Structural origins of the insulin-mimetic activity of bis(acetylacetonato)oxovanadium(IV)

We have investigated the interaction of bis(acetylacetonato)oxovanadium(IV) (VO(acac)(2)) with bovine serum albumin (BSA) by EPR and angle-selected electron nuclear double resonance, correlating results with assays of glucose uptake by 3T3-L1 adipocytes. EPR spectra of VO(acac)(2) showed no broadening in the presence of BSA; however, electron nuclear double resonance titrations of VO(acac)(2) in the presence of BSA were indicative of adduct formation of VO(acac)(2) with albumin of 1:1 stoichiometry. The influence of VO(acac)(2) on uptake of 2-deoxy-d-[1-(14)C]glucose by serum-starved 3T3-L1 adipocytes was measured in the presence and absence of BSA. Glucose uptake was stimulated 9-fold in the presence of 0.5 mm VO(acac)(2), 17-fold in the presence of 0.5 mm VO(acac)(2) plus 1 mm BSA, and 22-fold in the presence of 100 nm insulin. BSA had no influence on glucose uptake, on the action of insulin, or on glucose uptake in the presence of VOSO(4). The maximum insulin-mimetic effect of VO(acac)(2) was observed at VO(acac)(2):BSA ratios less than or equal to 1.0. Similar results were obtained also with bis(maltolato)oxovanadium(IV). These results suggest that the enhanced insulin-mimetic action of organic chelates of VO(2+) may be dependent on adduct formation with BSA and possibly other serum transport proteins.     

In vitro study of the insulin-mimetic behaviour of vanadium(IV, V) coordination compounds

A representative set of vanadium(IV and V) compounds in varying coordination environments has been tested in the concentration range 1 to 10(-6) mM, using transformed mice fibroblasts (cell line SV 3T3), with respect to their short-term cell toxicity (up to 36 hours) and their ability to stimulate glucose uptake by cells. These insulin-mimetic tests have also been carried out with non-transformed human fibroblasts (cell line F26). The compounds under investigation comprise established insulin-mimetic species such as vanadate ([H(2)VO(4)](-)), [VO(acetylacetonate)(2)], [VO(2)(dipicolinate)](-) and [VO(maltolate)(2)], and new systems and coordination compounds containing OO, ON, OS, NS and ONS donor atom sets. A vitality test assay, measuring the reduction equivalents released in the mitochondrial respiratory chain by intracellular glucose degradation, is introduced and the results are counter-checked with (3)H-labelled glucose. Most compounds are toxic at the 1 mM concentration level, and most compounds are essentially non-toxic and about as effective as or more potent than insulin at concentrations of 0.01 mM and below. V(V) compounds tend to be less toxic than V(IV)compounds, and complexes containing thio functional ligands are somewhat more toxic than others. Generally, ON ligation is superior in insulin-mimetic efficacy to OO or O/ NS coordination, irrespective of the vanadium oxidation state. There is, however, no striking correlation between the nature of the ligand systems and the insulin-mimetic potency in these cell culture tests, encompassing 41 vanadium compounds, the results on 22 of which are reported in detail here. The syntheses and characteristics of various new compounds are provided together with selected speciation results. The crystal and molecular structures of [[VO(naph-tris)](2)] [where naph-tris is the Schiff base formed between o-hydroxynaphthaldehyde and tris(hydroxymethyl)amine] are reported. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0311-5.     

Synthesis, structural properties and insulin-enhancing potential of bis(quercetinato)oxovanadium(IV) conjugate

An oxovanadium complex of quercetin (2), exhibits highly potent insulin-enhancing activity in streptozotocin-induced diabetic mice. It also mimics mitogenic potential of insulin as evaluated by [H(3)]thymidine uptake assay making an effective, orally active insulin-enhancing agent for the treatment of both type 1 and type 2 diabetes without any noticeable toxic effects.     

