Changes in the activity of enzymes,participating in glycogen metabolism of alloxan diabetic rats

Summary Alloxan diabetes induced in white rats by intraperitoneal injection of Aloxan-monohydrate (15 mg/100 g body weight) was used to study changes in the glycogen phosphorylase a and b, phosphoprotein phosphatases and hexokinase activities under insulin deficiency conditions. Among the enzymes studied, an increase in muscle phosphorylase a activity as well as the a/b ratio have been obtained. In diabetic muscle phosphoprotein phosphatases and hexokinase activities were diminished.AMP increased the liver glycogen phosphorylase activity twice in diabetic rats whereas in normal animals the enzyme was less sensitive to this effector. The changes in liver hexokinase activity at diabetes were not connected and correlated with the altered phosphorylase and protein phosphatase activities.The logical chain of probable molecular events taking place in muscle glycogen metabolism under the conditions of insulin deficiency is offered.     

Effect of experimental diabetes on the activity of hexokinase isoenzymes in tissues of the rat

The effect of experimental diabetes on the activity of hexokinase isoenzymes was studied in a wide range of tissues of the rat. In the tissues known to require insulin for glucose phosphorylation, the activity of hexokinase was markedly decreased; the fall being mainly in the Type IV (Glucokinase) in liver and Type II in other tissues, these tissues also exhibit glucose underutilization in diabetes. In the tissues which are commonly known not to require insulin, the activity of Type I hexokinase was significantly increased, these tissues exhibit aspects of glucose overutilization in diabetes in particular kidney and lens. These changes are discussed in relation to Spiro's hypothesis of glucose under and overutilization in tissues in diabetes.     

Hexokinase isoenzymes in diabetes

Hexokinase is present in the tissues in four isoenzymic forms. Cerebral tissue contains predominantly Type I hexokinase which is believed to be insulin-insensitive. In cerebral tissue about 60 to 70% of the hexokinase is bound to the particulate fraction. The changes in the distribution of hexokinase Type I and Type II together with the bound and free hexokinase have been studied in control, diabetic and diabetic animals treated with insulin. The results indicate that the presence of insulin is essential for the normal binding of the hexokinase to the particulate fraction. In heart tissue, Type II hexokinase bound to the pellet shows a significant decrease in diabetes, which is reversed on insulin administration.     

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.     

EFFECT OF FATTY ACIDS ON RAT BRAIN PARTICULATE HEXOKINASE

Abstract— The effect of free fatty acids on rat brain particulate hexokinase was studied in vitro. Hexokinase bound with brain mitochondrial fraction was found to be sensitive to the action of free fatty acids, resulting in the solubilization of at least part of bound enzyme activity into the supernatant. The decrease of total enzyme activity observed at the highest free fatty acid concentration was probably due to the inhibition of hexokinase. The physiological consequence of hexokinase solubilization by low concentrations of free fatty acids, similar to that observed in vivo , is discussed in relation to activity changes of soluble and particulate enzyme forms demonstrated previously under hypoxic conditions.     

Tubulin and microtubule are potential targets for brain hexokinase binding.

The metabolite-modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non-mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme-linked immunosorbent assay using specific anti-brain hexokinase serum (IC(50)=4.0+/-1.4 microM). This value virtually was not affected by specific ligands such as ATP or glucose 6-phosphate. Microtubule-bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell-free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.     

Effect of inorganic phosphate on the reverse reaction of bovine brain hexokinase

Kinetic studies were used to investigate the mode of brain hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) regulation by glucose 6-phosphate (glucose-6-P), ADP, and inorganic phosphate (Pi). A model for regulation of brain hexokinase by glucose-6-P and Pi had been proposed from initial-rate studies and binding experiments [Ellison, W. R., Lueck, J. D., & Fromm, H. J. (1975) J. Biol. Chem. 250, 1864-1871]. The results of the present investigation demonstrate that Pi is an activator of the brain hexokinase reaction when the reaction is studied in the nonphysiological direction. Evidence is presented which indicates that the back-reaction substrates and Pi can bind the enzyme simultaneously, and the suggestion is made that Pi binds to an allosteric site on the enzyme. These findings are in marked contrast to results obtained in the absence of ADP which convincingly demonstrate that glucose-6-P and Pi are mutually exclusive binding ligands for brain hexokinase. The kinetic data can be reconciled with the model for hexokinase regulation within the context of the well-established kinetic mechanism for brain hexokinase.     

Changes in the activity and isoenzymic spectrum of skeletal muscle and brain hexokinase under organism adaptation to intensive physical exercise

The activity of hexokinase and its isoenzymic spectrum were determined in skeletal muscles of different metabolic types and in brain of trained and untrained albino rats. It is shown that adaptation to intensive muscular activity accompanied by metabolic acidosis induces some changes in the hexokinase system, which consist in the increase of the total hexokinase activity not only under the optimal conditions but also with more acidic pH values of the medium and in the rise of the quantity of the muscular type isoenzyme which is more stable to the pH action.     

Glucose catabolism in brain. Intracellular localization of hexokinase

A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane.     

