in the presence of 1 mM EGTA) stimulated reassembly oftubulin in brain extracts from control rats; similar concentrations of nickel had no effect whilst cobalt was inhibitory. In the absence of EGTA 20–40 μM zinc stimulated reassembly in brain extracts from normal rats.
• 3.3. Zinc-induced changes in reassembly were associated with changes in the free sulphydryl group content of the assembled crude microtubule protein; increased assembly was associated with a higher free sulphydryl group content, decreased assembly with a lower content.
• 4.4. ⁶⁵Zn was found to bind to tubulin. This binding was partly inhibited by N-ethylmaleimide.

     

Carbohydrate metabolism in erythrocytes of copper deficient rats

Dietary copper deficiency is known to adversely affect the circulatory system of fructose-fed rats. Part of the problem may lie in the effect of copper deficiency on intermediary metabolism. To test this, weanling male Long-Evans rats were fed for 4 or 8 weeks on sucrose-based diets containing low or adequate copper content. Copper deficient rats had significantly lower plasma and tissue copper as well as lower plasma copper, zinc-superoxide dismutase activity. Copper deficient rats also had a significantly higher heart:body weight ratio when compared to pair-fed controls. Direct measurement of glycolysis and pentose phosphate pathway flux in erythrocytes using (13)C NMR showed no differences in carbon flux from glucose or fructose to pyruvate but a significantly higher flux through the lactate dehydrogenase locus in copper deficient rats (approximately 1.3 times, average of glucose and glucose + fructose measurements). Copper-deficient animals had significantly higher erythrocyte concentrations of glucose, fructose, glyceraldehyde 3-phosphate and NAD(+). Liver metabolite levels were also affected by copper deficiency being elevated in glycogen and fructose 1-phosphate content. The results show small changes in carbohydrate metabolism of copper deficient rats.     

Impaired microtubule assembly in brain from zinc-deficient pigs and rats

• 1.1. Microtubule reassembly was studied in brain extracts from pigs and rats deficient in zinc and from zinc-supplemented controls. Tubulin in extracts from zinc-deficient animals showed an impaired ability to repolymerize compared with extracts from control animals.
• 2.2. Crude microtubule protein isolated from zinc-deficient rat brain contained less free SH groups than that isolated from zinc-supplemented animals.
• 3.3. It is concluded that variation in zinc concentration close to the physiological range can influence microtubule assembly, and that zinc may have some function in microtubule polymerization in vivo.

     

The effect of zinc defficiency and food restriction on prostaglandin E2 and thromboxane B2 in saliva and plasma of rats

Zinc has been implicated in the regulation of prostaglandins and other arachidonic acid derivatives. Studies of zinc-deficient animals, however, are compromised by concomitant reduction in food intake that may also alter eicosanoid levels in body tissues and fluids. In this study, three groups of rats, designated as zinc-deficient, pair-fed and control, were fed diets containing 1 ppm, 15 ppm (in amounts paired to deficient rats) and 15 ppm Zn adlibitum, respectively, for 6 weeks. Saliva and blood were analyzed for PGE₂ adn TXB₂ by radioimmunoassay. Saliva concentrations of both eicosanoids were lower (p<0.05) in the pair-fed animals, but not significantly altered by zinc deficiency. Plasma levels of PGE₂ and TXB₂ were unchanged by either zinc deficiency or food restriction. The results of this study support the contention that the effect of zinc on these prostaglandins is not mediated by altered rates of synthesis or degradation but rather by effects on eicosanoid function.     

A COMPARATIVE STUDY OF BRAIN AND LIVER MITOCHONDRIA FROM NEW-BORN AND ADULT RATS

• 1 A method has been developed for the estimation of organelle number in subcellular fractions and applied to the estimation of the mitochondrial content of brain and liver from new-born and adult rats.
• 2 The respiratory enzyme content per mitochondrion of the adult brain was 3·5 times greater than that of the neonate. This increase in enzyme content was not correlated with an increase in the mean size of the organelle.
• 3 The succinate dehydrogenase activity per mitochondrion of the livers from neonatal and adult rats showed no obvious change. There was, however, an increase in the mean size of the organelle.
• 4 These findings are discussed with reference to the development of the brain and to the development of mitochondria.

