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PubMed2000

Activation of tyrosine kinase pathway by vanadate in gallbladder smooth muscle.

Vanadate, an inhibitor of tyrosine phosphatase activity, might induce gallbladder contraction through the stimulation of the tyrosine kinase pathway. The aim of this study was to characterize the effects of vanadate in the guinea pig gallbladder smooth muscle. Vanadate exerts contractile effects which are not mediated by neurotransmitter release. The tyrosine kinase inhibitor genistein nearly abolished vanadate contraction, suggesting that an increase in protein tyrosine phosphorylation mediates the actions of vanadate. This suggestion was confirmed by Western blot analysis. Vanadate contractions were reduced in the presence of methoxyverapamil or in Ca(2+)-free medium, suggesting that vanadate may induce Ca(2+) influx. Neither inactivation of the Na(+)/K(+) pump nor reversal of the Na(+)/Ca(2+) exchanger can account for vanadate's actions. Vanadate contractile effects were reduced by indomethacin, as well as mepacrine, the inhibitor of phospholipase A(2), but were not affected by phospholipase C inhibitors. Neither inhibitors of diacylglycerol lipase nor protein kinase C reduced the response induced by vanadate. These data indicate that the effects of vanadate on smooth muscle are mainly mediated by protein tyrosine phosphorylation and reveal a new link between tyrosine phosphorylation and arachidonic acid metabolism in the control of gallbladder smooth muscle contraction.

Animals↗
PubMed2002

Effect of pirfenidone against vanadate-induced kidney fibrosis in rats.

Renal fibrosis is a complication of kidney injury and can contribute to organ failure. Currently, there are no drugs for the treatment of renal fibrosis. Pirfenidone (PD) has been proven to have antifibrotic effects in animal models of fibrosis. We tested the ability of PD against vanadate-induced kidney fibrosis in rats. The rats were injected subcutaneously with vehicle or vanadate solution (1mg vanadate/kg/day) for 12 or 16 days to produce varying degrees of kidney fibrosis. The vanadate- and vehicle-treated rats were fed a laboratory diet or the same diet mixed with 0.6% PD ad lib. One vanadate-injected group was initially fed the same diet without PD and later switched to the diet containing PD 2 days after the last injection. The rats were killed at 12 and 25 days following the last dose. The changes found in the kidney of vanadate-treated rats included increases in RNA and DNA content and increases in kidney weight. Treatment with PD diminished the vanadate-induced increases in kidney weight and RNA content. The hydroxyproline content of the kidney in vanadate-treated animals was increased significantly (P< or =0.05) from the control level of 1452 microg/kidney to 1765 microg/kidney. Treatment with PD for 37 days caused significant reductions in the vanadate-induced increases in the hydroxyproline level. Similarly, treatment for 41 days also caused significant reductions (1744 microg/kidney) in vanadate-induced increases in the hydroxyproline level (1996 microg/kidney). The histological evaluation revealed that the severity of the lesions in the vanadate-treated group was moderate to severe, and treatment with PD for 41 days decreased the severity to a mild level. In addition, the delayed treatment with PD also minimized the vanadate-induced increases in the collagen content of the kidney. Although it is speculative, PD may potentially be therapeutic in the management of renal fibrosis.

Animals↗
PubMed1998

Stimulation of 125I-transferrin binding and 59Fe uptake in rat adipocytes by vanadate: treatment time determines apparent tissue sensitivity.