Bovine hexokinase type I: Full-length cDNA sequence and characterisation of the recombinant enzyme

This study reports the revised and full-length cDNA sequence of bovine hexokinase type I obtained from bovine brain. Since dissimilarities have been observed between the published bovine hexokinase type I coding sequence (GenBank accession no. M65140) (Genomics 11: 1014-1024, 1991) and an analysed portion of bovine hexokinase type I gene, the entire open reading frame was re-sequenced and the ends of cDNA isolated by rapid amplification of cDNA ends. The coding sequences, when compared with the published bovine hexokinase type I, contained a large number of mismatches that lead to changes in the resulting amino acid sequence. The revisions result in a hexokinase type I cDNA of 3619 bp that encodes a protein of 917 amino acids highly homologous to human hexokinase type I. The expression of the recombinant full-length enzyme demonstrated that it was a catalytically active hexokinase. When characterised for its kinetic and regulatory properties, it displayed the same affinity for glucose and MgATP as the human hexokinase type I and was inhibited by glucose 6-phosphate competitively versus MgATP. The production of the N- and C-terminal recombinant halves of the enzyme followed by comparison with the full-length hexokinase indicated that the catalytic activity is located in the C-terminal domain. (Mol Cell Biochem 268: 9–18, 2005)     

Expression of human brain hexokinase in Escherichia coli: purification and characterization of the expressed enzyme

Human brain hexokinase (hexokinase I) was produced in Escherichia coli from a synthetic gene under control of the bacteriophage T7 promoter. The expressed coding region derives from a human cDNA clone thought to specify hexokinase I based on amino acid sequence identity between the predicted translation product and hexokinase I from rat brain. The open reading frame from this cDNA was fused to the promoter and 5' flanking region of T7 gene 10, and expressed in E. coli by induction of T7 RNA polymerase. Induced cells contained a hexokinase activity and an abundant protein of apparent molecular weight 100,000, neither of which was present in cells lacking T7 RNA polymerase. Enzyme purified to near homogeneity consisted of a 100,000 Da protein, the size predicted from the nucleotide sequence of the expressed cDNA. The purified enzyme had Michaelis constants of 32 microM and 0.3 mM for glucose and ATP, respectively, and bound to rat liver mitochondria in the presence of MgCl2. Enzymatic activity was inhibited by glucose-6-P and this inhibition was relieved by inorganic phosphate. Deinhibition by phosphate is a property specific to brain hexokinase.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

Functioning of mitochondria-bound hexokinase in rat brain in accordance with generation of ATP inside the organelle.

The function of mitochondria-bound hexokinase, the enzymatic form peculiar to the brain, in utilization of ATP generated inside the organelles, was examined by incubating rat brain mitochondrial fraction with [14C]glucose under various conditions. Addition of succinate and ADP to the incubation medium increased glucose 6-phosphate formation by the mitochondrial hexokinase and caused a smaller increase in ATP concentration in the mitochondria. The glucose phosphorylation was markedly inhibited by the addition of dinitrophenol, potassium cyanide, and oligomycin, and the ATP concentration was decreased. On the other hand, addition of atractyloside suppressed the glucose phosphorylation without affecting the mitochondrial hexokinase activity, whereas addition of antiserum against the mitochondrial hexokinase inhibited both glucose 6-phosphate formation and hexokinase activity. A part of both the glucose phosphorylation and hexokinase activities, however, remained even in the presence of the maximum dose of the anti-hexokinase serum and atractyloside. These results indicate the active utilization of intrinsically generated ATP by the mitochondria-bound hexokinase, a part of which may be located away from the surface of the mitochondrial membrane.     

Competitive Inhibition of Rat Brain Hexokinase by 2-Deoxyglucose, Glucosamine, and Metrizamide

Abstract: The effects of metrizamide on the kinetics of rat brain hexokinase were compared in vitro with those of 2-deoxyglucose and glucosamine. Although metrizamide, 2-deoxyglucose, and glucosamine are known to be competitive inhibitors of approximately equal potency for glucose of yeast hexokinase ( K ₁ approximately 0.7 m m for all three), metrizamide is a much weaker competitive inhibitor ( K _i about 20 m m ) of rat brain hexokinase than either 2-deoxyglucose or glucosamine ( K _i about 0.3 m m for both). This indicates a greater active site specificity of rat brain hexokinase than of yeast hexokinase. Rat brain hexokinase activity is enhanced approximately threefold in the presence of 0.05, 0.2, and 0.8 mg/ml bovine serum albumin, while yeast hexokinase is only enhanced by 50% under these conditions. Despite the high K _i value for metrizamide, interference with glucose metabolism may occur whenever metrizamide is present in much higher concentrations than glucose. Myelography in humans is one such situation.     

Intracellular distribution of hexokinase in rabbit brain

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed, that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.     

