Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.     

Enzymic markers of thyroid C cells in some rodents.

The paper provides comparative data of the localization of histochemical reactions demonstrating the activities of alpha-glycerophosphate and succinate dehydrogenases, acid phosphatase, non-specific esterases and non-specific acetylcholinesterase in the C cells of thyroids of 26 animals belonging to 5 rodent species. The family Muridae is represented by the Wistar albino rat and albino mouse, the family Microtidae by the bank vole Clethrionomys glareolus (Schreber 1780), the field vole Microtus agrestis L. 1761, and the pine vole Pitymys subterraneus De Selys-Longchamps 1825. The observed enzyme activity differences were most conspicuous on comparing the rat and mouse thyroids and in a much less degree the Microtidae thyroids. Among the histochemical reactions tested that for succinate dehydrogenase proved to be least effective as a C cell marker, alpha-glycerophosphate dehydrogenase being better, and acid phosphatase and non-specific esterases the best (not in the rat thyroid). The reaction for non-specific cholinesterase (with some limitations) gave satisfactory results in the C cells of all animal's thyroids. The present paper continues earlier studies [19] on the morphology of the C cells in thyroid glands of the rodents of the families Muridae and Microtidae and aims at supplementing them with histochemical data of enzymic activities. It deals with enzyme reactions that are employed as C cell markers in Mammals other than Rodents.     

Role of the glycerophosphate shunt in regulating oxidation of cytoplasmic NADH and ATP synthesis by liver mitochondria during rat ontogeny

The activity of alpha-glycerophosphate dehydrogenase rate of alpha-glycerophosphate oxidation and ATP synthesis were determined in the liver mitochondria of 1, 3, 12 and 24-month old rats in norm and two days after thyroxin daily administration in a dose of 0.25 mg per 100 g of body mass. The value of the studied parameters decreases with the age, most considerably--from one to three months and then--from 3 to 24-month age. This evidence for the fact that the role of glycerophosphate shuttle in the energy supply of cells falls with ageing; thyroxin administration evoked almost a two-fold increase of the mentioned parameters in one-month rats and more than three-fold in 12 and 24-month animals. A decrease in the alpha-glycerophosphate dehydrogenase activity with ageing is supposed to be connected with the thyroid gland function.     

A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes

Differentiation of 3T3-L1 adipocytes, monitored by accumulation of neutral lipid and by increase in alpha-glycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3-L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its receptor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20-1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing alpha-glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8-phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6-(R phenyl-isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in alpha-glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3-L1 cells.     

Effect of somatotropin on the activity of mitochondrial alpha-glycerophosphate dehydrogenase in the liver of rats with hypo- and hyperthyroidism

The activity of mitochondrial alpha-glycerophosphate dehydrogenase in the liver of rats with hypothyrosis (two weeks after total thyroidectomy) is 36% of the normal level; in rats with hyperthyrosis (three weeks after subcutaneous implantation 2 mg of l-thyroxin on a kaolin base) it is 7.4 times as high. Bovine somatotropin (0.5 mg per 100 g of body mass) injected subcutaneously for 10 days has no effect on the enzyme activity in intact rats and in rats with hypothyrosis. In animals with hyperthyrosis somatotropin produces a 37% decrease in the enhance activity of the enzyme. Somatotropin fully normalizes a 25% increased amount of protein in the liver mitochondrial fractions of rats with hyperthyrosis.     

Some features of mitochondria and fluffy layer in regenerating rat liver

1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD–isocitrate dehydrogenase and NADP–isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18·4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4·7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD– and NADP–isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP–isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1·198, light mitochondria 1·193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed.     

A sensitive, continuous spectrophotometric method for assaying alpha-glycerophosphate dehydrogenase: activation by menadione

By use of a more sensitive method than standard assays, it is demonstrated that alpha-glycerophosphate dehydrogenase activity can be monitored continuously in rat liver mitochondria; maximum activity was obtained by sonication in the presence of Triton X-100. Vitamin K3 (menadione) seems to enhance the activity of the enzyme. The assay in the presence of menadione is linear over a much greater mitochondrial concentration (up to 250 micrograms of protein in the reaction mixture).     