Synthesis, characterisation and insulin-mimetic activity of oxovanadium(IV) complexes with amidrazone derivatives

The complexation of VO(2+) ion by ten acetamidrazone and 2-phenylacetamidrazone derivatives (L) was studied. Sixteen novel VO(2+) complexes were synthesised and characterised through the combined application of analytical and spectroscopic (EPR (electron paramagnetic resonance), FT-IR and diffuse reflectance electronic absorption) techniques. Eight are 1:2 species of composition [VOL(2)]SO(4) x xH(2)O and eight are 1:1 species with formula [VOL(SO(4))](n) x xH(2)O. The experimental data suggest a bidentate coordination mode for L with the donor set formed by the imine nitrogen and the carbonyl oxygen. EPR spectra indicate a square-pyramidal geometry for the 1:1 complexes and a penta-coordinated geometry intermediate between the square-pyramid and the trigonal-bipyramid for the 1:2 species. The hyperfine coupling constant along z axis, A(z), of the 1:2 complexes exhibits a marked reduction with respect to the predicted value (approximately 148x10(-4)cm(-1) vs. approximately 170x10(-4)cm(-1)). IR spectroscopic evidence supports the presence of sulphate as a counter-ion in the 1:2, and as a bridging bidentate ligand in the 1:1 complexes. Insulin-mimetic tests on modified fibroblasts, based on a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazoliumbromide) assay, performed on three of the bis-chelated and eight of the mono-chelated derivatives, indicate that they are biologically active. The similar hydro/lipophilicity and the lack of ligand substituents recognizable by cell membrane receptors prevent substantial differentiation in the insulin-mimetic action.     

An orally active antidiabetic vanadyl complex, bis(1-oxy-2-pyridinethiolato)oxovanadium(IV), with VO(S2O2) coordination mode; in vitro and in vivo evaluations in rats

According to Pearson's HSAB (hard and soft acids and bases) rule, the vanadyl ion is classified as a hard acid. However, vanadyl-cysteine methyl ester and dithiocarbamate complexes with VO(S2N2) and VO(S4) coordination modes, respectively, that contain bonds with a combination of hard acid (VO2+) and soft base (sulfur) have been found to form stable complexes and exhibit insulin-mimetic activities in in vitro and in vivo evaluations. Based on such observations, a purple bis(1-oxy-2-pyridinethiolato)oxovanadium(IV) (VO(OPT)) complex with VO(S2O2) coordination mode was prepared and found to have a strong insulin-mimetic activity in in vitro evaluation, which followed in vivo effectiveness on intraperitoneal injection and oral administration. Then, we examined the real-time ESR analysis of vanadyl species in the blood of live rats given VO(OPT) by use of the blood circulation monitoring-ESR method. The clearance of vanadyl species from the blood in terms of half-life (t(1/2)) was determined as 15 min in VO(OPT)-treated rats, while t(1/2) of VOSO4-treated rats was 5 min, indicating the long-term acting character of VO(OPT). On the basis of the results, VO(OPT) with VO(S2O2) coordination mode is proposed to be a potent orally active insulin-mimetic complex in treating insulin-dependent diabetes mellitus in experimental animals.     

In vivo effects of insulin and bis(maltolato)oxovanadium (IV) on PKB activity in the skeletal muscle and liver of diabetic rats

In this study, the in vivo effects of insulin and chronic treatment with bis(maltolato)oxovanadium (IV) (BMOV) on protein kinase B (PKB) activity were examined in the liver and skeletal muscle from two animal models of diabetes, the STZdiabetic Wistar rat and the fatty Zucker rat. Animals were treated with BMOV in the drinking water (0.75–1 mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKB activity more than 10fold and PKB activity more than 3fold in both animal models. Despite the development of insulin resistance, insulininduced activation of PKB was not impaired in the STZdiabetic rats up to 9 weeks of diabetes, excluding a role for PKB in the development of insulin resistance in type 1 diabetes. Insulin-induced PKB activity was markedly reduced in the skeletal muscle of fatty Zucker rats as compared to lean littermates (fatty: 7fold vs. lean: 14fold). In contrast, a significant increase in insulinstimulated PKBa activity was observed in the liver of fatty Zucker rats (fatty: 15.7fold vs. lean: 7.6fold). Chronic treatment with BMOV normalized plasma glucose levels in STZdiabetic rats and decreased plasma insulin levels in fatty Zucker rats but did not have any effect on basal or insulininduced PKB and PKB activities. In conclusion (i) in STZdiabetic rats PKB activity was normal up to 9 weeks of diabetes; (ii) in fatty Zucker rats insulininduced activation of PKB (but not PKB) was markedly altered in both tissues; (iii) changes in PKB activity were tissue specific; (iv) the glucoregulatory effects of BMOV were independent of PKB activity.     

