Glycogen content and glucose-6-phosphatase activity in the tissues of the adult frog and tadpole

Studies have been made on the content of glycogen and the activity of glucose-6-phosphatase in tissues of adult frog Rana temporaria (liver, brain, pia mater, n. ischiadicus, fast and slow muscles) and tadpoles (liver, tail muscles). It was found that the enzymic activity is somewhat higher in the liver of tadpoles than that in the liver of adult frogs, being low in the tail muscles. Pia mater and n. ischiadicus exhibit higher activity of glucose-6-phosphatase as compared to the liver. In tadpoles, glycogen content of the liver increases from the 40th stage of metamorphosis and decreases in the tail muscles to the 42nd-50th stages. Glycogen content in tissues other than liver of adult frogs is higher than in similar tissues of mammals.     

Phosphorylase and gluco-6-phosphatase activity and glycogen level in tissues of vertebrates

The activity of phosphorylase (EC 2.4.1.1), glucose-6-phosphatase (EC 3.1.3.9) and the content of glycogen have been determined in tissues of fish, amphibians, reptiles, mammalians. No differences in the activity of phosphorylase and glucose-6-phosphatase in the liver, myocardium, and brain of animals of the phylogenetic groups under study are found. The activity of glucose-6-phosphatase in the anaerobic muscles of poikilothermal animals is found to be rather high. The share of phosphorylase a in the skeletal muscles and brain as well as the glycogen content in the brain of these animals is essentially higher than that of adult mammalians.     

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.     

Phosphorylase and glucose-6-phosphatase activity in rat tissues during ontogenesis

The activity of phosphorylase and glucose-6-phosphatase was determined in the liver, cerebral hemispheres, musculus gastrocnemius and myocardium of uneven-aged rats. The phosphorylase activity was the highest in rats aged 14-30 days and the glucose-6-phosphatase activity--in rats aged one day. Considerable age changes are observed in the ratio of phosphorylases a and b.     

Effect of bemythyl on carbohydrate metabolism in cirrhotic rat liver

Effect of actoprotector bemitil (2-ethylthiobenzimidazole hydrobromide) on glycogen content and activities of glycogen synthase, glycogen phosphorylase, and glucose-6-phosphatase was studied in cirrhotically altered rat liver. The contents of glycogen and its fraction were determined a cytofluorimetrically (Kudryavtseva et al., 1974). In cirrhosis, the total glycogen content in hepatocytes increases by nearly 3 times, while the amount of a stable fraction of glycogen rises by 7.5 times. Glucose-6-phosphatase activity fell to the level of 25% compare to the norm. Activities of glycogen synthase and glycogen phosphorylase in the cirrhotic liver did not differ from the norm. In cirrhotically altered liver, bemitil produced a decrease in the total glycogen content due to a decrease in glycogen synthase activity in an increase in glucose-6-phosphatase and glycogen phosphorylase activities. The above results suggest a favorable effect of bemitil on cirrhotic liver.     

Studies on the effect of paraquat on glycogen mobilization in liver of common carp (Cyprinus carpio L.)

1. A herbicide, paraquat (1,1'dimethyl-4,4'-bipyridilium-dichloride) was administered to carp in 0.5-10.0 ppm concentrations, respectively, and blood sugar level, glucose-6-phosphatase and glycogen phosphorylase activities of liver were determined. 2. Paraquat treatment caused an increase of blood sugar level and enhanced phosphorylase and glucose-6-phosphatase activities. 3. Paraquat can induce alterations in endoplasmic reticulum that might contribute to the changes in glucose-6-phosphatase activity, resulting in an increase of blood glucose level and/or all the effects can be attributed to a high level of circulating epinephrine produced by paraquat toxicosis.     

