Enhanced hexose-6-phosphate dehydrogenase expression in adipose tissue may contribute to diet-induced visceral adiposity.

BACKGROUND: Visceral fat accumulation increases the risk of developing type 2 diabetes and metabolic syndrome, and is associated with excessive glucocorticoids (GCs). Fat depot-specific GC action is tightly controlled by 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) coupled with the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Mice with inactivation or activation of H6PDH genes show altered adipose 11ß-HSD1 activity and lipid storage. We hypothesized that adipose tissue H6PDH activation is a leading cause for the visceral obesity and insulin resistance. Here, we explored the role and possible mechanism of enhancing adipose H6PDH in the development of visceral adiposity in vivo. METHODS: We investigated the potential contribution of adipose H6PDH activation to the accumulation of visceral fat by characterization of visceral fat obese gene expression profiles, fat distribution, adipocyte metabolic molecules, and abdominal fat-specific GC signaling mechanisms underlying the diet-induced visceral obesity and insulin resistance in H6PDH transgenic mice fed a standard of high-fat diet (HFD). RESULTS: Transgenic H6PDH mice display increased abdominal fat accumulation, which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPα and PPARγ mRNA levels within adipose tissue. Transgenic H6PDH mice fed a high-fat diet (HFD) gained more abdominal visceral fat mass coupled with activation of GSK3ß and induction of XBP1/IRE1α, but reduced pThr308 Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls. HFD-fed H6PDH transgenic mice also had impaired insulin sensitivity and exhibited elevated levels of intra-adipose GCs with induction of adipose 11ß-HSD1. CONCLUSION: These data provide the first in vivo mechanistic evidence for the adverse metabolic effects of adipose H6PDH activation on visceral fat distribution, fat metabolism, and adipocyte function through enhancing 11ß-HSD1-driven intra-adipose GC action.

Direct protein-protein interaction of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen.

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11beta-HSD1 but not with the related enzymes 11beta-HSD2 and 17beta-HSD2, suggesting a specific interaction between H6PDH and 11beta-HSD1. The use of the 11beta-HSD1/11beta-HSD2 chimera indicates that the N-terminal 39 residues of 11beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins.

[Isolation of wood-decaying fungi and evaluation of their enzymatic activity (Quindío, Colombia)]. / Aislamiento y evaluación de la actividad enzimática de hongos descomponedores de madera (Quindío, Colombia).

White rot fungi (Ascomycota and Basidiomycota) were collected on fallen trunks with different decay stages, in a subandean forest (La Montaña del Ocaso nature reserve), and it was evaluated their ligninolitic activity. They were cultured on malt extract agar. Then it was performed semiquantitative tests for laccase and cellobiose dehydrogenase (CDH) activity using ABTS and DCPIP as enzymatic inducers. Based on the results of these tests, the fungi with higher activities from trunks with different decay stages were selected: Cookeina sulcipes (for stage 1), a fungus from the family Corticiaceae (for stage 2), Xylaria polymorpha (for stage 3) and Earliella sp. (for stage 4). A fermentation was performed at 28 degrees C, during 11 days, in a rotatory shaker at 150 rpm. Biomass, glucose, proteins and enzyme activities measurements were performed daily. The fungi that were in the trunks with decay states from 1 to 3, showed higher laccase activity as the state of decay increased. A higher DCH activity was also associated with a higher. Also, there was a positive relationship between both enzymes' activities. Erliella was the fungus which presented the highest biomass production (1140,19 g/l), laccase activity (157 UL(-1)) and CDH activity (43,50 UL(-1)). This work is the first report of laccase and CDH activity for Cookeina sulcipes and Earliella sp. Moreover, it gives basis for the use of these native fungi in biotechnological applications and the acknowledgment of their function in the wood decay process in native forest.

Identification of a novel lactose oxidase in Myrmecridium flexuosum NUK-21.

Lactobionic acid (O-ß-galactosyl-(1-4)-gluconic acid) (LBA) is a high-value lactose derivative, produced via oxidation of the reducing terminal of lactose. LBA can be produced by fermentation using certain microorganisms, although subsequent purification is challenging. Therefore, we have attempted to identify an enzyme for possible use in LBA production. Here, we purified a novel lactose oxidase (LOD) to homogeneity from a wheat bran culture of a soil-isolated fungal strain, Myrmecridium flexuosum NUK-21. Maximal activity was observed on the wheat bran solid culture after 3 days of NUK-21 growth, following release from cells at 0.66 unit·mL -1 culture filtrate. This new sugar oxidase was composed of a single polypeptide chain with a molecular mass of 47.2 kDa and was found to contain 2.0 zinc ions per mole of enzyme but no flavin adenine dinucleotide or heme. This enzyme was stable in the pH range 5.5-9.0, with an optimal reaction pH of 7.5. Its optimal reaction temperature was 40 °C, and it was stable up to 50 °C for 1 h at pH 7.5. LOD oxidized disaccharides with reducing-end glucosyl residues linked by an α or ß-1,4 glucosidic bond. The relative activity of LOD toward lactose, cellobiose and maltose was 100 : 83 : 4, respectively. To the best of our knowledge, this is the first report on the discovery of an LOD based on coenzyme moiety and enzyme substrate specificity.

Cooperativity between 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase is based on a common pyridine nucleotide pool in the lumen of the endoplasmic reticulum.

11Beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is a NADP(H)-dependent oxidoreductase of the ER lumen, which may have an important role in the pathogenesis of metabolic syndrome. Here, the functional coupling of 11beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase (H6PDH) was investigated in rat liver microsomal vesicles. The results demonstrate the existence of a separate intraluminal pyridine nucleotide pool in the hepatic endoplasmic reticulum and a close cooperation between 11betaHSD1 and H6PDH based on their co-localization and the mutual generation of cofactors for each other.

Properties of neutral cellobiose dehydrogenase from the ascomycete Chaetomium sp. INBI 2-26(-) and comparison with basidiomycetous cellobiose dehydrogenases.

The extracellular cellobiose dehydrogenase (CDH) obtained from Chaetomium sp. INBI 2-26(-) has a molecular mass of 95 kDa and an isoelectric point of 5. This novel CDH is highly specific for the oxidation of cellobiose (K(m,app) 4.5 microM) and lactose (K(m,app) 56 microM). With 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) (cyt c(3+)) as electron acceptors, CDH was most active at pH 6. The turnover number of the enzyme for cellobiose, lactose, DCIP and cyt c(3+) was in the range of 9-14s(-1) at 20 degrees C and pH 6. The UV-visible spectrum revealed the flavohemoprotein nature of the enzyme. The cytochrome b domain of the enzyme was reduced by ascorbate, dithionite, as well as specifically by cellobiose in a wide range of pH. The apparent first order rate constants of the spontaneous re-oxidation of the reduced heme domain were estimated as 0.01 and 0.00039 s(-1) at pH 4.5 and 6.5, respectively. The half-inactivation time of CDH at pH 6 and 55 degrees C was ca. 100 min; the stability at pH 8 and, particularly, pH 4 was remarkably lower. Cellobiose stabilized the enzyme against thermal inactivation, whereas DCIP in turn sensitized the enzyme. The new enzyme revealed low affinity for crystalline cellulose, but was capable of binding onto H(3)PO(4)-swollen filter paper. The results show significant differences to already known CDHs and perspectives for several biotechnological applications, where CDH with maximal activity at neutral pH and high affinity for cellobiose and lactose night have some advantages.

Intramembraneous localization of rat liver microsomal hexose-6-phosphate dehydrogenase and membrane permeability to its substrates.

A method for purifying hexose-6-phosphate dehydrogenase (beta-D-glucose: NAD(P) -oxidoreductase, EC 1.1.1.47) from rat liver microsomes is described. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. It is shown that the enzyme is bound to the inner surface of microsomal membranes, and that glucose 6-phosphate, but not NADP, penetrates almost freely into the membranes at 37 degrees C.

Electrochemical investigation of cellobiose dehydrogenase from new fungal sources on Au electrodes.

Following previous electrochemical investigations of cellobiose dehydrogenase (CDH), the present investigation reports on the initial screening of the electrochemistry of three new CDHs, two from the white rot basidiomycetes Trametes villosa and Phanerochaete sordida and one from the soft rot ascomycete Myriococcum thermophilum, for their ability to directly exchange electrons with 10 different alkanethiol-modified Au electrodes. Direct electron transfer (DET) between the enzymes and some of the modified Au electrodes was shown, both, in the presence and in the absence of cellobiose. However, the length and the head functionality of the alkanethiols drastically influenced the efficiency of the DET reaction and also influenced the effect of pH on the biocatalytic/redox currents, suggesting the importance of structural/sequence differences between these CDH enzymes. In this respect, the white rot CDHs exhibit excellent biocatalytic and redox currents, whereas for the soft rot CDH the DET communication is much less efficient. Cyclic voltammograms indicate that the heme domain of the CDHs is the part of the enzymes that most readily exchanges electrons with the electrode. However, for P. sordida CDH on 11-mercaptoundecanol or dithiopropionic acid-modified Au electrodes, a second voltammetric wave was noticed suggesting that for some orientations of the enzyme, DET communication with the FAD cofactor can also be obtained.

Mouse liver microsomal hexose-6-phosphate dehydrogenase. NADPH generation and utilization in monooxygenation reactions.

Hexose-6-phosphate dehydrogenase (H6PD) activity in washed hepatic microsomes from male ICR mice, when assayed with NADP+ and deoxyglucose-6-phosphate, was partially latent. Brief sonication or detergents activated H6PD causing an approximately 4- and 8.5-fold increase in NADPH generation respectively. The sonicated microsomes exhibited H6PD-linked N-demethylase activity toward aminopyrine. This activity was best sustained in the presence of deoxyglucose-6-phosphate, while galactose-6-phosphate, glucose-6-phosphate, and glucose were less effective. Reaction media containing sonicated microsomes, NADP+ and deoxyglucose-6-phosphate also catalyzed N-demethylation of p-chloro-N-methylaniline, N,N-dimethylaniline and nicotine, O-demethylation of p-nitroanisole, p-hydroxylation of aniline, ring hydroxylation of biphenyl at the 2- and 4-positions, dearylation of parathion, and the N-oxidation of N,N-dimethylaniline. In general, the hexose-6-phosphate dehydrogenase-linked monooxygenation rates were 60% or more of those observed in the presence of exogenous NADPH.

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.

Glucose dehydrogenase of bovine heart: activity, purification and properties of the enzyme.

The activity of glucose dehydrogenase (EC 1.1.1.47; GDH; glucose NAD(P) oxidoreductase) was demonstrated in the supernatant obtained after centrifugation (1500 g) of bovine heart homogenate. More than 50% of GDH activity was found in the microsomal fraction. The optimum pH for the microsomal enzyme was 8.9. About 200-fold purification of GDH was achieved by successive application of ultracentrifugation in 0.25 M mannitol, treatment with solid ammonium sulphate, and CM-32 cellulose chromatography. The purified preparation oxidized not only glucose but also D-glucosamine, N-acetylglucosamine and xylose in 87, 63 and 23%, respectively. Purified GDH was inhibited by p-chloromercuribenzoate and glucose 6-phosphate.

Anatomy and fiber type composition of human interarytenoid muscle.

Intrinsic laryngeal muscle investigations, especially those of the interarytenoid (IA) muscle, have been primarily teleologically based. We determined IA muscle anatomy and histochemical and immunohistochemical classification of extrafusal and intrafusal (muscle spindle) fibers in 5 patients. Extrafusal fibers were oxidative type I and glycolytic types IIA and IIX. Intrafusal fibers of muscle spindles were identified by the presence of tonic and neonatal myosin. The results demonstrate that the IA muscle has a phenotype similar to that of limb skeletal muscle. Myosin coexpression, the absence of intrafusal fibers, and fiber type grouping were unusual features found previously in the thyroarytenoid and posterior cricoarytenoid muscles, but they were not present in the IA muscle. These findings lead to the conclusion that the IA muscle has functional significance beyond its assumed importance in maintaining vocal fold position during phonation. The presence of spindles demonstrates differences in motor control as compared to the thyroarytenoid and posterior cricoarytenoid muscles. Further, extrafusal fiber characteristics implicate IA muscle involvement in muscle tension dysphonia and adductor spasmodic dysphonia. Given the unique physiologic characteristics of the human IA muscle, further research into the role of the IA muscle in voice disorders is warranted.

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.

[Degradation of the herbicide atrazine by the soil mycelial fungus INBI 2-26(-)--a producer of cellobiose dehydrogenase]. / Razlozhenie gerbitsida atrazina pochvennym mitselial'nym gribom INBI 2-26(-)--produtsentom tsellobiozodegidrogenazy.

Nonsporulating mycelial fungi producing cellobiose dehydrogenase (CDH) and isolated from soils of South Vietnam with high residual content of dioxins are capable of growing on a solid medium in the presence of high atrazine concentrations (to 500 mg/l). At 20 and 50 mg/l atrazine, the area of fungal colonies was 1.5-1.2-fold larger, respectively, compared with control colonies of the same age, whereas development of the colonies at 500 mg/l atrazine was delayed by 5 days, compared with controls grown in the absence of atrazine. Surface cultivation of the fungus on a minimal medium with glucose as a sole source of carbon and energy decreased the initial concentration of atrazine (20 mg/l) 50 times in 40 days; in addition, no pronounced sorption of atrazine by mycelium was detected. This was paralleled by accumulation in the culture medium of extracellular CDH; atrazine increased the synthesis of this enzyme two- to threefold. Accumulation of beta-glucosidase (a mycelium-associated enzyme) and cellulases preceded the formation of CDH.

High-throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation.

Extracellular fungal flavocytochrome cellobiose dehydrogenase (CDH) is a promising enzyme for both bioelectronics and lignocellulose bioconversion. A selective high-throughput screening assay for CDH in the presence of various fungal oxidoreductases was developed. It is based on Prussian Blue (PB) in situ formation in the presence of cellobiose (<0.25 mM), ferric acetate, and ferricyanide. CDH induces PB formation via both reduction of ferricyanide to ferrocyanide reacting with an excess of Fe³âº (pathway 1) and reduction of ferric ions to Fe²âº reacting with the excess of ferricyanide (pathway 2). Basidiomycetous and ascomycetous CDH formed PB optimally at pH 3.5 and 4.5, respectively. In contrast to the holoenzyme CDH, its FAD-containing dehydrogenase domain lacking the cytochrome domain formed PB only via pathway 1 and was less active than the parent enzyme. The assay can be applied on active growing cultures on agar plates or on fungal culture supernatants in 96-well plates under aerobic conditions. Neither other carbohydrate oxidoreductases (pyranose dehydrogenase, FAD-dependent glucose dehydrogenase, glucose oxidase) nor laccase interfered with CDH activity in this assay. Applicability of the developed assay for the selection of new ascomycetous CDH producers as well as possibility of the controlled synthesis of new PB nanocomposites by CDH are discussed.

A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

Effect of diabetes on enzymes involved in rat hepatic corticosterone production.

BACKGROUND: Numerous studies have explored the etiologic or permissive role of 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) in obesity and Type 2 diabetes, biochemical conditions often with concurrent hyperinsulinism. In contrast, there are limited data on the effect of insulin deficiency (i.e. Type 1 diabetes) on 11ß-HSD1 or endoplasmic reticulum enzymes that generate the reduced pyridine cofactor NADPH. Thus, the aim of the present study was to examine the effect of insulin-deficient, streptozotozin diabetes on key microsomal enzymes involved in rat hepatic corticosterone production. METHODS: After rats had been rendered diabetic with streptozotocin and some had been treated with insulin (2-6 units, s.c., long-acting insulin once daily) for 7 days, hepatic microsomes were isolated. Serum corticosterone and fructosamine were obtained premortem. Intact microsomes were incubated in vitro and 11ß-HSD1, hexose-6-phosphate dehydrogenase (H6PDH), and isocitrate dehydrogenase (IDH) measured. RESULTS: Although diabetes markedly altered body weight gain and serum protein glycosylation (assessed by fructosamine), there was no significant change in hepatic 11ß-HSD1 reductase activity, with or without insulin treatment. However, serum corticosterone levels were significantly correlated with 11ß-HSD1 reductase activity when all groups were analyzed together (P < 0.05). Untreated diabetes modified (P < 0.01) two hepatic microsomal NADPH-generating enzymes, namely H6PDH and IDH, resulting in a 37% decrease and 14% increase in enzyme levels, respectively. CONCLUSIONS: Consistent with most in vivo studies, chronic insulin deficiency with attendant hyperglycemia does not significantly modify hepatic 11ß-HSD1 reductase activity, but does alter the activity of two microsomal enzymes coupled with pyridine cofactors.

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications.

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 172], the variant oxidizes both sugars equally well [(k(cat)/K(M))(Glc)/(k(cat)/K(M))(Gal) = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K(M) values and approximately ten-fold higher k(cat) values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60 degrees C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D-glucose/D-galactose mixtures at both 30 and 50 degrees C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 A resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.