Amelioration of insulin resistance in diabetic ob/ob mice by a new type of orally active insulin-mimetic vanadyl complex: bis(1-oxy-2-pyridinethiolato)oxovanadium(IV) with VO(S(2)O(2)) coordination mode.

Recently, we have shown that a newly synthesized vanadyl complex, bis(1-oxy-2-pyridinethiolato)oxovanadium(IV), VO(opt)(2), is a potent orally active insulin-mimetic in treating streptozotocin-induced diabetes in rats, with long-term action. In the present study, the anti-diabetic effect of VO(opt)(2) and its mechanism in ob/ob mice, an obese non-insulin-dependent diabetes mellitus (NIDDM) animal model, was investigated. In ob/ob mice, 15-day oral treatment with VO(opt)(2) resulted in a dose-dependent decrease in the levels of glucose, insulin and triglyceride in blood. VO(opt)(2) was also effective in ameliorating impaired glucose tolerance in ob/ob mice, when an oral glucose tolerance test was performed after treatment with VO(opt)(2). Tumor necrosis factor-alpha (TNF-alpha) is a key component of obesity-diabetes link, we therefore examined the attenuating effect of VO(opt)(2) on impaired insulin signal transduction induced by TNF-alpha. Elevated expression of TNF-alpha was observed in the epididymal and subcutaneous fat tissues of ob/ob mice. Incubation of 3T3-L1, mouse adipocytes, with TNF-alpha reduced the phosphorylation of insulin receptor substrate-1 (IRS-1), whereas VO(opt)(2) treatment resulted in an enhancement of IRS-1 phosphorylation, irrespective of the presence or absence of TNF-alpha. Overall, the present study demonstrates that VO(opt)(2) exerts an anti-diabetic effect in ob/ob mice by ameliorating impaired glucose tolerance, and furthermore, attenuates the TNF-alpha-induced decrease in IRS-1 phosphorylation in adipocytes. These results suggest that the anti-diabetic action of VO(opt)(2) is derived from an attenuation of a TNF-alpha induced impaired insulin signal transduction via inhibition of protein tyrosine phosphatase, providing a potential clinical utility for VO(opt)(2) in the treatment of NIDDM.     

Inhibition of protein tyrosine phosphatase 1B and alkaline phosphatase by bis(maltolato)oxovanadium (IV)

Vanadate has been recognized as a specific and potent phosphatase inhibitor since its structure is similar to that of phosphate. In this study, we measured the inhibition of glutathione S-transferase-tagged protein tyrosine phosphatase 1B (GST-PTP1B) and alkaline phosphatase (ALP) by the insulin enhancing compounds, bis(maltolato)oxovanadium(IV) (BMOV). The results showed that the activity of GST-PTP1B was reversibly inhibited by solutions of BMOV with an IC₅₀ value of 0.86 ± 0.02 μM. Steady state kinetic studies showed that inhibition of GST-PTP1B by BMOV was of a mixed competitive and noncompetitive type. In addition, incubation of GST-PTP1B with BMOV showed a time-dependent biphasic inactivation of the protein. On the other hand, the inhibitory behavior of BMOV on ALP activity was reversible and competitive with an IC₅₀ value of 32.1 ± 0.6 μM. Incubation with BMOV did not show biphasic inactivation of ALP. The reversible inhibition of GST-PTP1B by BMOV is more potent than that of ALP, but solutions of BMOV inhibited both enzymes. This data support the suggestion that mechanisms for the inhibitory effects of BMOV on GST-PTP1B and ALP are very different.     