Glycogen metabolism in the developing accessory lobes of Lachi in the nerve cord of the chick: metabolic correlations with the avian glycogen body

Glycogen synthase, glycogen phosphorylase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphatase were determined for the first time in the necessary lobes of Lachi from late embryonic chicks. The activities of these enzymes were compared with those found in other glycogen-metabolizing tissues, specifically the glycogen body, liver, and skeletal muscle, obtained from the same embryos. The data show that, as in the glycogen body, the accessory lobes of Lachi lack glucose-6-phosphatase, but contain relatively high activity levels of glycogen synthase I, total and active glycogen phosphorylase, and the dehydrogenases of glucose-6-phosphate and 6-phosphogluconate. The percent of glycogen synthase I activity in the Lachi lobes is from two- to 20-fold greater than observed in the glycogen body, liver, or muscle, whereas the percent of glycogen phosphorylase a activity is comparable to that of the liver, but greater than that in the glycogen body or muscle. The activity of each dehydrogenase of the pentose phosphate cycle in the Lachi lobes is similar to that noted in the glycogen body, but is over two- or fivefold greater than that activity found in muscle or liver. Our data, together with other recent evidence, suggest that the role of glycogen in these functionally enigmatic tissues may be to support the precocious process of myelin synthesis in the developing bird, as well as possibly to provide alternate sources of energy for the avian central nervous system.     

Exposure of cells to a cell number-counting factor decreases the activity of glucose-6-phosphatase to decrease intracellular glucose levels in Dictyostelium discoideum

The development of Dictyostelium discoideum is a model for tissue size regulation, as these cells form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). CF signal transduction involves decreasing intracellular CF glucose levels. A component of CF, countin, has the bioactivity of the entire CF complex, and an 8-min exposure of cells to recombinant countin decreases intracellular glucose levels. To understand how CF regulates intracellular glucose, we examined the effect of CF on enzymes involved in glucose metabolism. Exposure of cells to CF has little effect on amylase or glycogen phosphorylase, enzymes involved in glucose production from glycogen. Glucokinase activity (the first specific step of glycolysis) is inhibited by high levels of CF but is not affected by an 8-min exposure to countin. The second enzyme specific for glycolysis, phosphofructokinase, is not regulated by CF. There are two corresponding enzymes in the gluconeogenesis pathway, fructose-1,6-bisphosphatase and glucose-6-phosphatase. The first is not regulated by CF or countin, whereas glucose-6-phosphatase is regulated by both CF and an 8-min exposure to countin. The countin-induced changes in the Km and Vmax of glucose-6-phosphatase cause a decrease in glucose production that can account for the countin-induced decrease in intracellular glucose levels. It thus appears that part of the CF signal transduction pathway involves inhibiting the activity of glucose-6-phosphatase, decreasing intracellular glucose levels and affecting the levels of other metabolites, to regulate group size.     

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.     

A protein in crude cytosol regulates glucose-6-phosphatase activity in crude microsomes to regulate group size in Dictyostelium

Dictyostelium discoideum form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). The CF signal transduction pathway involves CF-repressing internal glucose levels by increasing the K(m) of glucose-6-phosphatase. Little is known about how this enzyme is regulated. Glucose-6-phosphatase is associated with microsomes in both Dictyostelium and mammals. We find that the activity of glucose-6-phosphatase in crude microsomes from cells with high, normal, or low CF activity had a negative correlation with the amount of CF present in these cell lines. In crude cytosols (supernatants from ultracentrifugation of cell lysates), the glucose-6-phosphatase activity had a positive correlation with CF accumulation. The crude cytosols were further fractionated into a fraction containing molecules greater than 10 kDa (S>10K) and molecules less than 10 KDa (S<10K). S>10K from wild-type cells strongly repressed the activity of glucose-6-phosphatase in wild-type microsomes, whereas S>10K from countin(-) cells (cells with low CF activity) significantly increased the activity of glucose-6-phosphatase in wild-type microsomes by decreasing K(m). The regulatory activities in the wild-type and countin(-) S>10Ks are heat-labile and protease-sensitive, suggesting that they are proteins. S<10K from both wild-type and countin(-) cells did not significantly change glucose-6-phosphatase activity. Together, the data suggest that, as a part of a pathway modulating multicellular group size, CF regulates one or more proteins greater than 10 KDa in crude cytosol that affect microsome-associated glucose-6-phosphatase activity.     

Maximum activities and properties of glucose 6-phosphatase in muscles from vertebrates and invertebrates.

