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Enhanced Sensitivity of Insulin-resistant Adipocytes to Vanadate Is Associated with Oxidative Stress and Decreased Reduction of Vanadate (+5) to Vanadyl (+4)*

  • Bing Lu
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • David Ennis
    Footnotes
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Robert Lai
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Elena Bogdanovic
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Rinna Nikolov
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Lisa Salamon
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Claire Fantus
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Hoang Le-Tien
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • I. George Fantus
    Correspondence
    To whom correspondence should be addressed: Mount Sinai Hospital, 600 University Ave., Suite 780, Toronto, Ontario M5G 1X5, Canada. Tel.: 416-586-8665; Fax: 416-586-8785
    Affiliations
    Department of Medicine, Mount Sinai Hospital, Banting and Best Diabetes Centre and Department of Physiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada
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  • Author Footnotes
    * This work was supported by a grant from the Canadian Institutes of Health Research (to I. G. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Supported in part by a Banting and Best Center summer studentship.
Open AccessPublished:September 21, 2001DOI:https://doi.org/10.1074/jbc.M106783200
      Vanadate (sodium orthovanadate), an inhibitor of phosphotyrosine phosphatases (PTPs), mimics many of the metabolic actions of insulin in vitro and in vivo. The potential of vanadate to stimulate glucose transport independent of the early steps in insulin signaling prompted us to test its effectiveness in an in vitro model of insulin resistance. In primary rat adipocytes cultured for 18 h in the presence of high glucose (15 mm) and insulin (10−7m), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the insulin-resistant cells (EC50 for control, 325 ± 7.5 μm; EC50 for insulin-resistant, 171 ± 32 μm; p < 0.002). Enhanced sensitivity was also present for vanadate stimulation of insulin receptor kinase activity and autophosphorylation and Akt/protein kinase B Ser-473 phosphorylation consistent with more effective PTP inhibition in the resistant cells. Investigation of this phenomenon revealed that 1) depletion of GSH with buthionine sulfoximine reproduced the enhanced sensitivity to vanadate while preincubation of resistant cells withN-acetylcysteine (NAC) prevented it, 2) intracellular GSH was decreased in resistant cells and normalized by NAC, 3) exposure to high glucose and insulin induced an increase in reactive oxygen species, which was prevented by NAC, 4) EPR (electron paramagnetic resonance) spectroscopy showed a decreased amount of vanadyl (+4) in resistant and buthionine sulfoximine-treated cells, which correlated with decreased GSH and increased vanadate sensitivity, while total vanadium uptake was not altered, and 5) inhibition of recombinant PTP1Bin vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the parodoxical increased sensitivity to vanadate in hyperglycemia-induced insulin resistant adipocytes is due to oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4). Thus, sensitivity of PTP inhibition and glucose transport to vanadate is regulated by cellular redox state.
      IR
      insulin receptor
      IRS
      insulin receptor substrate
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PKB
      protein kinase B
      PTP
      protein-tyrosine phosphatase
      2-DG
      2-deoxyglucose, BSO, buthionine sulfoximine
      Tyr(P)
      phosphotyrosine, WGA, wheat germ agglutinin
      ECL
      enhanced chemiluminescence
      EPR
      electron paramagnetic resonance
      DCF
      dichorofluorescein
      DCF-DA
      dichorofluorescein-diacetate
      NAC
      N-acetylcysteine
      PNPP
      para-nitrophenyl phosphate
      DTT
      dithiothreitol
      GST
      glutathioneS-transferase
      ROS
      reactive oxygen species
      SH2
      Src homology 2
      PAGE
      polyacrylamide gel electrophoresis
      DMEM
      Dulbecco's modified Eagle's medium
      dd
      double distilled
      G
      gauss
      PBS
      phosphate-buffered saline
      BSA
      bovine serum albumin
      high G/I
      high concentrations of glucose and insulin
      Insulin resistance of glucose transport is a well documented characteristic of type 2 diabetes mellitus (
      • Kahn C.R.
      ,
      • Olefsky J.M.
      • Nolan J.J.
      , ). The etiology of the insulin resistance appears to be multifactorial since it is found in subjects with obesity, those with impaired glucose tolerance, as well as in those with overt type 2 diabetes. There is evidence that both genetic and acquired factors contribute to the insulin resistance (,
      • Beck-Nielsen H.
      • Groop L.C.
      ,
      • O'Doherty R.
      • Stein D.
      • Foley J.
      ). In type 2 diabetic subjects, peripheral target tissue insulin resistance is characterized by defects in both sensitivity, manifested as a rightward shift in the insulin dose-response curve, and responsiveness, a decrease in maximum response (
      • Zierath J.R.
      • Galuska D.
      • Nolte L.A.
      ,
      • Ciaraldi T.P.
      • Kolterman O.G.
      • Scarlett J.A.
      • Kao M.
      • Olefsky J.M.
      ,
      • Shepherd P.R.
      • Kahn B.B.
      ). This combination of defects has been reproduced in vitro in isolated rat adipocytes incubated for several hours in the presence of a combination of high glucose and high insulin concentrations (
      • Garvey W.T.
      • Olefsky J.M.
      • Matthaei S.
      • Marshall S.
      ). In this in vitro model, defects in insulin stimulation of glucose transport and glucose transporter translocation are prominent, similar to that found in adipocytes isolated from subjects with type 2 diabetes (
      • Shepherd P.R.
      • Kahn B.B.
      ,
      • Garvey W.T.
      • Olefsky J.M.
      • Matthaei S.
      • Marshall S.
      ,
      • Traxinger R.R.
      • Marshall S.
      ,
      • Kashiwagi A.
      • Verso M.A.
      • Andrews J.
      • Vasquez B.
      • Reaven G.
      • Foley J.E.
      ).
      The signaling pathway by which insulin stimulates glucose transport is only partially understood. Insulin binds to its receptor (IR)1 and activates its intrinsic Tyr kinase, resulting in phosphorylation of the receptor and its substrates, IRS-1 and -2 (
      • Cheatham B.
      • Kahn C.R.
      ,
      • White M.F.
      ,
      • Holman G.D.
      • Kasuga M.
      ). These, in turn, act as docking proteins for several SH2 domain-containing proteins such as Grb2, SH2 domain-containing phosphatase-2, and p85 (
      • Cheatham B.
      • Kahn C.R.
      ,
      • White M.F.
      ,
      • Holman G.D.
      • Kasuga M.
      ,
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun X.J.
      • Shoelson S.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ). Binding of the SH2 domains of the p85 subunit of PI 3-kinase results in activation of the p110 catalytic subunit and the generation of the lipid products phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (
      • Myers Jr., M.G.
      • Backer J.M.
      • Sun X.J.
      • Shoelson S.
      • Hu P.
      • Schlessinger J.
      • Yoakim M.
      • Schaffhausen B.
      • White M.F.
      ,
      • Rameh L.E.
      • Cantley L.C.
      ). These lipids facilitate phosphoinositide-dependent kinase-1- and -2-mediated phosphorylation of several substrate enzymes such as PKB (Akt/RAC-PK) and atypical protein kinase C ζ and λ (
      • Alessi D.R.
      • James S.R.
      • Downes C.P.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • Cohen P.
      ,
      • Stephens L.
      • Anderson K.
      • Stokoe D.
      • Erdjument-Bromage H.
      • Painter G.F.
      • Holmes A.B.
      • Gaffney P.R.
      • Reese C.B.
      • McCormick F.
      • Tempst P.
      • Coadwell J.
      • Hawkins P.T.
      ,
      • Kohn A.D.
      • Summers S.A.
      • Birnbaum M.J.
      • Roth R.A.
      ,
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ). The requirement for the IR, IRS 1/2, PI 3-kinase, and either or both PKB and atypical protein kinase Cs for glucose transport stimulation by insulin is well documented, whereas less is known about the more distal signaling events (
      • Shepherd P.R.
      • Kahn B.B.
      ,
      • Cheatham B.
      • Kahn C.R.
      ,
      • White M.F.
      ,
      • Holman G.D.
      • Kasuga M.
      ,
      • Wang Q.
      • Somwar R,.
      • Bilan P.J.
      • Liu Z.
      • Jin J.
      • Woodgett J.R.
      • Klip A.
      ,
      • Bandyopadhyay G.
      • Standaert M.L.
      • Zhao L., Yu, B.
      • Avignon A.
      • Galloway L.
      • Karnam P.
      • Moscat J.
      • Farese R.V.
      ,
      • Pessin J.E.
      • Thurmond D.C.
      • Elmendorf J.S.
      • Coker K.J.
      • Okada S.
      ).
      Vanadate (+5) and vanadyl (+4) compounds have been well documented to mimic many of the actions of insulin (
      • Shechter Y.