Ligand-induced conformations of rat brain hexokinase: effects of glucose-6-phosphate and inorganic phosphate

Binding of glucose-6-P induces conformational change in rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) as indicated by decreased susceptibility to digestion by chymotrypsin and an increased sedimentation coefficient on sucrose density gradients. These effects are competitively reversed by P_i, as are solubilization (of the mitochondrial form of hexokinase) and inhibition by glucose-6-P. Thus, the observed conformational changes are likely to be directly related to the effect of these ligands on catalytic activity and the interaction of the hexokinase with the mitochondrial membrane.Both glucose-6-P and P_i stabilize the enzyme against heat inactivation; this effect, as well as the effect of glucose-6-P on inactivation by chymotrypsin, have been used to estimate the dissociation constants for the complexes of hexokinase with glucose-6-P and P_i; the values are 7–8 μm, and 0.25 mm, respectively.These observations are consistent with a model in which brain hexokinase may exist in two distinct conformations, rapidly and reversibly interconvertible. The effect of glucose-6-P and P_i are explained by highly preferential binding to one or the other of these conformations.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

Hexokinase Levels in Discrete Regions of Rat Brain: Evaluation by Quantitative Immuno fluoresence Using Interactive Laser Cytometry and Comparison with Basal Rates of Glucose Utilization

Relative levels of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been determined in 16 discrete regions of adult rat brain by a quantitative immunofluorescence method. The distribution of immunofluorescence in brain sections was determined by interactive laser cytometry and related to hexokinase content by comparison with standard sections containing known amounts of the enzyme. In many of these regions, referred to here as group I regions, hexokinase content was correlated with previously reported basal rates of glucose utilization. However, several regions (group II regions) in which hexokinase content exceeded that expected from basal rates of glucose utilization were also detected. Compared with the corresponding regions from albino rat brain, higher hexokinase levels were found in the dorsal and ventral lateral geniculate (group I regions) of pigmented Norway rats, a result reflecting previously reported increased glucose utilization by these regions in pigmented rats. There was no difference in hexokinase levels in the superior colliculus, a group II structure, from albino and pigmented rats, a finding implying that a reported increase in rate of glucose utilization in the superior colliculus of pigmented rats is effected without an increase in hexokinase content. It is suggested that group II regions may be adapted to sustain increases in rates of glucose utilization that are, relative to basal rates, considerably greater than those experienced by group I regions.     

Mitochondrial hexokinase from small-intestinal mucosa and brain

1. The submitochondrial localization of hexokinase activity in preparations of mitochondria from the small intestine of the guinea pig was studied by conventional methods. 2. Hexokinase activity in this tissue was predominantly associated with the outer mitochondrial membrane. 3. The inactivation of mitochondrial enzymes by trypsin in iso-osmotic and hypo-osmotic conditions was also used to determine the submitochondrial localization of hexokinase activity. 4. Hexokinase activity was found to be on the outside of the outer mitochondrial membrane. 5. It was shown that both type I and type II hexokinase activities are bound to the outside of the outer mitochondrial membrane. The types are present in the same ratio as that in which they occur in the cytosol of the cell. 6. Mitochondrial hexokinase from the small intestine did not show the latency phenomenon demonstrated by mitochondrial hexokinase from brain when subjected to a variety of treatments. However, hexokinase activity was solubilized from preparations of mitochondria from the small intestine by the same treatments as for mitochondrial hexokinase from brain. 7. The submitochondrial distribution of hexokinase activity in mitochondrial preparations from rat brain was determined by the trypsin inactivation method. 8. Hexokinase activity in preparations of mitochondria from rat brain was found on the outside of the outer membrane, between the mitochondrial membranes, and within the inner mitochondrial membrane. 9. Hexokinase from rat brain showed latency properties irrespective of its submitochondrial location.     

Daily changes in parameters of energy metabolism in brain of rainbow trout: dependence on feeding

We assessed the daily patterns of parameters involved in energy metabolism in plasma and brain of rainbow trout. Where daily rhythms were found, we analyzed the potential influence of feeding. Immature rainbow trout were randomly distributed in 3 groups: fish fed for 7 days, fish fasted for 7 days, and fish fasted for 7 days and refed for 4 days. On sampling day, fish of fed and refed groups were fed at 11.00 h, and all fish were sampled from each treatment group using the following time schedule: 14.00, 18.00, 21.00, 00.00, 04.00, 07.00, 10.00 and 14.00 h. The results obtained from metabolic parameters assessed in plasma and brain can be grouped into three different categories, such as (i) those displaying no 24 h changes in fed fish such as plasma lactate, protein or acetoacetate levels, as well as brain amino acid and protein levels, and lowKm(glucose) hexokinase, and aspartate aminotransferase activities, (ii) those displaying 24 h changes that were apparently dependent on feeding since they disappeared in fasted fish such as the case of plasma cortisol, glucose and triglyceride levels, as well as brain glycogen, glucose, and lactate levels, and pyruvate kinase and hexokinase IV activities, and (iii) those parameters displaying 24 h changes apparently not dependent on feeding such as plasma amino acids, brain acetoacetate levels as well as several enzyme activities measured in brain such as glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, glutamate dehydrogenase, and lactate dehydrogenase-oxidase. In general, 24 h changes dependent on feeding indicate an increased use of glucose in brain several hours post-feeding whereas those changes not dependent on feeding were characterized by reduced levels/activity at the night period suggesting a metabolic depression in brain during darkness.