1. The maximum catalytic activities of glucose 6-phosphatase were measured in a large number of muscles from vertebrates and invertebrates. The activities range from less than 0.1 to 8.0 mumol/min per g fresh wt. at 30 degrees C: the highest activity, observed in the flight muscle of the wasp (Vespa vulgaris), is similar to that in rat liver. The hydrolytic activity was shown to be specific towards glucose 6-phosphate. 2. The pH optimum was 6.8 and the Km was approx. 0.6 mM (flight muscle of a moth). 3. Almost all of the glucose 6-phosphatase activity from extracts of the flight muscle of a moth and the pectoral muscle of a pigeon were recovered in the cytosolic fraction (i.e. 150,000 g supernatant). 4. During development of the locust (Schistocerca gregaria), the activity of the phosphatase in the flight muscle increased during the first 3 days after the final moult. 5. The activity of glucose 6-phosphatase from insect and avian muscle was separated from that of non-specific phosphatase on a Bio-Gel P-100 column. 6. For the activities from 63 muscles, there was a strong positive correlation between those of glucose 6-phosphatase and hexokinase, but no correlation between the activities of glucose 6-phosphatase and fructose bisphosphatase. It is suggested that the role of glucose 6-phosphate in muscle is either to produce glucose from glucose 6-phosphate derived from glycogen or to provide the enzymic basis for a substrate ("futile") cycle between glucose and glucose 6-phosphatase in muscle to improve the sensitivity of the mechanism that regulates the rate of glucose phosphorylation.     

A time course study of the effect of repetitive doses of estradiol-17 beta on serum glucose and lipids, liver glycogen and some carbohydrate metabolizing enzymes in liver of male flounder (Platichtys flesus L.)

1. Repeated treatment of male flounder with 5 and 100 microgram doses of estradiol-17 beta increases the level of phospholipid, triglyceride, free fatty acids and total lipid in serum as a function of time and dose, during a period of 17 days. 2. The glucose level in serum is increased and decreased respectively by doses of 5 and 100 micrograms. 3. Five and 100 microgram doses decrease the level of glycogen in liver. 4. Five microgram doses do not affect the activity of the measured enzymes considerably, with the exception of phosphorylase a. 5. One hundred microgram doses increase the activity of glucose-6-phosphate dehydrogenase after 11 days. 6. One hundred microgram doses increase the activity of pyruvate kinase continuously during the experimental period and decrease phosphorylase a and glucose-6-phosphatase activity.     

On the nature of the interaction between 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid and microsomal glucose-6-phosphatase. Evidence for the involvement of sulfhydryl groups of the phosphohydrolase

The effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) on microsomal glucose 6-phosphate hydrolysis has been reinvestigated and characterized in order to elucidate the topological and functional properties of the interacting sites of the glucose-6-phosphatase. The studies were performed on microsomal membranes, partially purified and reconstituted glucose-6-phosphatase preparations and show the following. (a) DIDS inhibits activity of the glucose-6-phosphatase of native microsomes as well as the partially purified glucose-6-phosphatase. (b) Inhibition is reversed when the microsomes and the partially purified phosphohydrolase, incorporated into asolectin liposomes, are modified with Triton X-114. (c) Treatment of native microsomes with DIDS and the following purification of glucose-6-phosphatase from these labeled membranes leads to an enzyme preparation which is labeled and inhibited by DIDS. (d) Preincubation of native microsomes or partially purified glucose-6-phosphatase with a 3000-fold excess of glucose 6-phosphate cannot prevent the DIDS-induced inhibition. (e) Inhibition of glucose-6-phosphatase by DIDS is completely prevented when reactive sulfhydryl groups of the phosphohydrolase are blocked by p-mecuribenzoate. (f) Reactivation of enzyme activity is obtained when DIDS-labeled microsomes are incubated with 2-mercaptoethanol or dithiothreitol. Therefore, we conclude that inhibition of microsomal glucose 6-phosphate hydrolysis by DIDS cannot result from binding of this agent to a putative glucose-6-phosphate-carrier protein. Our results rather suggest that inhibition is caused by chemical modification of sulfhydryl groups of the integral phosphohydrolase accessible to DIDS attack itself. An easy interpretation of these results can be obtained on the basis of a modified conformational model representing the glucose-6-phosphatase as an integral channel-protein located within the hydrophobic interior of the microsomal membrane [Schulze et al. (1986) J. Biol. Chem. 261, 16,571-16,578].     