Improvement of diabetes, obesity and hypertension in type 2 diabetic KKAy mice by bis(allixinato)oxovanadium(IV) complex

Previously, we found that bis(allixinato)oxovanadium(IV) (VO(alx)(2)) exhibits a potent hypoglycemic activity in type 1-like diabetic mice. Since the enhancement of insulin sensitivity is involved in one of the mechanisms by which vanadium exerts its anti-diabetic effects, VO(alx)(2) was further tested in type 2 diabetes with low insulin sensitivity. The effect of oral administration of VO(alx)(2) was examined in obesity-linked type 2 diabetic KKA(y) mice. Treatment of VO(alx)(2) for 4 weeks normalized hyperglycemia, glucose intolerance, hyperinsulinemia, hypercholesterolemia and hypertension in KKA(y) mice; however, it had no effect on hypoadiponectinemia. VO(alx)(2) also improved hyperleptinemia, following attenuation of obesity in KKA(y) mice. This is the first example in which a vanadium compound improved leptin resistance in type 2 diabetes by oral administration. On the basis of these results, VO(alx)(2) is proposed to enhance not only insulin sensitivity but also leptin sensitivity, which in turn improves diabetes, obesity and hypertension in an obesity-linked type 2 diabetic animal.     

Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase

The actions of insulin, vanadate (V) and hydrogen peroxide (H2O2) on IGF-II binding and insulin receptor tyrosine kinase activity were studied in rat adipocytes. Incubating adipocytes with a combination of V plus H2O2 resulted in a potent synergistic effect on both the increase in IGF-II binding and the activation of the insulin receptor kinase. Catalase, which removes H2O2, abolished this synergism if added at the time of mixing of V plus H2O2 but not if added 10 min. later, suggesting that the formation of peroxide(s) of vanadate generated a potent insulin mimicker. The data support a critical role for the insulin receptor kinase in insulin action. The novel insulin-mimetic compound, a presumed peroxide of vanadate, could prove useful for investigating insulin action and may be valuable for treating insulin resistance.     

A nonspecific phosphotyrosine phosphatase inhibitor, bis(maltolato)oxovanadium(IV), improves glucose tolerance and prevents diabetes in Zucker diabetic fatty rats

The molecular basis of insulin resistance, a major risk factor for development of Type II diabetes, involves defective insulin signaling. Insulin-mediated signal transduction is negatively regulated by the phosphotyrosine phosphatase, PTP1B, and numerous studies have demonstrated that organo-vanadium compounds, which are nonselective phosphotyrosine phosphatase inhibitors, have insulin-mimetic properties. However, whether or not vanadium compounds can prevent the transition from insulin resistance to overt diabetes is unknown. We compared the ability of bis(maltolato)oxovanadium(IV) (BMOV), an orally bioavailable organo-vanadium compound, and rosiglitazone maleate (RSG), a known insulin sensitizer, to prevent development of diabetes in Zucker diabetic fatty (ZDF) rats. Treatment began at 6 weeks of age when animals are insulin resistant and hyperinsulinemic, but not yet hyperglycemic, and ended at 12 weeks of age, which is 4 weeks after ZDF rats typically develop overt diabetes. BMOV-treated ZDF rats did not develop hyperglycemia, showed significant improvement in insulin sensitivity, and retained normal pancreatic islet morphology and endocrine cell distribution, similar to RSG-treated animals. BMOV and RSG treatment also prevented the hyper-phagia and polydipsia present in untreated ZDF rats; however, BMOV-treated ZDF rats gained much less weight than did RSG-treated animals. Circulating levels of adiponectin decreased in untreated ZDF rats compared to lean controls, but these levels remained normal in BMOV-treated ZDF rats. In contrast, in RSG-treated ZDF rats, plasma adiponectin levels were nearly 4-fold higher than in lean control rats, primarily as a result of a large increase in the amount of low-molecular weight forms of adiponectin in circulation. These data demonstrate that phosphatase inhibition offers a new approach to diabetes prevention, one that may have advantages over current approaches.     

A new halogenated antidiabetic vanadyl complex, bis(5-iodopicolinato)oxovanadium(IV): in vitro and in vivo insulinomimetic evaluations and metallokinetic analysis

A new vanadyl complex, bis(5-iodopicolinato)oxovanadium(IV), VO(IPA)2, with a VO(N2O2) coordination mode, was prepared by mixing 5-iodopicolinic acid and VOSO4 at pH 5, with the structure characterized by electronic absorption, IR, and EPR spectra. Introduction of the halogen atom on to the ligand enhanced the in vitro insulinomimetic activity (IC50 = 0.45 mM) compared with that of bis(picolinato)oxovanadium(IV) (IC50 = 0.59 mM). The hyperglycemia of streptozotocin-induced insulin-dependent diabetic rats was normalized when VO(IPA)2 was given by daily intraperitoneal injection. The normoglycemic effect continued for more than 14 days after the end of treatment. To understand the insulinomimetic action of VO(IPA)2, the organ distribution of vanadium and the blood disposition of vanadyl species were investigated. In diabetic rats treated with VO(IPA)2, vanadium was distributed in almost all tissues examined, especially in bone, indicating that the action of vanadium is not peripheral. Vanadyl concentrations in the blood of normal rats given VO(IPA)2 remain significantly higher and longer than those given other complexes because of its slower clearance rate. VO(IPA)2 binds with the membrane of erythrocytes, probably owing to its high hydrophobicity in addition to its binding with serum albumin. The longer residence of vanadyl species shows the higher normoglyceric effects of VO(IPA)2 among three complexes with the VO(N2O2) coordination mode. On the basis of these results, VO(IPA)2 is indicated to be a preferred agent to treat insulin-dependent diabetes mellitus in experimental animals.     

An electron spin-echo envelope modulation (ESEEM) study of the QA binding pocket of PS II reaction centers from spinach and Synechocystis

We have used electron spin-echo envelope modulation spectroscopy (ESEEM) to characterize the protein-cofactor interactions present in the QA- binding pocket of PS II centers isolated from spinach and Synechocystis. We conclude that the ESEEM spectrum of QA- is the result of interactions of the S = 1/2 electron spin of QA- with the I = 1 nuclear spins of the peptide nitrogens of two different amino acids. One peptide nitrogen has ESEEM peaks near 0.7, 2.0, 2.85, and 5.0 MHz with isotropic and dipolar hyperfine couplings of Aiso = 2.0 MHz and Adip = 0.25 MHz, respectively. On the basis of these hyperfine couplings we predict the existence of a strong hydrogen bond between QA- and the peptide nitrogen with a hydrogen bond distance of about 2 A. We have not identified the amino acid origin of this peptide nitrogen. By using amino acid specific isotopic labeling in conjunction with site-directed mutagenesis, we demonstrate that the second peptide nitrogen is that of D2-Ala260, with ESEEM peaks near 0.6 and 1.5 MHz and an isotropic hyperfine coupling, Aiso, less than 0.2 MHz. This small isotropic coupling suggests that the D2-Ala260 peptide nitrogen at best forms a weak hydrogen bond with QA-.     

Synthetic analogs for oxovanadium(IV/V)-glutathione interaction: an NMR, EPR, synthetic and structural study of oxovanadium(IV/V) compounds with sulfhydryl-containing pseudopeptides and dipeptides

The reaction of [VO(CH3COO)2(phen)] (phen = 1,10-phenanthroline) with the sulfhydryl-containing pseudopeptides (scp), N-(2-mercaptopropionyl)glycine (H3mpg), N-(2-mercaptopropionyl)cysteine (H4m2pc), N-(3-mercaptopropionyl)cysteine (H4m3pc) and the dipeptides glycylglycine (H2glygly) and glycyl-L-alanine (H2glyala), in the presence of triethylamine, results in the formation of the compounds Et3NH[VO(mpg)(phen)] (1), (Et3NH)2[VO(m2pc)] (4), [(Et3NH)2[VO(m3pc) (5), [VO(glygly)(phen)] x 2CH3OH (2 x 2CH3OH) and [VO(glyala)(phen)] x CH3OH (3 x CH3OH). Evidence for the molecular connectivity in 2 x CH3OH was established by X-ray crystallography, showing the vanadium(IV) atom ligated to a tridentate glygly2- ligand at the N(amine), N(peptide) and O(carboxylato) atoms. Combination of the correlation plot of the EPR parameters gz versus Az, together with the additivity relationship supported the prediction of the equatorial donor atom sets of the V(IV)O2+ center at various pH values for the V(IV)O2+-glutathione system considered in this study. Model NMR studies (interaction of vanadium(V) with the scp H3mpg) showed that there is a possibility of vanadium(V) ligation to glutathione.     

Observation of manganese(II)-ligand superhyperfine couplings in complexes with proteins by electron spin-echo spectroscopy

Pulsed electron paramagnetic resonance spectroscopy has been used to detect Mn(II)-ligand superhyperfine couplings in complexes with creatine kinase and in the Mn(II) metalloprotein concanavalin A. Electron spin-echo envelopes from Mn(II), bound in these complexes, are modulated by superhyperfine interactions between Mn(II) and nearby, weakly coupled nuclear spins. The characteristic frequencies of the modulations were obtained by Fourier transformation of the three-pulse, spin-echo envelopes. In transition-state analogue complexes of creatine kinase (enzyme-MnIIADP-anion-creatine), superhyperfine interactions from the directly coordinated nitrogen of the thiocyanate ligand give envelope modulations. The source of the modulations was confirmed by measurements with the 14N and 15N forms of thiocyanate. On the other hand, the nitrogen of coordinated nitrate, which is two bonds removed from the paramagnetic center, does not produce detectable modulations. In spectra for Mn(II) concanavalin A, envelope modulations are detected due to the nitrogen of the coordinated histidine residue. Complexes prepared in 2H2O give strong signals due to weakly coupled 2H. For Mn(II)-doped single crystals of sodium pyrophosphate, signals are observed in the frequency domain spectra that are due to coupling from 31P. Phosphorus signals from the ADP ligand in complexes with creatine kinase show approximately the same coupling constant but have a much broader line width.     

Synthesis,characterization and catalytic oxidations of oxovanadium(IV), oxotitanium(IV) and dioxomolybdenum(VI) complexes with chiral imines of L-amino acids

Representative oxovanadium(IV), oxotitanium(IV), and dioxomolybdenum(VI) complexes of N-salicylidene-L-amino acids (the amino acids are: valine, leucine, except for Mo(VI), and histidine) have been synthesized and characterized by various spectroscopic techniques. It has been found that while in the histidine complexes of titanium(IV) and vanadium(IV) the amino acid residue is bound in the expected glycine-like mode, in the molybdenum(VI) complex it is bound in the unusual histamine-like mode. Also, the structure of this molybdenum(VI) complex contains imidazolate-bridged polymeric units in the solid state, while the carboxyl group of the amino acid residue is protonated. In solution the polymeric structure is cleaved and the monomers contain carboxylate and protonated imidazole groups at the histidine residue. The histamine-like structure of the histidine complex was probcd by comparison with that of the chiral dioxomolybdenum(VI) complex of N-salicylidene-L-histidinol. While the structure of the metal centers is six-coordinate for the dioxomolybdenum(VI) complexes and the histidine complexes of oxotitanium(IV) and oxovanadium(IV), it is likely that the complexes of the latter metals derived from nonpolar amino acids do not achieve coordination numbers higher than five. The present oxometal complexes are catalytically active in the sulfoxidation of sulfides and in the epoxidations of activated olefins by tert-butyl hydroperoxide, but in general exhibit a low degree of asymmetric induction in these reactions.     

In vivo modulation of a DnaJ homolog, CbpA, by CbpM

CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.