      ,
      • Orvig C.
      • Thompson K.H.
      • Battell M.
      • McNeill J.H.
      ,
      • Brichard S.M.
      • Henquin J.-C.
      ,
      • Tsiani E.
      • Fantus I.G.,.
      ). Glucose uptake and metabolism are stimulated in fat and muscle tissue (
      • Tolman E.L.
      • Barris E.
      • Burns M.
      • Pansini A.
      • Partridge R.
      ,
      • Shechter Y.
      • Karlish S.J.
      ), lipolysis is inhibited (
      • Fantus I.G.
      • Kadota S.
      • Deragon G.
      • Foster B.
      • Posner B.I.
      ), and hepatic glucose output is suppressed (
      • Gil J.
      • Miralpeix M.
      • Carreras J.
      • Bartrons R.
      ,
      • Bruck R.
      • Prigozin H.
      • Krepel Z.
      • Rotenberg P.
      • Shechter Y.
      • Bar-Meir S.
      ). Oral administration of vanadate and vandyl resulted in lowering of glucose concentrations in rodent models of both type 1 and type 2 diabetes (
      • Heyliger C.E.
      • Tahiliani A.G.
      • McNeill J.H.
      ,
      • Brichard S.M.
      • Assimacopoulos-Jeannet F.
      • Jeanrenaud B.
      ,
      • Meyerovitch J.
      • Rothenberg P.
      • Shechter Y.
      • Bonner-Weir S.
      • Kahn C.R.
      ,
      • Brichard S.M.
      • Okitolonda W.
      • Henquin J.C.
      ). Recent studies in human diabetic subjects suggest that these vanadium compounds can lower glycemia and improve lipid levels (
      • Cohen N.
      • Halberstam M.
      • Shlimovich P.
      • Chang C.J.
      • Shamoon H.
      • Rossetti L.
      ,
      • Goldfine A.B.
      • Simonson D.C.
      • Folli F.
      • Patti M.E.
      • Kahn C.R.
      ), whereas this treatment was less effective in improving insulin resistance in nondiabetic obese subjects (
      • Halberstam M.
      • Cohen N.
      • Shlimovich P.
      • Rossetti L.
      • Shamoon H.
      ).
      The mechanism by which vanadate mimics insulin is not completely clear. Although protein-tyrosine phosphatase (PTP) inhibition appears to be important, the role of the IR and/or other Tyr kinases remains controversial (
      • Green A.
      ,
      • D'Onofrio F.
      • Le M.Q.
      • Chiasson J.L.
      • Srivastava A.K.
      ,
      • Pugazhenthi S.
      • Khandelwal R.L.
      ). In some studies, IR kinase activation by vanadate has been documented (
      • Tamura S.
      • Brown T.A.
      • Whipple J.H.
      • Fujita-Yamaguchi Y.
      • Dubler R.E.
      • Cheng K.
      • Larner J.
      ,
      • Fantus I.G.
      • Ahmad F.
      • Deragon G.
      ). However, it has been found that insulin-mimetic effects can be stimulated independent of any IR activation (
      • Green A.
      ,
      • D'Onofrio F.
      • Le M.Q.
      • Chiasson J.L.
      • Srivastava A.K.
      ,
      • Strout H.V.
      • Vicario P.P.
      • Saperstein R.
      • Slater E.E.
      ). The activation of cytosolic (
      • Shisheva A.
      • Shechter Y.
      ) and membrane-associated (
      • Ida M.
      • Imai K.
      • Hashimoto S.
      • Kawashima H.
      ) low molecular weight tyrosine kinases have been linked with some of these metabolic effects. However, since these associations were based on inhibition by staurosporine, which inhibits a wide spectrum of kinases, their role remains unclear. In other studies, glucose transport stimulation by vanadate, in contrast to that stimulated by insulin, was found to be independent of the enzymes PI 3-kinase (
      • Ida M.
      • Imai K.
      • Hashimoto S.
      • Kawashima H.
      ,
      • Tsiani E.
      • Bogdanovic E.
      • Sorisky A.
      • Nagy L.
      • Fantus I.G.
      ) and PKB (
      • Tsiani E.
      • Bogdanovic E.
      • Sorisky A.
      • Nagy L.
      • Fantus I.G.
      ). These data raised the possibility that vanadium compounds may have a unique ability to stimulate and/or enhance insulin metabolic effects in some states of insulin resistance.
      To determine the efficacy of vanadate to stimulate glucose transport in an insulin resistant target tissue, we examined its actions in the insulin-resistant rat adipocyte model induced by high glucose and insulin. We found that there was a paradoxical enhanced sensitivity of glucose uptake to vanadate in the insulin-resistant cells,i.e. a leftward shift in the vanadate dose-response curve. This was associated with a concomitant enhanced sensitivity of the insulin-resistant cells to vanadate-stimulated Tyr phosphorylation of the IR and of its intrinsic Tyr kinase activity as well as Ser-473 phosphorylation of the downstream kinase PKB. Investigation of the possible mechanism showed that there was a greater intracellular concentration of vanadate (+5) relative to vanadyl (+4) in the insulin-resistant adipocytes. In vitro studies showed that inhibition of PTP1B phosphatase activity was more sensitive to vanadate (+5) than vanadyl (+4). These data demonstrate that vanadate (+5) is a more potent PTP inhibitor than vanadyl (+4) and that there is decreased reduction of vanadate (+5) to vanadyl (+4) in the insulin-resistant adipocytes. They are also consistent with our observations of increased generation of reactive oxygen species (ROS) in adipocytes exposed to high glucose and insulin and of the influence of altered cellular levels of GSH (glutathione) on vanadate sensitivity

      DISCUSSION

      The mechanism of the insulin-mimetic effects of vanadium compounds is not completely understood. Although well documented to inhibit PTPs (
      • Shechter Y.
      ,
      • Fantus I.G.
      • Kadota S.
      • Deragon G.
      • Foster B.
      • Posner B.I.
      ,
      • Swarup G.
      • Speeg Jr., K.V.
      • Cohen S.
      • Garbers D.L.
      ), vanadium compounds have been suggested to directly activate tyrosine kinases (
      • Stankiewicz P.J.
      • Tracey A.S.
      ), inhibit glucose-6-phosphatase (
      • Schulz L.O.
      ), inhibit protein degradation (
      • Fantus I.G.
      • George R.
      • Tang S.
      • Chong P.
      • Poznansky M.J.
      ), alter phosphoinositide metabolism (
      • Atkinson T.P.
      • Lee C.W.
      • Rhee S.G.
      • Hohman R.J.
      ,
      • Blake R.A.
      • Walker T.R.
      • Watson S.P.
      ), and bind to a variety of small molecules, such as ADP, GDP, and NADH (
      • Crans D.C.
      • Mahroof-Tahir M.
      • Keramidas A.D.
      ,
      • Crans D.C.
      ), which may all in turn influence cell signaling. However, the predominant effect on PTPs and the ability of general Tyr kinase inhibitors to block the insulin-like actions of vanadium (
      • Shisheva A.
      • Shechter Y.
      ,
      • Elberg G.
      • He Z.
      • Li J.
      • Sekar N.
      • Shechter Y.
      ,
      • Ida M.
      • Imai K.
      • Hashimoto S.
      • Kawashima H.
      ) indicate that PTP inhibition is the primary mechanism. This concept is also supported by our previous observation that peroxovanadium (pervanadate) is a more potent insulin-mimetic agent and a correspondingly more powerful PTP inhibitor (
      • Fantus I.G.
      • Kadota S.
      • Deragon G.
      • Foster B.
      • Posner B.I.
      ).
      Whether the more oxidized form vanadate (+5) or reduced form, vanadyl (+4) is the more important and relevant insulin-mimetic form in intact cells and in vivo has been controversial (
      • Elberg G.
      • Li J.
      • Shechter Y.
      ,
      • Shaver A.
      • Ng J.B.
      • Hall D.A.
      • Posner B.I.
      ,
      • Li J.
      • Elberg G.
      • Crans D.C.
      • Shechter Y.
      ). Thus, in some studies it was demonstrated that a cytosolic Tyr kinase was activated by vanadate (+5) while vanadyl (+4) directly inhibited receptor Tyr kinases including the IR kinase (
      • Elberg G.
      • Li J.
      • Shechter Y.
      ). In a subsequent report, the same group suggested that vanadyl (+4) had unique properties to activate a cytosolic protein-tyrosine kinase and that adipocytes were more sensitive to vanadyl than vanadate (
      • Li J.
      • Elberg G.
      • Crans D.C.
      • Shechter Y.
      ). The complex chemistry of vanadium in solution and particularly in living cells (
      • Crans D.C.