Vanadium compounds have been documented to stimulate a number of insulin biological effects in vitro and in vivo. We previously demonstrated stimulation of glucose transport and insulin-like growth factor-II (IGF-II) binding in rat adipocytes. These actions are associated with translocation of glucose transporters and IGF-II receptors from an intracellular compartment to the plasma membrane. The transferrin receptor is also recruited to the plasma membrane in response to insulin. Freshly isolated rat adipocytes were incubated with vanadate and insulin at 37 degrees C, and after treating the cells with KCN to inhibit further receptor movement, diferric 125I-transferrin binding was assayed. Vanadate stimulated a dose- and time-dependent increase in 125I-transferrin binding, reaching maximum (approximately threefold) stimulation at 1 mmol/L after a 4-hour incubation. This was equivalent to the maximum insulin effect that was obtained with 10(-8) mol/L after 30 minutes. A similar degree of stimulation was achieved with 0.1 mmol/L vanadate after 8 hours of exposure. Dose-response data showed that the apparent sensitivity to vanadate was time-dependent and increased with the duration of exposure (EC50: 30 minutes, 1 mmol/L; 3 hours, 0.35 mmol/L). Scatchard analysis of 125I-transferrin binding showed that both insulin and vanadate increased receptor binding capacity with no effect on receptor affinity. Total cellular transferrin receptor content measured by immunoblotting with monoclonal anti-transferrin receptor antibody (OX-26) was not altered by insulin or vanadate, consistent with receptor translocation. Assessment of 59Fe uptake from 59Fe-labeled diferric transferrin showed that vanadate augmented 59Fe uptake in a dose-dependent manner to an extent similar to insulin, demonstrating the functional activity of the receptors (percent of control: 10(-8) mol/L insulin, 175% +/- 23.8%, P < .02; 0.3 mmol/L vanadate, 188% +/- 17.3%, P < .01). We conclude that vanadate mimics insulin to augment cell surface transferrin receptors and increase Fe uptake in rat adipocytes. The time-dependent apparent increase in sensitivity is consistent with the effectiveness of very low concentrations of vanadate in vivo after several days of administration, and suggests a requirement for vanadate entry into cells to mediate this biological response.

Adipocytes↗
PubMed1992

Interaction of rabbit muscle aldolase at high ionic strengths with vanadate and other oxoanions.

Reductive, nonreductive, and photolytic interactions of vanadate with fructose-1,6-bisphosphate aldolase were examined and used to explore the interactions of oxoanions with aldolase. Aldolase is known to interact strongly with oxoanions at low ionic strength and weakly at higher ionic strength. Oxoanions inhibit aldolase competitively with respect to fructose 1,6-bisphosphate although the location of the oxoanion binding site on aldolase remains elusive. In this work, the interaction of aldolase with a series of oxoanions was compared at ionic strength approaching physiologic levels. The size and shape of the anion were important for the effective binding to aldolase, and no significant increase in affinity for aldolase was observed by the addition of alkyl groups to the oxoanions. Vanadate competitively inhibits aldolase in a manner analogous to the other oxoanions. Since vanadate solutions contain a mixture of vanadate oxoanions, the nature of the inhibition was determined using a combination of enzyme kinetics and 51V NMR spectroscopy. Aldolase contains a significant number of thiol functionalities, and as expected, vanadate undergoes redox chemistry with them, generating an irreversibly inhibited aldolase. This oxidative chemistry was attributed to the vanadate tetramer, whereas vanadate dimer was a reversible inhibitor. Vanadate monomer does not significantly interact with aldolase reversibly or irreversibly. Vanadyl cation has the lowest inhibition constant under these high ionic strength conditions. Using Yonetani-Theorell analysis, it appears that phosphate, pyrophosphate, and sulfate bind to the same site on aldolase, whereas vanadate, arsenate, and molybdate bind to another site. UV light-induced photocleavage of aldolase by vanadate was examined, and the loss of aldolase activity was correlated with cleavage of the aldolase subunit. Further studies using vanadium as a probe should reveal details on the location of the vanadate and vanadyl cation binding sites. This study suggests several sites on aldolase will accommodate oxoanions, and one of these sites also accommodates vanadyl cation.

Animals↗
PubMed2002

Mechanism of inhibition of the beta-lactamase of Enterobacter cloacae P99 by 1:1 complexes of vanadate with hydroxamic acids.

