Change in the activity of the key gluconeogenesis enzymes in the rat liver and kidneys during the action of subextreme and extreme factors on the body]

The activity of glucogenesis key enzymes (phosphoenolpyruvate carboxinase, fructoso-1,6-siphosphatase, glucoso-6-phosphatase) of the rat liver and kidneys was studied simultaneously under the effect of extreme and subextreme factors on the organism. The low initial phosphoenolpyruvate carboxikinase activity in the liver and its high inductivity under extreme conditions suggest a role of this enzyme as limiting link in glyconeogenesis. The activity of phosphoenolpyruvate carboxinase in the kidneys is comparable to that of fructoso-1,6-diphosphatase; it is considerably higher than the activity of glucoso-6-phosphatase. The phosphoenolpyruvate carboxinase activity in the kidneys is 5--6 times higher than in the liver. The activity of phosphoenolpyruvate carboxinase and glucoso-6-phosphatase is increased under the effect of extreme factors, and that of fructoso-1,6-diphosphatase remains unchanged. The lack of clear synchronous changes in the activity of glucogenesis key enzymes in the liver and kidneys indicates that the cells of these organs do not provide the united operon for phosphoenolpyruvate carboxinase, fructoso-1,6-diphosphatase and glucoso-6-phosphatase with common regulation mechanism.     

Early stages of hepatic carcinogenesis after vagotomy

Initial stages of hepatocarcinogenesis have been studied in nonoperated and vagotomized animals. As a carcinogenic substance diethylnitrosamine (DENA) has been used. In order to estimate manifestation of the changes, the histochemical method for revealing glucoso-6-phosphatase activity, adenosine triphosphatase and gamma-glutamyltranspeptidase in the liver has been applied. The disturbance of vagus innervation is stated to delay the course of early stages of hepatocarcinogenesis, induced with DENA.     

Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.     

Lamellar structures in rat ova and their chemical composition.

The paper represents a study of the chemical composition of lamellar structures in rat ova during cleavage based on the morphology of their submicroscopic structure after the action of various fixatives and various methods of contrasting ultrathin sections and on the employment of cytochemical methods. Reactions to RNA, polysaccharides, acid and alkaline phosphatase, non-specific esterase, glucoso-6-phosphatase and succinate dehydrogenase were tested on a submicroscopic level; lipids were tested on a light-microscopic level. The results have shown that the lamellae are composed of proteins. No RNA, polysaccharides, lipids or any of the investigated enzymes were detected in lamellar structures. Lamellar structures, therefore, are considered to be storage material chiefly used in the second half of the cleavage for developmental processes in the rat ovum.     

Toxicologic evaluation of nitrates entering the body with plant products

An acute toxicological experiment on rats has been performed. During 30 days the animals got nitrates with water and vegetable food. The activity of glucoso-6-phosphatase, malate dehydrogenase, beta-galactosidase, the content of N-acetylneuraminic acid was determined in liver, kidney and blood serum; the level of methemoglobin in blood was estimated. It was found that the changes of studied indexes are less expressed during nitrate introduction with food than with water; on the level of minimum concentrations the changes in non-specific symptom-complex were more expressed. The maximum innocuous intake of nitrates with vegetables is 500 mg in 24 hours taking into consideration the average weight of an individual (50 kg).     

Properties of Some Enzymes of Gluconeogenesis in Platyhelminths

Activities and properties of some enzymes of gluconeogenesis, the malic enzyme, phosphoenolpyruvate carboxykinase, glucoso-6-phosphatase, alanine aminotransferase, and aspartate aminotransferase, were studied in turbellarians Phagocata sibirica and trematodes Eurytrema pancreaticum. It was shown that adaptation of trematodes to the parasitic mode of life affected essentially the biosynthetic processes and led to formation of different metabolic pathways.     

Comparison of the Inactivation of Microsomal Glucose-6-Phosphatase by in Situ Lipid Peroxidation-Derived 4-Hydroxynonenal and Exogenous 4-Hydroxynonenal

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied.

2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes.

3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E.

4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5μM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150μM or 300μM was needed with a preincubation time of 30 and 60 min, respectively.

5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynbnenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.     

Localization of oxidized nocotinamide--adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope.