Biorhythm study of the alpha-glycerophosphate dehydrogenase activity of the peripheral blood lymphocytes in rats with Walker carcinosarcoma

In the series of experiments alpha-glycerophosphate dehydrogenase activity was daily determined at the same time by cytochemical method in the peripheral lymphocyte of normal rats and those with Walker carcinosarcoma. Using an original algorithm a hierarchy of biorhythms was singled out on a computer according to the individual changes in alpha-glycerophosphate dehydrogenase activity in each rat. It was shown that tumor development resulted in the reduction of the mean level of alpha-glycerophosphate dehydrogenase activity and considerable changes in diverse rhythms. Particular attention is paid to the phase changes, which can reveal changes in phase coordination of glycerophosphate shuttle cycle components.     

Alpha-Glycerophosphate dehydrogenase (insoluble) and lactic dehydrogenase activities in the skeletal muscles of two insects.

The flight muscle preparations of the dragonfly Pantala flavescens and the aquatic beetle Cybister confusus showed extremely low levels of lactic dehydrogenase activity and high levels of alpha-glycerophosphate dehydrogenase (insoluble) activity. The activities of these two enzymes in the leg muscle of the beetle were approximately the same (1:1), but lactic dehydrogenase activity was several times higher than that in the flight muscles of both Insects. These results have been interpreted as indicating the high energy-yielding demands of the flight muscles during continuous sustained activity, while the leg muscles of the beetle which are involved in swimming activity derive their energy predominantly through anaerobic glycolysis.     

Mitochondrial alpha-glycerophosphate dehydrogenase activity in active and torpid ground squirrels, Spermophilus richardsoni.

1. Mitochondrial alpha-glycerophosphate dehydrogenase (GPD) activity was assayed in liver homogenates from active and torpid ground squirrels. 2. Arrhenius plots of GPD activity were linear in non-hibernating animals and discontinuous in hibernators. Compared with non-hibernators, the energy of activation in hibernators was reduced between 37 and 25 degrees C, but increased between 25 and 6 degrees C. 3. A dose-response relation between GPD activity and injected thyroxine was determined in active animals. No correlation was found between enzyme activity at 37 or 6 degrees C and circulating titres of thyroid hormones, in ground squirrels sampled during the preparative and hibernating phases.     

Effect of triiodothyronine and thyroxine on mitochondrial alpha-glycerophosphate dehydrogenase activity and mitochondrial protein content of liver, muscle and brain of toad, Bufo melanostictus

The responsiveness of the adult toad to triiodothyronine (T3) and thyroxine (T4) was studied by measuring the mitochondrial alpha-glycerophosphate dehydrogenase activity and mitochondrial protein content of liver, muscle and brain of toad. Both T3 and T4 increased the alpha-GPD activity and mitochondrial protein content of liver and muscle of toad. The extent of increase in the alpha-GPD activity and mitochondrial protein content were more pronounced with T3 than with T4. Further that the muscle exhibited more alpha-GPD activity than liver, whenever liver showed greater mitochondrial protein content than that of muscle. Brain showed no significant change in the alpha-GPD activity and mitochondrial protein content. Injections of cycloheximide showed inhibition of T3 induced changes in liver and muscle. Injection of propylthiouracil also counteracted the T4 induced effects of liver and muscle.     

Glucose-6-phosphate dehydrogenase activity and NADPH/NADP+ ratio in liver and pancreas are dependent on the severity of hyperglycemia in rat

Hyperglycemia is associated with metabolic disturbances affecting cell redox potential, particularly the NADPH/NADP+ ratio and reduced glutathione levels. Under oxidative stress, the NADPH supply for reduced glutathione regeneration is dependent on glucose-6-phosphate dehydrogenase. We assessed the effect of different hyperglycemic conditions on enzymatic activities involved in glutathione regeneration (glucose-6-phosphate dehydrogenase and glutathione reductase), NADP(H) and reduced glutathione concentrations in order to analyze the relative role of these enzymes in the control of glutathione restoration. Male Sprague-Dawley rats with mild, moderate and severe hyperglycemia were obtained using different regimens of streptozotocin and nicotinamide. Fifteen days after treatment, rats were killed and enzymatic activities, NADP(H) and reduced glutathione were measured in liver and pancreas. Severe hyperglycemia was associated with decreased body weight, plasma insulin, glucose-6-phosphate dehydrogenase activity, NADPH/NADP+ ratio and glutathione levels in the liver and pancreas, and enhanced NADP+ and glutathione reductase activity in the liver. Moderate hyperglycemia caused similar changes, although body weight and liver NADP+ concentration were not affected and pancreatic glutathione reductase activity decreased. Mild hyperglycemia was associated with a reduction in pancreatic glucose-6-phosphate dehydrogenase activity. Glucose-6-phosphate dehydrogenase, NADPH/NADP+ ratio and glutathione level, vary inversely in relation to blood glucose concentrations, whereas liver glutathione reductase was enhanced during severe hyperglycemia. We conclude that glucose-6-phosphate dehydrogenase and NADPH/NADP+ were highly sensitive to low levels of hyperglycemia. NADPH/NADP+ is regulated by glucose-6-phosphate dehydrogenase in the liver and pancreas, whereas levels of reduced glutathione are mainly dependent on the NADPH supply.     

