Studies on cartilage formation. XX. Histochemical investigation of some enzymes of glycogen metabolsim in regenerative articular surfaces.

In 28 dogs the distal articular cartilage of the femur was removed and the regenerating articular surface on the 70th postoperative day was studied histochemically for hexokinase, glucose-6-phosphatase, phosphohexose-isomerase, fructose-1, 6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, lactate dehydrogenase isoenzymes, phosphoglucomutase, phosphorylase, glycogen synthetase, UDP--glucose dehydrogenase, and UDP-glucuronic acid-4-epimerase. The articular surface consisted of fibrous tissue and of cartilage islets. The latter contained cells differentiating into cartilage and young chondrocytes. The glycolytic enzymes reacted positively in the regenerative articular surface. Enzyme activities were higher in the cells (particularly the chondroblasts and young chondrocytes) of the cartilage islets than in the connective tissue. In the cells differentiations into cartilage, beside the LDH isoenzymes characteristic of glycolysis, a significant LDH1 and LDH2 activity was observed. At the same site the presence of fructose-1, 6-diphosphatase-activity could be assumed, but there was no glucose-6-phosphatase activity. Glycogen synthesis proceeded in the cells of the cartilage islets and UDP-glucuronic acid-4-epimerase activity was observed in the differentiated cells. UDP-glucose dehydrogenase activity was positive in every section of the articular surface.     

Cytochemical observations on glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and -glycerophosphate dehydrogenase in chick liver cell cultures infected with Trichomonas vaginalis

Cytochemical reactions specific for glucose-6-phosphatase, glucosan phosphorylase, glucose-6-phosphate dehydrogenase, and α-glycero-phosphate dehydrogenase were observed in the epithelial cells and macrophages of chick liver cell cultures; α-glycerophosphate dehydrogenase activity was observed also in the fibroblasts. Distribution of three of the enzymes was limited to the cytoplasm, their activity being localized primarily in cytoplasmic inclusions. Weak staining of the nuclei and strong staining of the nucleoli occurred in addition to the cytoplasmic reaction in cells treated for glucose-6-phosphatase. In cell cultures inoculated with Trichomonas vaginalis, the activity of three of the enzymes decreased progressively in the course of infection, but that of α-glycerophosphate dehydrogenase increased.     

Undifferentiated patterns of key carbohydrate-metabolizing enzymes in injured livers. I. Acute carbon-tetrachloride intoxication of rat.

In acute CCL4 intoxication of rats significantly increased activities of hepatic low-Km hexokinases, glucose-6-phosphate dehydrogenase, phosphofructokinase, aldolase A and pyruvate kinase M2 with concurrently decreased activities of glucokinase, glucose-6-phosphatase, fructose-1,6-diphosphatase, aldolase B and pyruvate kinase L were observed. The resulting enzyme pattern was apparently different from that in dietary induction. Principal component analysis revealed that the degree of enzyme deviation in the injured liver was much greater than that in the regenerating liver after partial hepatectomy and was closer to that in fetal liver or hepatoma tissue.     

The effects of growth hormone, thyroxine and insulin on the activities of reduced nicotinamide adenine dinucleotide phosphate dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase in fetal rat liver.

Growth hormone (GH), thyroxine (T4) and insulin were injected, in utero into 20.5 day-old rat fetuses to study the effects of these hormones on the activities of liver NADPH dehydrogenase, glucose-6-phosphatase and glycogen phosphorylase. It was found that at 21.5 days of gestation, GH increases the fetal liver glucose-6-phosphatase activity and decreases the liver glycogen phosphorylase activity. T4 treatment augments the activity of NADPH dehydrogenase even at 0.3% of the dose shown previously to produce premature elevation of activity. Prior to this experiment T4 in large doses has been shown to be capable of elevating glucose-6-phosphatase. However, at the lower T4 dose used, no treatment effect was observed. The fetal rat liver is responsive to insulin at 21.5 days and insulin was able to depress glucose-6-phosphatase activity. Thereby, showing that the influence of insulin on this enzyme begins prior to birth instead of just subsequent to birth.     