The class C beta-lactamase of Enterobacter cloacae P99 is competitively inhibited by low concentrations of 1:1 complexes of vanadate and hydroxamic acids. Structure-activity studies indicated that the hydroxamic acid functional group was essential to this inhibition. Both aryl and alkyl hydroxamic acids form inhibitory ternary complexes with vanadate and the enzyme, although, in certain cases of the latter, the inhibition may not be seen because of the low formation constants of the vanadate-hydroxamic acid complex. After all of the vanadate species present in solution had been taken into account, "real" K(i) values for the vanadate complexes could be determined. The K(i) value of the best of the inhibitors that were investigated, the 1:1 complex of vanadate with 4-nitrobenzohydroxamic acid, was 0.48 microM. Kinetics studies showed that the association and dissociation rate constants of this complex with the enzyme were 1.48 x 10(6) s(-1) M(-1) and 0.73 s(-1), respectively; the magnitude of the latter indicates covalent interaction of the complex with the enzyme. (51)V NMR and UV-vis spectra suggest that the structure of the vanadate complex bound to the enzyme may be very similar to that in solution. A (13)C NMR spectrum of the enzyme complex with 4-nitrobenzo[(13)C]hydroxamic acid and vanadate yields a coordination-induced shift (CIS) of 7.74 ppm. This is significantly larger than that of the vanadate complex in free solution (3.62 ppm), suggesting either, somewhat contrary to the (51)V and UV-vis spectra, greater interaction between vanadium and the hydroxamate carbonyl oxygen in the enzyme complex than in free solution or, more likely, polarization of the hydroxamate by interaction, e.g., hydrogen bonding, with the enzyme. Molecular modeling indicates that a pentacoordinated vanadate complex may well be able to snugly occupy the enzyme active site; Asn 152 is suitably placed to hydrogen bond to the hydroxamic acid oxygen atom. The experimental results are in accord with a model whereby the vanadate-hydroxamate-enzyme complex is a moderately good analogue of the transition state of the reaction of the beta-lactamase with phosphonate inhibitors.

Dose-Response Relationship, Drug↗
PubMed1986

The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level.

Sodium vanadate has several insulin-like effects. To determine whether vanadate acts via the insulin receptor, I investigated the effect of vanadate on glucose transport (2-deoxyglucose uptake) in adipocytes that had been treated to decrease the number of insulin receptors. Trypsin (100 micrograms/ml) caused greater than 95% loss of 125I-insulin binding and rendered glucose transport resistant to both insulin and an anti-insulin-receptor antibody. However, vanadate caused an 8-fold increase in the transport rate [EC50 (concn. giving 50% of maximum effect) 0.2 mM] in both control and trypsin-treated cells, demonstrating that the insulin receptor does not have to be intact for vanadate to stimulate glucose transport. Insulin receptors were depleted by treatment of adipocytes with insulin (100 ng/ml) in the presence of Tris (which blocks receptor recycling). A 2 h treatment caused 60% loss of receptors, and a shift to the right in the dose-response curve for insulin stimulation of glucose transport (EC50 0.3 ng of insulin/ml in controls, 1.2 ng/ml in treated cells). The response to vanadate was again unaffected. Treatment with insulin for 4 h caused a 67% decrease in insulin binding and, in addition to the rightward shift in the insulin dose-response curve, a decrease in basal and maximal transport rates (which cannot be explained by decreased insulin receptor number). The EC50 of vanadate was again equal in control and treated cells, but glucose transport in the presence of a maximally effective concentration of vanadate (1 mM) was decreased. I conclude that the effect of vanadate on glucose transport is independent of the insulin receptor. Induction of a post-receptor defect (which may be a decrease in the total number of cellular glucose transporters) by prolonged exposure to insulin decreases the potency of a maximally effective concentration of vanadate. The findings demonstrate that vanadate stimulates glucose transport by an effect at a level distal to the insulin receptor.

Adipose Tissue↗
PubMed2000

Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H(2)O(2) by N-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H(2)O(2) inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H(2)O(2) generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53-/-) cells. These results demonstrate that vanadate induces p53 activation mainly through H(2)O(2) generation, and this activation is required for vanadate-induced apoptosis.

Acetylcysteine↗
PubMed1995

Vanadate inhibition of hepatocytic autophagy. Calcium-modulated and osmolality-modulated antagonism by asparagine.