NAD+ glycohydrolase activity located in the nuclear envelope was maximally solubilized by treatment with 0.1--0.2% Triton X-100. The residual activity largely represents the chromatin-associated NAD+ glycohydrolase. Under these conditions the phospholipids were extensively solubilized (over 90%) while leaving the nuclei physically stable, although the nuclear membranes were removed, as shown by electron microscopy. After Triton X-100 treatment, deoxyribonuclease I did not significantly affect the residual NAD+ glycohydrolase activity, although the DNA was completely broken down. This enzyme activity can be released from the nuclear pellet by incubation with phospholipase C. For comparative studies, the glucose 6-phosphatase activity, known to be present in the nuclear envelope, was investigated. Treatment with 0.01% Triton X-100 released 10--20% of the phospholipids, but without solubilizing either glucose 6-phosphatase or NAD+ glycohydrolase. Higher Triton X-100 concentrations (0.1--1.0%) inhibited glucose 6-phosphatase, but not NAD+ glycohydrolase activity. NAD+ glycohydrolase is apparently present in a latent form in the nuclear envelope. Glucose 6-phosphatase, However, shows no such latency.     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

Cytochemical localization of glucose-6-phosphatase activity in cerebral endothelial cells

Glucose-6-phosphatase (G6Pase) activity, with glucose-6-phosphate and mannose-6-phosphate as substrates, was examined by cytochemistry in capillary and arteriole endothelial cells of the mouse brain. G6Pase activity was observed ultrastructurally in the lumen of the nuclear envelope and endoplasmic reticulum (ER) of these cells. The reactive ER and nuclear membrane appeared to be in continuity. Nucleoside diphosphatase activity, also a marker for the ER in some cell types, was not seen within the ER of the cerebral microvasculature. The ER of arterioles and capillaries did not bind lead nonspecifically when incubated in a substrate-free medium. Speculation is raised concerning the involvement of G6Pase in glucose metabolism of cerebral endothelial cells and in making blood-borne glucose available to brain parenchyma.     

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.     

Molecular pathology of glucose-6-phosphatase

It was known in the 1950s that hepatic microsomal glucose-6-phosphatase plays an important role in the regulation of blood glucose levels. All attempts since then to purify a single polypeptide with glucose-6-phosphatase activity have failed. Until recently, virtually nothing was known about the molecular basis of glucose-6-phosphatase or its regulation. Recent studies of the type 1 glycogen storage diseases, which are human genetic deficiencies that result in impaired glucose-6-phosphatase activity, have greatly increased our understanding of glucose-6-phosphatase. Glucose-6-phosphatase has been shown to comprise at least five different polypeptides, the catalytic subunit of glucose-6-phosphatase with its active site situated in the lumen of the endoplasmic reticulum; a regulatory Ca2+ binding protein; and three transport proteins, T1, T2, and T3, which respectively allow glucose-6-phosphate, phosphate, and glucose to cross the endoplasmic reticulum membrane. Purified glucose-6-phosphatase proteins, immunospecific antibodies, and improved assay techniques have led to the diagnosis of a variety of new type 1 glycogen storage diseases. Recent studies of the type 1 glycogen storage diseases have led to a much greater understanding of the role and regulation of each of the glucose-6-phosphatase proteins.     

Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue

Nuclei, nuclear membranes and rough endoplasmic reticulum (rER) were isolated from onion root tips and stems. Structural preservation and purity of the fractions was determined by electron microscopic and biochemical methods. Gross compositional data (protein, phospholipid, nonpolar lipids, sterols, RNA, DNA), phospholipid and fatty acid patterns, enzyme activities (ATPases, ADPase, IDPase, glucose-6-phosphatase, 5'-nucleotidase, acid phosphatase, and NADH- and NADPH-cytochrome C reductases), and cytochrome contents were determined. A stable, high salt-resistant attachment of some DNA with the nuclear membrane was observed as well as the association of some RNA with high salt-treated nuclear and rER membranes. The phospholipid pattern was identical for both nuclear and rER membranes and showed a predominance of lecithin (about 60%) and phosphatidyl ethanolamine (20-24%). Special care was necessary to minimize lipid degradation by phospholipases during isolations. Nonpolar lipids, mostly sterols and triglycerides, accounted for 35-45% of the membrane lipids. Sterol contents were relatively high in both membrane fractions (molar ratios of sterols to phospholipids ranged from 0.12 to 0.43). Sitosterol accounted for about 80% of the total sterols. Palmitic, oleic, and linoleic acids were the most prevalent acids in membrane-bound lipids as well as in storage lipids and occurred in similar proportions in phospholipids, triglycerides and free fatty acids of the membrane. About 80% of the fatty acids in membrane phospholipids and triglycerides were unsaturated. A cytochrome of the b5 type was characterized in these membranes, but P-450-like cytochromes could not be detected. Both NADH and NADPH-cytochrome c reductases were found in nuclear and rER membranes and appeared to be enriched in rER membranes. Among the phosphatases, Mg2+-ATPase and, to lesser extents, ADPase, IDPase and acid phosphatase activities occurred in the fractions, but significant amounts of monovalent ion-stimulated ATPase, 5'-nucleotidase and glucose-6-phosphatase activities did not. The results obtained emphasize that the close biochemical similarities noted between rER and nuclear membranes of animal cells extend to these fractions from plant cells.     