Enzymes of glycolysis and the pentose phosphate pathway during development of the rabbit stomach worm Obeliscoides cuniculi

The activities of glycolytic and other enzymes of carbohydrate metabolism were measured in free-living and parasitic stages of the rabbit stomach worm Obeliscoides cuniculi. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphatase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoenol pyruvate carboxykinase, lactate dehydrogenase, alcohol dehydrogenase, and glucose-6-phosphatase activities were present in worms recovered 14, 20 and 190 days postinfection.The presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase indicates the possible function of a pentose phosphate pathway and a capacity for gluconeogenesis, respectively, in these worms.The ratio of pyruvate kinase (PK) to phosphoenol pyruvate carboxykinase (PEPCK) less than I in parasitic stages suggests that their most active pathway is that fixing CO₂ into phosphoenol pyruvate to produce oxaloacetate.Low levels of glucose-6-phosphate dehydrogenase, triosephosphate isomerase, PEPCK and PK were recorded in infective third-stage larvae stored at 5°C for 5 and 12 mos. The ratio of PK to PEPCK greater than 1 indicates that infective larvae preferentially utilize a different terminal pathway than the parasitic stages.     

Glycogen metabloism in the developing chick glycogen body: functional significance of the direct oxidative pathway.

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase were quantitatively determined for the first time in glycogen body tissue from late embryonic and neonatal chicks. For comparative purposes, the activities of these enzymes were examined also in liver and skeletal muscle from pre- and post-hatched chicks. The present data show that both the embryonic and neonatal glycogen body lack glucose-6-phosphatase, but contain relatively high levels of glucose-6-phosphate dehydrogenase. The activity of each dehydrogenase in either embryonic or neonatal glycogen body tissue is two- to five-fold greater than that found in muscle or liver from pre- or post-hatched chicks. The relatively high activities observed for both dehydrogenases in the glycogen body, together with the absence of glucose-6-phosphatase activity in that tissue, suggest that the direct oxidative pathway (pentose phosphate cycle) of glucose metabolism is a functionally significant route for glycogen utilization in the glycogen body. It is hypothesized that the glycogen body is metabolically linked to lipid synthesis and myelin formation in the central nervous system of the avian embryo.     

Phosphatidylcholine-induced repair of damaged hepatocyte membranes in heliotrine poisoning

Hepatocyte membranes destruction in experimental toxic hepatitis caused by heliotrine administration was accompanied by a 10-fold increase in blood serum activity of aldolase fructose-I-monophosphate, a decrease in cytochrome P-450 content, an increase in the rate of cytochrome P-450 inactivation, as well as a decrease in microsomal glucose-6-phosphatase activity. Administration of phosphatidylcholine liposomes decreased the activity of aldolase twofold, which indirectly shows partial reconstitution of liver cell membranes. Phosphatidylcholine protective action is also manifested in an increase in the activity of glucose-6-phosphatase, a microsomal marker enzyme, up to its control level and in a 20% reduced rate of cytochrome P-450 inactivation. It has been shown that destroyed liver cell membranes may be repaired by the introduction of phosphatidylcholine in the form of multilayer liposomes.     

Activity of glucose-6-phosphate metabolism enzymes in the livers of rats with experimental valekson poisoning]

The activity of hexokinase, glucose-6-phosphatase and glucose-6-phosphoric dehydrogenase was studied in the liver of rats after one hour, one and five days after a single oral administration of organic phosphorus insecticide valekson. It was determined that administration of the preparation led to an increase of activity in the homogenate and solubilization of glucose-6-phosphatase, activation of glucose-6-phosphoric dehydrogenase and inhibition of hexokinase. The changes were maximum one hour after the administration of the compound. The results show that a decrease of the intensity of glucose-6-phosphate formation and metabolism is one of the pathogenetic factors in the development of valekson-induced intoxication.     

Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.     

Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.     

Histochemical demonstration of enzymes in rat liver during post-natal development

Summary Male and female rat liver were studied during post-natal development. A correlation was found between biochemically determined hydroxylations and enzymhisto-chemically determined NADPH-nitro-BT reductase and Naphthol-AS-D esterase. No correlation was found between glucose-6-phosphate dehydrogenase or iso-citric acid dehydrogenase activity and hydroxylations. The difference in hydroxylating capacity between male and female rats may be caused by the fact that the number of cells with hydroxylating activity in the liver lobule, as judged by the NADPH-nitro-BT reductase and Naphthol-AS-D esterase activity, is higher in male than in female rats.List of Abbreviations NADH reduced nicotinamide adenine dinucleotide - NADPH reduced nicotinamide adenine dinucleotide phosphate - G6PD glucose-6-phosphate dehydrogenase - ICD iso-citric acid dehydrogenase - G6Pase glucose-6-phosphatase - NADPH -nitro-BT red - NADPH Nitro-blue tetrazolium reductase - SDH succinic acid dehydrogenase - TCA trichloracetic acid     

Effect of sodium molybdate on carbohydrate metabolizing enzymes in alloxan-induced diabetic rats

We evaluated the effect of sodium molybdate on carbohydrate metabolizing enzymes and mitochondrial enzymes in diabetic rats. Diabetic rats showed a significant reduction in the activities of glucose metabolising enzymes like hexokinase, glucose-6-phosphate dehydrogenase, glycogen synthase and in the level of glycogen. An elevation in the activities of aldolase, glucose-6-phosphatase, fructose 1,6- bisphosphatase, glycogen phosphorylase and in the level of blood glucose were also observed in diabetic rats when compared to control rats. The activities of mitochondrial enzymes isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH-dehydrogenase and cytochrome-C-oxidase were also significantly lowered in diabetic rats. Molybdate administration to diabetic rats reversed the above changes in a significant manner. From our observations, we conclude that administration of sodium molybdate regulated the blood sugar levels in alloxan-induced diabetic rats. Sodium molybdate therapy not only maintained the blood glucose homeostasis but also altered the activities of carbohydrate metabolising enzymes. Molybdate therapy also considerably improved the activities of mitochondrial enzymes, thereby suggesting its role in mitochondrial energy production.     

Energy metabolism in subcellular fractions of normal and acute ischemic kidneys

The level of oxidative phosphorylation, activity of phosphofructokinase, fructose-1,6-diphosphate aldolase, ketose-1-phosphate aldolase, glucose-6-phosphatase and lactate dehydrogenase are determined in subcellular fractions in the kidney cortex layer of rabbits which have suffered from acute ischemia (for 15, 30, 60, 120 min). Ischemia inhibits the oxidative processes in mitochondria which is proportional to the duration of the effect. An increase in the activity of glycolytic chain enzymes in microsomes and soluble fraction for 15-30 min of ischemia evidences for a compensation of the energy losses at the expense of glycolysis with short periods of ischemia. Glycolysis is inhibited with a more prolonged effect. It is established that the anti-ischemic protection of the organ viability is to be conducted not only with allo- but also with auto-transplantation of the kidney in case of short acute ischemia.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

Some ultrastructural observations on the microfilaria of Breinlia sergenti—The excretory complex,rectal cells and anal vesicle

The present investigations describe the fine structure of the excretory complex, rectal cells and the anal vesicle of the microfilaria of Breinlia sergenti. The structure of the excretory cell and rectal cells is found to be similar to nerve cells. Axonal (digitiform) processes in the excretory and anal vesicles are described and a possible sensory function is ascribed to these structures.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

Regulation of carbohydrate metabolism by indole-3-carbinol and its metabolite 3,3′-diindolylmethane in high-fat diet-induced C57BL/6J mice