The phosphate analogue vanadate, at 10 mM, strongly (approximately 90%) inhibited the autophagic sequestration of endogenous lactate dehydrogenase in isolated rat hepatocytes. The effect of vanadate was markedly (approximately 80%) antagonized by asparagine (20 mM), and to a lesser extent by glutamine, glycine, and alanine. The antagonism was only observed in the presence of Ca2+ when an isotonic standard incubation medium was used, but by increasing the medium osmolality this Ca2+ requirement could be eliminated. Asparagine induced a cell swelling (17% at 20 mM) that might account for at least part of its vanadate antagonism, since hypotonic cell swelling by itself stimulated autophagy (with a maximal effect at approximately 200 mosM). Conversely, hypertonic media inhibited autophagy and were additive to vanadate. In a strongly hypotonic medium (less than 200 mosM), both asparagine and vanadate were inhibitory. However, since vanadate alone had no effect on cell volume, the vanadate-asparagine antagonism could not be exerted exclusively at the level of cell volume regulation. An additional mechanism might be a partial deamination of asparagine, generating ammonia, which was found to oppose the vanadate inhibition of autophagy while having no effect on cell volume. Other metabolizable amino acids, like alanine and glycine, were moderately vanadate-antagonistic while failing to induce cell swelling. These results are compatible with a vanadate-antagonistic effect of asparagine mediated partly through an unknown mechanism (possibly pH change) by its deamination product, ammonia, partly through cell swelling and a secondary Ca2+ influx that could compensate for a vanadate-induced depletion of intracellular calcium stores.

Adenosine Triphosphate↗
PubMed1980

The effects of vanadate on the fluxes of sodium and potassium ions through the sodium pump.

1. The effects of sodium orthovanadate on the fluxes of sodium and potassium (or rubidium) ions through the sodium pump have been investigated in intact human red cells and in resealed ghosts prepared from them. Sodium-potassium exchange, potassium-potassium exchange, pump reversal, sodium-sodium exchange and uncoupled sodium efflux have each been studied.2. When intact human red cells were incubated in high-sodium media containing vanadate in low concentrations, inhibition of potassium or rubidium influx was marked only if the potassium or rubidium concentration in the medium was sufficiently high to cause nearly maximal influx in the absence of vanadate. The absence of inhibition at lower potassium or rubidium concentrations cannot be explained by supposing that the onset of inhibition by vanadate is slower in these conditions.3. Lowering the extracellular sodium concentration, or raising the vanadate concentration, decreased the minimum concentration of extracellular potassium or rubidium at which inhibition by vanadate was detected.4. Experiments on potassium influx into intact red cells treated with the ionophore A23187 showed that magnesium ions act at intracellular sites to promote inhibition by vanadate.5. Measurements of potassium efflux from intact red cells incubated in high-sodium media, with or without potassium, showed that potassium-potassium exchange was inhibited by vanadate at low concentrations whereas reversal of the pump was not.6. Measurements of sodium efflux from intact red cells or resealed ghosts incubated in high-sodium media, with or without potassium, showed that vanadate had little or no effect on sodium-sodium exchange at concentrations at which sodium-potassium exchange was markedly reduced. Much higher concentrations of vanadate did cause partial inhibition of sodium-sodium exchange.7. Experiments to determine whether vanadate in low concentrations inhibited uncoupled sodium efflux were inconclusive, but suggested that the flux was inhibited. Measurements of the ATP hydrolysis that is thought to be associated with the uncoupled sodium efflux showed that this hydrolysis was strongly inhibited.8. The different effects of vanadate on the different fluxes are discussed, and related to the way in which vanadate is thought to act on the sodium pump.

Biological Transport, Active↗
PubMed1991

Characterization of vanadate-dependent NADH oxidation stimulated by Saccharomyces cerevisiae plasma membranes.