Effect of diet on linoleic acid desaturation and on some enzymes of carbohydrate metabolism

The effect of diet on the desaturation of linoleic acid to gamma-linolenic acid by liver microsomal preparations, on blood glucose and insulin levels, and on activities of glucokinase, hexokinase, pyruvate kinase, and alpha-glycerophosphate dehydrogenase have been studied. The female rats used in these experiments were maintained on one of the following dietary regimes: (a) fasted, (b) fasted for 96 hr and refed glucose, (c) balanced diet, (d) carbohydrate-free diet, (e) lipid-free diet, or (f) protein-free diet. Fasting for 96 hr caused a decrease of both linoleic acid desaturation and glucokinase and pyruvate kinase activity together with a slight decrease of the blood insulin level. Alpha-glycerophosphate dehydrogenase activity was not modified. Refeeding of glucose for 50 hr increased the conversion of linoleic acid to linolenic acid as well as the activities of all the enzymes studied except alpha-glycerophosphate dehydrogenase. The increase in desaturation, however, was transient. The feeding of a lipid-free diet did not modify the tested parameters. Feeding a carbohydrate-free diet for 96 hr resulted in increased linoleic acid desaturation but decreased glucokinase and pyruvate kinase activity, thus apparently eliminating a putative correlation between the fatty acid desaturating activity and glycolytic activity or blood insulin levels under these experimental conditions. The findings suggest that dietary proteins may play an important role in determining the level of fatty acid desaturation.     

Sequential changes in rat liver nuclear tri-iodothyronine receptors and mitochondrial α-glycerophosphate dehydrogenase activity after administeration of tri-iodothyronine

The dynamics of the induction of nuclear tri-iodothyronine receptors and mitochondrial alpha-glycerophosphate dehydrogenase were studied in rat liver after a single injection of tri-iodothyronine. The maximal binding capacity (C(max.)) and association constant (K(a)) of the nuclear receptors were determined by Scatchard analyses with and without correction for the endogenous tri-iodothyronine measured by radioimmunoassay. The administration of tri-iodothyronine induced sequential increases in the concentration of nuclear receptors and alpha-glycerophosphate dehydrogenase activity in the liver. The nuclear-receptor concentration was increased to 2.5 times that in the hypothyroid rat 1 day after the administration of hormone, and then decreased, with a half-life of about 2 days. alpha-Glycerophosphate dehydrogenase activity changed in parallel with the nuclear-receptor concentration, showing a delayed response. The total amount of non-histone protein in the liver was significantly increased 3 days after the administration. It seems likely therefore that the tri-iodothyronine-induced increase in nuclear-receptor concentration is responsible, at least in part, for the induction of this enzyme. The possibility is also suggested that nuclear receptors may be one of the non-histone proteins selectively synthesized at an early stage of the hormonal stimulation. Throughout the time course, the K(a) values of the nuclear receptors for tri-iodothyronine remained unchanged, when corrected for endogenous tri-iodothyronine bound to the non-histone proteins, although they were apparently changed when the correction was not made. The results obtained provide further evidence for hormonal modulation of the nuclear receptors which is closely linked with the hormonal effect.     

Cytochemical Analysis of Enzyme Activity during Regeneration in Blepharisma intermedium Bhandary

SYNOPSIS. Transection and repair in Blepharisma intermedium modify the distribution of several enzymes. The number of active phagosomes is reduced and there are increased staining reactions for acid phosphatase, the nucleases and type-B esterases in the ground cytoplasm. 5'-Nucleotidase activity is localized in the cytoplasmic matrix. Except for ribonuclease, the enzymes may have a perimacronuclear deposition at some period during regeneration. Acid phosphatase may be involved in development of the oral membranelles. Adenosine triphosphatase and lactic dehydrogenase have a moderate staining intensity whereas malic and succinic dehydrogenases have a strong reaction in the cytoplasm; in addition, these enzymes may be localized in the kinetics involved in the neoformation of the oral concrescence. Nuclear reorganization is attended by an increased frequency of peripheral dehydrogenase activity in the late stages of regeneration. These observations are compared with catalytic systems known to function in protozoan metabolism and in vertebrate regeneration.     