Glucose-6-phosphatase as a marker for tumors of liver and kidney origin

Glucose-6-phosphatase is primarily a liver and kidney enzyme. This enzyme was studied in various tumors, however, glucose-6-phosphatase activity was found only in tumors of liver, kidney, or adrenal origin. Glucose-6-phosphatase activity was useful in identifying the tissue origin of extrarenal Wilms'. Metastatic tumors within the liver or kidney that originated from other tissues did not have glucose-6-phosphatase activity. Therefore, it is suggested that glucose-6-phosphatase can be used as a specific enzyme marker for tumors of liver and kidney origin.     

The mechanism of histone activation of the hepatic microsomal glucose-6-phosphatase system: a novel method to assay glucose-6-phosphatase activity

The mechanism of activation of hepatic microsomal glucose-6-phosphatase (EC 3.1.3.9) by histone 2A has been investigated in both intact and disrupted microsomes. Histone 2A increased the Vmax and decreased the Km of glucose-6-phosphatase in intact microsomes but had no effect on glucose-6-phosphatase activity in disrupted microsomes. Histone 2A was shown to activate glucose-6-phosphatase in intact microsomes by disrupting the membrane vesicles and thereby allowing the direct measurement of the activity of the latent glucose-6-phosphatase enzyme. The study demonstrated that disrupting microsomes with histone 2A is an excellent method for directly assaying glucose-6-phosphatase activity as it poses none of the problems encountered with all of the previously used methods.     

Glycogen-enzymes containing bodies in type 2 fibres of tenotomized muscles in the rat

Inclusion bodies containing glycogen-enzymes were found in 30 to 60% of type 2 fibres of tenotomized calf muscles (m. gastrocnemius, m. soleus, m. plantaris) in rats, using histochemical reactions. The bodies appeared within 1 week after the tenotomy and were localized both in the central and the subsarcolemmal regions and rarely extruded into the extracellular space. These aggregates are 3 to 15 microns in length and 2 to 11 microns in diameter. In addition to glycogen, these bodies also contained various enzymes of the glycogen metabolism such as phosphorylase, a branching enzyme, and glucose-6-phosphatase, but showed no NADH-reductase, lactate dehydrogenase, or myofibrillar ATP-ase activity. The results indicate that glycogen-enzymes containing bodies are a degenerative phenomenon, which occurs only in type 2 fibres of the tenotomized muscles.     

Effect of dietary fiber from banana (Musa paradisiaca) on metabolism of carbohydrates in rats fed cholesterol free diet

Effect of feeding isolated dietary fiber from M. paradisiaca on the metabolism of carbohydrates in the liver has been studied. Fiber fed rats showed significantly lower levels of fasting blood glucose and higher concentration of liver glycogen. Activity of glycogen phosphorylase, glucose-1-phosphate, uridyl transferase and glycogen synthase was significantly higher while phosphoglucomutase activity showed lower activity. Activity of some glycolytic enzymes, viz. hexokinase and pyruvic kinase was lower. Glucose-6-phosphatase showed higher activity while fructose 1-6 diphosphatase activity was not affected. Glucose-6-phosphate dehydrogenase on the other hand showed higher activity. The changes in these enzyme activities have been attributed due to the effect of higher concentration of bile acids produced in the liver as a result of feeding fiber. Evidence for this has been obtained by studying the in vitro effect of cholic acid and chenodeoxy cholic acid.     

Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes

The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase.     

Effect of thyroxine on the hepatic glycogen and glucose-6-phosphatase activity of developing rats

The influence of exogenous thyroxine was studied on the hepatic glycogen content and glucose-6-phosphatase activity of rats of different age groups. The glycogen content and glucose-6-phosphatase activity were found to be decreased in the livers of 5, 15, 30 and 60-day-old rats after thyroxine treatment. In normal rats of 5, 15, 30 and 60-day-old, a gradual rise in both the hepatic glycogen content and glucose-6-phosphatase activity was noted as the age advanced from immature to adult.