Plasma membrane-stimulated vanadate-dependent NADH oxidation has been characterized in Saccharomyces cerevisiae. This activity is specific for vanadate, because molybdate, a similar metal oxide, did not substitute for vanadate in the reaction. Vanadate-dependent plasma membrane-stimulated NADH oxidation activity was dependent on the concentrations of vanadate, NADH, and NADPH and required functional plasma membranes; no stimulation occurred in the presence of boiled membranes or bovine serum albumin. The dependence of membrane-stimulated vanadate-dependent NADH oxidation was not linearly dependent on added membrane protein. The activity was abolished by the superoxide anion scavenger superoxide dismutase and was stimulated by paraquat and NADPH. These data are consistent with the previously proposed chain reaction for vanadate-dependent NADH oxidation. The role of the plasma membrane appears to be to stimulate superoxide radical formation, which is coupled to NADH oxidation by vanadate. 51V-nuclear magnetic resonance studies are consistent with the hypothesis that a phosphovanadate anhydride is the stimulatory oxyvanadium species in the phosphate buffers used at pHs 5.0 and 7.0. In phosphate buffers, compared with acetate buffers, the single vanadate resonance was shifted upfield at both pH 5.0 and pH 7.0, which is characteristic of the phosphovanadate anhydride. Since the cell contains an excess of phosphate to vanadate, the phosphovanadate anhydride may be involved in membrane-mediated vanadate-dependent NADH oxidation in vivo.

Cell Fractionation↗
PubMed1983

Vanadate effect on the Na,K-ATPase and the Na-K pump in in vitro-grown rat vascular smooth muscle cells.

The impact of vanadate on the Na,K-ATPase system in the vascular smooth muscle cell is poorly understood. The present study describes the kinetics of the effect of vanadate on Na,K-ATPase and the Na-K pump in in vitro grown rat VSMC's. Vanadate interaction with the Na,K-ATPase system in vascular smooth muscle cells was examined by observing its influence on ouabain-sensitive adenosine triphosphate hydrolysis in disrupted cells rendered permeable by osmotic shock, and the uptake of rubidium by intact cells. The I50 for vanadate inhibition of ouabain-sensitive hydrolysis of adenosine triphosphate occurred at vanadate concentrations of 10(-6) to 10(-7) M. This inhibition was potassium dependent. The maximal inhibitory effect of vanadate occurred at potassium concentrations of 10-20 mEq/liter. Sodium exerted a moderate antagonistic influence on vanadate inhibition of ouabain-sensitive adenosine triphosphate hydrolysis. Rubidium uptake by vascular smooth muscle cells was not altered within 120 minutes when 10(-5) M vanadate was added to the medium containing intact vascular smooth muscle cells. Yet, vanadium concentrations in the vascular smooth muscle cells within this incubation period reached levels 1.48-fold higher than the extracellular vanadate concentrations of 10(-5) M. These observations indicate that vanadate is a potent inhibitor of the VSMC Na,K-ATPase in disrupted vascular smooth muscle cells. However, in intact vascular smooth muscle cells vanadium gaining access into the vascular smooth muscle cell's interior does not inhibit the Na-K pump, probably because of its binding to intracellular proteins and/or conversion from the vanadate to the vanadyl ion.

Animals↗
PubMed1992

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads.

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Antilipolytic actions of vanadate and insulin in rat adipocytes mediated by distinctly different mechanisms.

Vanadate, which mimics the biological effects of insulin, also inhibits lipolysis in rat adipocytes. Here we demonstrate that the antilipolytic effect of vanadate differs from that of insulin at least by the five following criteria: 1) vanadate inhibits lipolysis mediated by high (supraphysiological) concentrations of catecholamines; 2) vanadate antagonizes (Bu)2cAMP-mediated lipolysis; 3) vanadate antagonizes isobutylmethylxanthine-dependent lipolysis, 4) vanadate inhibits lipolysis mediated by okadaic acid; and 5) wortmannin, which blocks the antilipolytic effect of insulin, fails to block vanadate-mediated antilipolysis. Vanadate does activate phosphoinositol 3-kinase, and wortmannin blocks this activation. Our working hypothesis assumes that all of the insulin-like effects of vanadate, including antilipolysis, are initiated by the inhibition of protein phosphotyrosine phosphatases (PTPases). Among documented PTPase inhibitors we found that VOSO4 (oxidation state +4), several organic vanadyl compounds (+4), zinc (Zn2+), tungstate (W), and molybdate (Mo) also had antilipolytic activity. The order of potency was vanadyl acetylacetonate > or = VOSO4 > or = NaVO3 > or = vanadyl-dipicolinate > Zn2+ >> W > Mo, and it correlated better with the inhibition of adipose membranal-PTPases in cell-free experiments. We have concluded that the antilipolytic effect of vanadate is 1) mechanistically distinct from that of insulin, 2) independent of phosphoinositol 3-kinase activation, and 3) independent of the lipolytic cascade. We also strongly suggest that the antilipolytic effect of vanadate emanates from inhibiting adipose membranal, rather than cytosolic PTPases, and present preliminary data showing distinct differences in catalysis between these two PTPase categories. Overall, the study indicates that antilipolysis can be manifested via alternative, insulin-independent, signal-transducing pathways.