     

PROPERTIES OF THIAMINE DI- AND TRIPHOSPHATASES IN RAT BRAIN MICROSOMES: EFFECTS OF CHLORPROMAZINE

Abstract— The mechanism of the action of chlorpromazine on rat brain thiamine phosphatases were studied to clarify the properties of these enzymes in the CNS. Chlorpromazine at concentrations of 0.25-1.0 m m caused marked decrease of microsomal and soluble thiamine triphosphatase (TTPase) activities and marked increase of microsomal thiamine diphosphatase (TDPase) activity. Imipramine and desipramine also inhibited TTPase but did not cause any marked change in TDPase activities. Addition of chlorpromazine (0.5 m m ) decreased the V_max of microsomal TTPase by about one-half, increased that of TDPase about 3-fold, and lowered the K _m value for TDP but not for TTP.
Acetone treatment of the microsomal fraction lowered the TTPase activity and markedly enhanced the TDPase activity. In acetone-treated microsomes, chlorpromazine also inhibited TTPase activity but did not activate TDPase. Deoxycholate had similar effects to chlorpromazine on these enzyme activities.     

Effect of thiamine deficiency on the filarial infection of albino rats with Litomosoides carinii

Induction of thiamine deficiency in albino rats led to greater susceptibility to infection with the filarial parasite, Litomosoides carinii. The patency of the infection was prolonged and there was a greater worm burden and a higher peak microfilaraemia in the deficient animals. The haemagglutinating antibody response to the infection was significantly reduced. The mitogenic response of the lymphocytes to PHA and Con A decreased progressively in infected, pair-fed and deficient animals in that order, suggesting the immunosuppressive effect of the infection and the synergistic role of thiamine deficiency on this effect. At the onset of latency to the infection, the serum from animals of all groups promoted antibody-dependent adhesion of splenic cells to microfilariae.     

Vitamin B6 deficiency decreases the glucose utilization in cognitive brain structures of rats

The effects of vitamin B(6) deficiency on metabolic activities of brain structures were studied. Male Sprague-Dawley weanling rats received one of the following diets: (1) 7 mg pyridoxine HCl/kg (control group); (2) 0 mg pyridoxine HCl/kg (vitamin B(6)-deficient group); or (3) 7 mg pyridoxine HCl/kg with food intake restricted in quantity to that consumed by the deficient group (pair-fed control group). After 8 weeks of dietary treatment, rats in all three groups received an intravenous injection of 2-deoxy-[(14)C] glucose (100 microCi/kg). Vitamin B(6) status was evaluated by plasma pyridoxal 5'-phosphate concentrations. The vitamin B(6)-deficient group had significantly lower levels of plasma pyridoxal 5'-phosphate than did the control and pair-fed groups. The local cerebral glucose utilization rates in structures of the limbic system, basal ganglia, sensory motor system, and hypothalamic system were determined. The local cerebral glucose utilization rates in each of the four brain regions in the deficient animals were approximately 50% lower (P < 0.05) than in the control group. Results of the present study suggest that serious cognitive deficit may occur in vitamin B(6)-deficient animals.     

Trypanosoma lewisi: pyruvate and transketolase activity in normal and thiamine deficient rats

Transketolase and pyruvate changes were studied in rats infected with Trypanosoma lewisi and fed complete, thiamine-deficient and pair-fed control diets. Regardless of the dietary group, marked increases in pyruvate levels were observed in the infected animals. There were no significant differences in erythrocyte transketolase activity of rats given a full complement diet. Significant decreases, however, were observed in the transketolase activity of pair-fed and thiamine deficient rats. The greater decreases occurred in the infected animals.