Evidence for protein phosphorylation as a regulatory mechanism for hepatic microsomal glucose 6 phosphatase

Incubation of microsomal vesicles with ATP and protein kinase results in a fivefold increased glucose-6-phosphatase activity. Evidence is presented that this effect is mediated via a moiety of the outer membrane surface. Evidence is also presented for the presence of an endogenous, peripheral membrane protein also capable of activating glucose-6-phosphatase in an ATP dependent reaction. It is suggested that the glucose-6-phosphate transmembrane carrier system may be the target of phosphorylation.     

Content of ATP and glucoso-6-phosphate in the dogs' bone marrow cells after cryopreservation

The content of ATP and glucoso-6-phosphate in the dogs' bone marrow cells before and after cryopreservation in liquid nitrogen have been studied. It has been found that ATP and glucoso-6-phospate levels in the dog bone marrow cells after cryopreservation in liquid nitrogen without cryoprotectors was very low. Solutions of DMSO and PEO-400 were used as cryoprotectors. The DMSO was more effective than PEO-400, because no metabolic changes were observed in incubated and freezed-thawed cells there.     

Protease inhibitors but not proteases inhibit the glucose-6-phosphatase of native rat liver microsomes.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.     

Nicotinamide adenine dinucleotide splitting enzyme: a plasma membrane protein of murine macrophages.

The subcellular distribution of NADase in splenic and peritoneal macrophages of the mouse has been studied. Conventional procedures for fractionation and isolation of subcellular components demonstrated that the NADase of murine macrophages was localized in the microsomal fraction. By using the diazonium salt of sulfanilic acid, a nonpenetrating reagent known to inactivate ecto-enzymes in intact cells, purified plasma membrane preparations, and marker enzymes, 5′-nucleotidase for plasma membrane and glucose 6-phosphatase for the microsomal fraction, we have shown that: (i) NADase of murine macrophages is a plasma membrane ecto-enzyme and (ii) the microsomal fraction is a mixture of endoplasmic reticulum and plasma membrane elements. At 5 × 10^?4 M concentration, the diazonium salt of sulfanilic acid drastically decreased NADase in intact splenic and peritoneal macrophages of the mouse. 5′-Nucleotidase was similarly inhibited by this reagent, whereas the activity of glucose 6-phosphatase remained unaffected. There was a good recovery of NADase of high specific activity in plasma membrane preparations that were characterized by high 5′-nucleotidase and low glucose 6-phosphatase activity.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.     

O-glycosylation of FoxO1 increases its transcriptional activity towards the glucose 6-phosphatase gene

Mono-O-glycosylations post-translationally regulate the activity of nucleocytoplasmic proteins. We showed that glucosamine and an inhibitor of deglycosylation (PUGNAc) induced O-glycosylation of FoxO1, resulting in increased expression of a glucose-6-phosphatase reporter gene. This effect was independent of FoxO1 re-localisation, since it was also observed with constitutively nuclear FoxO1-AAA mutant. Moreover, in HepG2 cells, glucosamine and PUGNAc have a synergistic effect on the glucose-6-phosphatase reporter gene, and this effect was inhibited by FoxO1 siRNAs. Since glucose-6-phosphatase plays a key role in hepatic glucose production, our observation may be of importance with regard to glucotoxicity associated with chronic hyperglycaemia in diabetes.     

Glucose-6-phosphatase inactivation and peroxidation of phosphatidylcholine in microsomal membranes.

Peroxidation induced by ascorbate on phospholipids of isolated rat liver microsomes were accompanied by losses in glucose-6-phosphatase activity (EC 3.1.3.9.). The existence of marked differences in the degradation rate for each phospholipid suggests a relationship between the alteration of phosphatidylcholine containing one saturated and one unsaturated fatty acid and the decrease in activity of glucose-6-phosphatase; the inactivation of this enzyme was unrelated to the alteration of other phospholipids. These results support the idea that glucose-6-phosphatase and molecules of phosphatidylcholine having one saturated and one unsaturated fatty acid are in close apposition within the microsomal membrane.