Adipocytes↗
PubMed1991

The influence of vanadate on insulin counter-regulatory hormones in obese fa/fa rats.

Vanadate has been shown to improve glucose homoeostasis in mildly glucose-intolerant and severely insulin-resistant fa/fa rats. The present study examined whether changes in insulin counter-regulatory hormones contribute to this beneficial effect of vanadate. Since oral administration of Na3VO4 caused a decrease in food intake and stopped the increase in body weight, vanadate-treated fa/fa rats were compared with both controls with food available ad libitum and pair-fed rats. Slightly lower plasma glucose levels were maintained in conjunction with markedly lower plasma insulin levels in vanadate-treated rats, and this effect was not simply due to the smaller body weight of the animals. Compared with control rats, treatment with vanadate affected neither basal plasma glucagon levels nor the increase in glucagon levels observed after insulin-induced hypoglycaemia or after i.v. injection of arginine. Compared with pair-fed rats, treatment with vanadate prevented the fall in basal plasma glucagon and its exaggerated rise in response to insulin that mere food restriction produced. Plasma corticosterone levels were high in fa/fa rats. Vanadate and pair-feeding similarly decreased basal plasma levels of corticosterone as well as nocturnal corticosteronuria. Thus the attenuation of the hypercorticism of fa/fa rats results from the reduction in body weight gain rather than from a specific action of vanadate. Vanadate did not influence urinary excretion of noradrenaline, an index of neural sympathetic activity, but prevented the increase in adrenaline excretion, an index of adrenal medulla activity, that was produced by food restriction in pair-fed rats. In conclusion, vanadate administration has no or little specific effects on three major insulin counter-regulatory hormones. This reinforces the suggestion that the beneficial effects of vanadate on glucose homoeostasis in fa/fa rats are mainly due to a correction of insulin resistance in peripheral tissues.

Animals↗
PubMed1994

Vanadate augments insulin-stimulated insulin receptor kinase activity and prolongs insulin action in rat adipocytes. Evidence for transduction of amplitude of signaling into duration of response.

Vanadate, a protein tyrosine phosphatase inhibitor, preserves insulin-stimulated lipogenesis after removal of insulin. To investigate the mechanism of this action of vanadate, lipogenesis was studied in isolated rat adipocytes exposed to vanadate for 60 min followed by insulin for 15 min at 37 degrees C. Vanadate (10-50 microM) prolonged insulin-stimulated lipogenesis. The half-time (t1/2) of the decay in insulin (0.34 nM)-stimulated lipogenesis after removal of insulin by washing in pH 7.0 followed by pH 7.6 buffer was 21 min in the absence and 59 min in the presence of vanadate. During these conditions, vanadate did not alter insulin binding nor the removal of insulin by the series of washes. In contrast to lipogenesis, the t1/2 of the decay in insulin receptor tyrosine kinase (IRK) activity, assayed with the artificial substrate Poly[Glu:Tyr] (4:1), was not significantly prolonged by vanadate (6 vs. 6.8 min). However, insulin-stimulated IRK activity was markedly augmented by vanadate to 319 +/- 19% of insulin alone, associated with a similar augmentation of phosphotyrosine incorporation into the insulin receptor beta-subunit determined by Western blotting with antiphosphotyrosine antibodies. To determine the relationship between prolongation of lipogenesis and the increase in IRK, adipocytes were exposed to 17.2 nM insulin to activate the IRK to the same extent as insulin (0.34 nM) plus vanadate (maximum activation). During these two conditions, the decay of lipogenesis was similar and after stimulation with 17.2 nM insulin was not prolonged by vanadate. We conclude that vanadate prolongs insulin action at insulin concentrations that do not maximally activate the IRK by augmenting IRK activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocytes↗
PubMed1996

The insulin-mimetic agent vanadate promotes receptor endocytosis and inhibits intracellular ligand-receptor degradation by a mechanism distinct from the lysosomotropic agents.