Indole glucosinolates, present in cruciferous vegetables have been investigated for their putative pharmacological properties. The current study was designed to analyse whether the treatment of the indole glucosinolates—indole-3-carbinol (I3C) and its metabolite 3,3′-diindolylmethane (DIM) could alter the carbohydrate metabolism in high-fat diet (HFD)-induced C57BL/6J mice. The plasma glucose, insulin, haemoglobin (Hb), glycosylated haemoglobin (HbA1c), glycogen and the activities of glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were analysed in liver and kidney of the treated and HFD mice. Histopathological examination of liver and pancreases were also carried out. The HFD mice show increased glucose, insulin and HbA1c and decreased Hb and glycogen levels. The elevated activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase and subsequent decline in the activity of glucokinase and glucose-6-phosphate dehydrogenase were seen in HFD mice. Among treatment groups, the mice administered with I3C and DIM, DIM shows decreased glucose, insulin and HbA1c and increased Hb and glycogen content in liver when compared to I3C, which was comparable with the standard drug metformin. The similar result was also obtained in case of carbohydrate metabolism enzymes; treatment with DIM positively regulates carbohydrate metabolic enzymes by inducing the activity of glucokinase and glucose-6-phosphate dehydrogenase and suppressing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase when compared to I3C, which were also supported by our histopathological observations.     

Histochemical studies on the albino rat pancreas in different periods following vagotomy and simultaneous pyloromyotomy.

The effect of complete subphrenic vagotomy and simultaneous pyloromyotomy on the morphological state and the activities of some intracellular enzymes of the albino rat was studied histochemically. Within the first weeks after vagotomy, the pancreatic acini were found to diminish in size, and the beta-cells in the islets of Langerhans became oedematous. In the acini, the activities of succinate dehydrogenase, cytochrome oxidase, AS naphthol acetate esterase, and glucose-6-phosphatase were observed to decline, but the reactions for beta-glucuronidase and aryl sulphatase showed intensifications and polymorphic behaviour both in acinar and in islet cells. The latter also and particularly the beta-cells simultaneously revealed enhanced activities of succinate dehydrogenase, cytochrome oxidase, beta-glucuronidase, and aryl sulphatase, and an entire disappearance of the reaction for glucose-6-phosphatase. The alpha-cells increased their AS naphthol acetate esterase activity. After 5 weeks following vagotomy, morphological and enzymatic changes in the acini and islets were negligible, and after 5 and 9 months no differences were noted between the vagotomized rats and the control animals.     

Thermal stability of microsomal glucose-6-phosphatase

The thermal stability of glucose-6-phosphatase in rat liver microsomes was examined in untreated and cholate-treated microsomes. Activity of the enzyme was measured with both glucose-6-P and mannose-6-P as substrates. Heat treatment did not cause glucose-6-phosphatase activity to decline to zero with a single rate constant in untreated microsomes. Instead, heat treatment produced an enzyme with a small residual activity that was stable. The residual level of activity was not stimulated by addition of detergent. In untreated microsomes the energies of activation for the processes of decay were different for glucose-6-phosphatase and mannose-6-phosphatase activities, suggesting that the rate-limiting steps for the hydrolysis of these compounds were different. Treatment of microsomes with detergent increased the rate constants for the thermal decay of glucose-6-phosphatase by about 150 times, and, in contrast to untreated microsomes, glucose-6-phosphatase and mannose-6-phosphatase decayed to zero with a single rate constant in cholate-treated microsomes. Also, rate constants for thermal inactivation of glucose-6-phosphatase and mannose-6-phosphatase were the same in cholate-treated microsomes. Removal of cholate increased the stability of glucose-6-phosphatase but did not regenerate the form of the enzyme present in untreated microsomes. The data for the stability of glucose-6-phosphatase under different conditions provide evidence that the enzyme can exist in at least five different stable states that are enzymatically active.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight