Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells.

Insulin stimulation of hydrogen peroxide production by rat epididymal fat cells was investigated by studying the oxidation of formate to CO2 by endogenous catalase. Under optimal concentrations of formate (0.1 to 1 mM) and glucose (0.275 mM), insulin stimulated formate oxidation 1.5- to 2.0-fold. Inhibitors of catalase activity, including nitrite and azide, inhibited both basal and insulin-stimulated formate oxidation at concentrations that did not interfere with insulin effects on glucose C-1 oxidation or glucose H-3 incorporation into lipids. The addition of exogenous catalase increased formate oxidation only slightly, while exogenous H2O2 (0.5 mM) stimulated formate oxidation by endogenous catalase strongly. These data indicate that the insulin-stimulated H2O2 production was intracellular. Insulin dose-response curves for formate oxidation were identical with those for glucose H-3 incorporation into lipids. The dependence of relative insulin effects on the logarithm of the glucose concentration was bell-shaped for formate oxidation and correlated highly with the coresponding dependences of glucose C-1 oxidation and glucose H-3 incorporation into lipids. This suggests that insulin stimulation of intracellular H2O2 production is linked to glucose metabolism. Since it is known that extracellular H2O2 can mimic insulin in several respects, these observations suggest that H2O2 may act as a "second messenger" for the observed effects of insulin.     

The insulin-like effect of hydrogen peroxide on pathways of lipid synthesis in rat adipocytes.

In addition to the well known insulin-like effects of certain concentrations of H2O2 on glucose transport and oxidation in isolated rat adipocytes, the present work demonstrates that lipid synthesis from glucose is also enhanced over a narrow range of H2O2 concentrations (0.15 to 0.5 mM) added to the incubation medium. As in the case of insulin, H2O2 was found to stimulate greater glucose incorporation into glyceride-fatty acid than incorporation into glyceride-glycerol. As part of a multifaceted regulation of lipogenesis, H2O2, like insulin, increased the amount of pyruvate dehydrogenase in the active form without increasing the total amount of pyruvate dehydrogenase. Pyruvate dehydrogenase activity increased within 5 min of H2O2 incubation, reached a maximum at 15 min and declined thereafter as the H2O2 disappeared from the incubation medium. While medium glucose per se was found to activate the enzyme, it is unlikely that the effect of H2O2 was mediated by the known enhancement of glucose transport since the effects on the enzyme were maximal in the absence of glucose in the incubation medium. These findings add to the growing list of insulin effects that are reproduced by H2O2, and strengthen the hypothesis that assigns H2O2 the role of "second messenger" of insulin.     

N epsilonB29-(+)-biotinylinsulin and its complexes with avidin. Synthesis and biological activity

Insulin was modified with d-biotin-N-hydroxysuccinimide ester in dimethylformamide. Mono-, di-, and triacylated insulins were separated by preparative isoelectric focusing. Monoacylated derivatives (isoelectric point 5.1) were fractionated twice on DEAE-cellulose to yield pure N epsilonB29-biotinylinsulin. The structure of the product was established by amino acid analysis before and after deamination. N epsilonB29-biotinylinsulin had biological activity indistinguishable from insulin on glucose oxidation and lipid synthesis assays using isolated rat epididymal fat cells. Complexes of N epsilonB29-biotinylinsulin with avidin, having essentially all but one binding site filled with biotin, were prepared in order to obtain a 1:1 insulin:avidin ration. The elicited identical maximal biological responses, but showed a potency decreased to 5% of that of insulin. Such complexes conjugated with ferritin will provide a useful tool in the development of electron microscopic stains of insulin receptors.     

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae

The bacterial second messenger bis-(3′-5′)-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain–containing diguanylate cyclases and degraded by HD-GYP domain–containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain–containing PDE-As to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.     

TNP and its analogs: Modulation of IP6K and CYP3A4 inhibition

Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N²-(m-trifluorobenzyl), N⁶-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.     

Phosphatidylinositol 3-kinase regulates differentiation of H9c2 cardiomyoblasts mainly through the protein kinase B/Akt-independent pathway.

Phosphatidylinositol 3-kinase (PI3-kinase) is known to be a crucial regulator of muscle differentiation. However, its downstream pathway for this function is quite obscure. In this experiment we demonstrated the regulatory mechanism of the differentiation of H9c2 cardiomyoblasts, focusing on PI3-kinase, protein kinase B/Akt (PKB/Akt) and p42/44 mitogen-activated protein kinase (p42/44 MAPK). When H9c2 cells stably transfected with a constitutively active p110 (H9c2-p110*), a constitutively active PKB/Akt (H9c2-Akt), and an empty vector (H9c2-con) were induced to differentiate, H9c2-p110* cells differentiated fastest, followed by H9c2-Akt cells. H9c2-con cells differentiated at the slowest rate. Consistent with this result, LY294002 completely blocked differentiation of all these transfected cell lines, whereas PD098059 had no effect on their differentiation. When H9c2-p110* cells were transiently transfected with a dominant negative form of PKB/Akt, differentiation was not affected. Taken together, we concluded that PI3-kinase, but not p42/44 MAPK, regulates differentiation of H9c2 cardiomyoblasts mainly through the PKB/Akt-independent pathway.     

Roles of RhoA and phospholipase A2 in the elevation of intracellular H2O2 by transforming growth factor-beta in Swiss 3T3 fibroblasts.

We have investigated the mechanisms by which transforming growth factor-beta (TGF-beta) increased intracellular H2O2 in Swiss 3T3 fibroblasts. Increase of intracellular H2O2 by TGF-beta was maximal at 30 min and blocked by catalase from Aspergillus niger. Scrape-loading of C3 transferase, which down-regulated RhoA, inhibited the production of H2O2 in response to TGF-beta. TGF-beta stimulated release of arachidonic acid, which was completely inhibited by mepacrine, a phospholipase A2 inhibitor. Mepacrine also blocked the increase of H2O2 by TGF-beta. In addition, arachidonic acid increased intracellular H2O2. Furthermore, TGF-beta stimulated stress fibre formation, which was blocked by catalase, without membrane ruffling. Catalase also inhibited stimulation of thymidine incorporation by TGF-beta. These results suggested that TGF-beta increased intracellular H2O2 through RhoA and phospholipase A2, and also suggested that intracellular H2O2 was required for the stimulation of stress fibre formation and DNA synthesis in response to TGF-beta.     

Chemical Constituents of the Culture Broth of Panus rudis

In our ongoing search for new secondary metabolites from fungal strains, one novel compound (1) and nine known compounds (2-10) were isolated from the EtOAc-soluble layer of the culture broth of Panus rudis. The culture broth of P. rudis was extracted in acetone and fractionated by solvent partition; column chromatography using silica gel, Sephadex LH-20, and Sephadex G-10; MPLC; and HPLC. The structures of isolated compounds were elucidated by one- and two-dimensional NMR and LC-ESI-mass measurements. One new compound, panepoxydiol (1), and nine known compounds, (E)-3-(3-hydroxy-3-methylbut-1-en-1-yl)-7-oxabicyclo[4.1.0]hept-3-ene-2,5-diol (2), isopanepoxydone (3), neopanepoxydone (4), panepoxydone (5), panepophenanthrin (6), 4-hydroxy-2,2-dimethyl-6-methoxychromane (7), 6-hydroxy-2,2-dimethyl-3-chromen (8), 2,2-dimethyl-6-methoxychroman-4-one (9), 3,4-dihydroxy-2,2-dimethyl-6-methoxychromane (10), were isolated from the culture broth of P. rudis. This is the first report of isolation of a new compound panepoxydiol (1) and nine other chemical constituents (2-5, 7-10) from the culture broth of P. rudis.