Vanadate (sodium orthovanadate) is an insulin-mimetic agent and phosphotyrosine phosphatase inhibitor that has been proposed as a potential therapeutic agent for diabetes. We previously reported that vanadate decreased the number of cell-surface insulin receptors but inhibited receptor degradation in cultured lymphocytes (IM-9) (1). To determine whether vanadate affected receptors without intrinsic tyrosine kinase activity, its effects on LDL and transferrin receptors and their ligands were examined. Vanadate exposure resulted in a dose- and time-dependent decrease in LDL binding to cultured human fibroblasts associated with a decrease in cell surface receptor number while total solubilized cell LDL receptors increased. Vanadate also inhibited the LDL-mediated downregulation of total cellular LDL receptors in the absence and presence of cycloheximide consistent with an inhibition of LDL receptor degradation. In the case of the ligand, vanadate augmented the accumulation of intact 125I-LDL associated with an inhibition of up to 80% of the ability of LDL to decrease cholesterol synthesis. Since these actions were similar to the effects of lysosomotropic agents, we examined the effect of vanadate on intraendosomal pH using the fluorescent probe acridine orange. In contrast with chloroquine and NH4Cl, vanadate did not neutralize the pH of the acidic intracellular compartment. Furthermore, after a transient insulin-like effect, chronic exposure to vanadate diminished 125I-diferric transferrin binding to rat adipocytes. In contrast with the inhibitory action of NH4Cl, intracellular 59Fe uptake remained unaffected and was proportional to cell-surface binding capacity in the presence of vanadate. These data demonstrate a chronic effect of vanadate to promote the accumulation of intracellular receptors and to inhibit ligand and receptor degradation. The latter effect is not mediated by pH changes, appears to be localized to a late endosomal/lysosomal compartment, and suggests a possible role for tyrosine dephosphorylation in the regulation of receptor-ligand degradation.

Adipocytes↗
PubMed1994

Stimulation of Cl- secretion by AlF4- and vanadate in T84 cells.

We investigated the mechanism of Cl- secretion by fluoroaluminate(AlF4-) and sodium orthovanadate(vanadate) using the human colonic T84 cell line. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure short circuit current(ISC). Serosal addition of AlF4- or vanadate to T84 monolayers produced a sustained increase in ISC. Removal of Ca2+ from the serosal bathing solution partially inhibited AlF4-(-)and vanadate-induced ISC, and readministration of Ca2+ restored AlF4-(-)and vanadate-induced ISC. Carbachol application in the presence of forskolin, AlF4- or vanadate induced a synergistic increase of ISC. Forskolin and vanadate significantly increased cellular cAMP level, while carbachol and AlF4- did not. Carbachol, AlF4- and vanadate significantly increased [Ca2+]i. After Na+ in mucosal bathing solution was replaced with K+, and the mucosal membrane of T84 cell was permeabilized with amphotericin B, AlF4-, vanadate, and carbachol increased K+ conductance, but forskolin did not. After sodium chloride in serosal bathing solution was replaced with sodium gluconate and the serosal membrane was permeabilized with nystatin, forskolin, AlF4-, and vanadate increased Cl- conductance, but carbachol did not. AlF4-(-)induced ISC was remarkably inhibited by the pretreatment of pertussis toxin(2 micrograms/ml) for 2 hours. These results indicate that AlF4- and vanadate can increase Cl- secretion via simultaneous stimulation of Cl- channel and K+ channel in T84 cells. However, the AlF4- action is mostly attributed to stimulation of pertussis toxin-sensitive G-proteins, whereas the vanadate action mostly results from G protein-independent mechanisms.

Aluminum↗
PubMed1990

Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells.

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.

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