Effects of some antitumor agents on growth and glycolytic enzymes of the flagellate Crithidia

Some antitumor agents known to specifically inhibit certain tumor cell enzymes were examined for activity against glycolytic enzymes and growth of the insect trypanosomatid, Crithidia fasciculata. The cytoplasmic enzymes hexokinase, alpha-glycerophosphate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were tested. Agaricic acid (2-hydroxy-1,2,3-nonadecane tricarboxylic acid) was highly inhibitory (50 to 100%) to malic and alpha-glycerophosphate dehydrogenases at approximately 3 x 10(-5)m; 2-(p-hydroxyphenyl)-2-phenylpropane (2 x 10(-4)m), and 5,6-dichloro-2-benzoxazolinone (5 x 10(-4)m) were less effective (50% inhibition) against them. The antiprotozoal agents primaquine (4 x 10(-4)m) and Melarsoprol (8 x 10(-4)m) were 30 to 40% inhibitory. Agaricic acid, 2-(p-hydroxyphenyl)-2-phenylpropane, and 5,6-dichloro-2-benzoxazolinone inhibited growth of Crithidia at less than 10(-4)m. Eight other test compounds from the Cancer Chemotherapy National Service Center (CCNSC) were not toxic to cell growth, although two (4-biphenylcarboxylic acid and 1-[p-chlorobenzyl]-2-ethyl-5-methyl-indole-3-acetic acid) inhibited Crithidia alpha-glycerophosphate dehydrogenase below 1 mm. All of the compounds used specifically inhibited cancer cell alpha-glycerophosphate dehydrogenase. The corresponding enzyme in pathogenic African trypanosomes is important in their terminal respiration. C. fasciculata may be useful in preliminary evaluation of chemotherapeutic agents as potential trypanocides.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

Development of rat liver and colon substrate cycle enzymes in offspring with and without prenatal exposure to a carcinogenic dose of 1,2-dimethylhydrazine

Pregnant F-344 rats were exposed by intubation to a single dose (35 mg/kg) of 1,2-dimethylhydrazine dihydrochloride or to an acetate buffer on day 14 of gestation. A detailed examination of the effects of this dose of 1,2-dimethylhydrazine on brush border enzymes in the offspring was performed. Changes in the liver and colon levels of the alpha-glycerophosphate and malate/aspartate substrate cycle enzymes were measured during the development; at days 17 and 20 of gestation and at 2, 6, 13, 20, 27, 55, 110 days and 1 year after birth. It is concluded that metabolic energy enzymes, glutamate oxaloacetate transaminase and malate dehydrogenase, are more sensitive to 1,2-dimethylhydrazine treatment than are the brush border hydrolases or NAD- and Fp-linked alpha-glycerophosphate dehydrogenases.     

The components of an α-glycerophosphate cycle and their relation to oxidative metabolism in the lens

1. The concentration of ATP in a lens brei is maintained when the brei is incubated in oxygen with alpha-glycerophosphate. Lack of alpha-glycerophosphate or incubation in nitrogen causes the concentration to decrease. alpha-Glycerophosphate has some effect under anaerobic conditions but this is not sufficient to account for the maintenance in oxygen. 2. Manometric experiments show that alpha-glycerophosphate enhances the respiration of lens preparations. This respiration can be further increased by the addition of ADP and is abolished by cyanide and antimycin. The inference from these experiments is that a mitochondrial system able to oxidize alpha-glycerophosphate is present, i.e. the particulate half of the alpha-glycerophosphate cycle. 3. More than the calculated proportion of NADH is used when limiting amounts of dihydroxyacetone phosphate are added to lens tissue in spectrophotometric experiments. Dihydroxyacetone phosphate is therefore regenerated and an alpha-glycerophosphate cycle is operative. 4. A preparation of a particulate alpha-glycerophosphate dehydrogenase that takes up oxygen with methylene blue as electron acceptor is described. 5. Methods for obtaining mitochondria from lens are compared, and a useful extraction medium is defined. 6. Mitochondria with activities of the same order of magnitude as those obtained from liver, with alpha-glycerophosphate and glutamate as substrates, are prepared from epithelium detached from the capsule; some respiratory control is observed.     

Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat.

The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.