      • Mahroof-Tahir M.
      • Keramidas A.D.
      ,
      • Crans D.C.
      ), along with the uncertainty about which Tyr kinase(s) is responsible for the insulin-like bioeffects, has made this a difficult question to resolve.
      In this study we found that the dose-dependent stimulation of glucose uptake in adipocytes was paradoxically more sensitive to vanadate in cells rendered insulin-resistant by exposure to high glucose and insulin. The increased sensitivity of glucose uptake was paralleled by an increased sensitivity of IR phosphorylation and Tyr kinase activity. Although the role of the IR in mediating the glucose transport stimulated by vanadate is not clear and the IR may not be the kinase involved, its Tyr phosphorylation serves as a cellular marker of PTP inhibition. Furthermore, we found an enhanced sensitivity of PKB Ser-473 phosphorylation in response to vanadate in the insulin-resistant adipocytes. This Ser kinase has been demonstrated to be a necessary component of the signaling pathway of insulin to stimulate glucose uptake. We noted that the concentrations of vanadate that achieved maximum Ser-473 phosphorylation were higher (∼5–10 mm) than those required to achieve maximum IR activation (1 mm). Since vanadate is a nonspecific PTP inhibitor, this observation is consistent with activation of other Tyr kinases and recruitment of additional pools of PKB not activated by insulin. Similar results were observed in muscle cells with pervanadate (
      • Tsiani E.
      • Bogdanovic E.
      • Sorisky A.
      • Nagy L.
      • Fantus I.G.
      ). Taken together, these results are consistent with an enhanced activity of vanadate to inhibit PTPs in insulin-resistant cells.
      One possible explanation for the enhanced sensitivity to vanadate could have been an elevated PTP activity in the insulin-resistant cells, since the apparent efficacy of vanadium compounds to mimic insulin depends on PTP activity. However, in this model of insulin resistance, we have recently found that IR-related PTP activity is diminished rather than elevated (
      • Tang S.
      • Le-Tien H.
      • Goldstein B.
      • Shin P.
      • Lai R.
      • Fantus I.G.
      ). These findings are in agreement with otherin vitro (
      • Kroder G.
      • Bossenmaier B.
      • Kellerer M.
      • Capp E.
      • Stoyanov B.
      • Muhlhofer A.
      • Berti L.
      • Horikoshi H.
      • Ullrich A.
      • Haring H.
      ) and in vivo (
      • Kusari J.
      • Kenner K.A.
      • Suh K.I.
      • Hill D.E.
      • Henry R.R.
      ,
      • Worm D.
      • Vinten J.
      • Staehr P.
      • Henriksen J.E.
      • Handberg A.
      • Beck-Nielsen H.
      ) studies demonstrating that hyperglycemia-induced insulin resistance is not associated with increased IR Tyr dephosphorylation or PTP activity. An alternative explanation was postulated based on several phenomena.
      First, it is known that, upon entering cells, vanadate (+5) is reduced to vanadyl (+4). This may be a mechanism to limit cell toxicity, as vanadate and particularly, pervanadate, have been found to cause oxidative stress (
      • Krejsa C.M.
      • Nadler S.G.
      • Esselstyn J.M.
      • Kavanagh T.J.
      • Ledbetter J.A.
      • Schieven G.L.
      ), DNA damage (
      • Hiort C.
      • Goodisman J.
      • Dabrowiak J.C.
      ), and induce neoplastic transformation in cultured fibroblasts (
      • Sabbioni E.
      • Pozzi G.
      • Devos S.
      • Pintar A.
      • Casella L.
      • Fischbach M.
      ). In the latter study, vanadyl (+4) alone did not induce neoplastic transformation whereas vanadate (+5) had minimal activity. However, depletion of GSH with diethylmaleate sensitized cells to vanadate-induced transformation. These results, combined with the proposal that vanadate (+5) was the major inhibitor of PTPs rather than vanadyl (+4) (see above) suggested that, in our resistant cells, there was decreased reduction of vanadate to vanadyl and/or increased re-oxidation of vanadyl to vanadate. Finally, exposure of various tissues to high concentrations of glucose has been documented to result in oxidative stress (
      • Wolff S.P.
      • Jiang Z.Y.
      • Hung J.V.
      ,
      • Baynes J.W.
      ) and depletion of GSH (
      • Kashiwagi A.
      • Asahina T.
      • Ikebuchi M.
      • Tanaka Y.
      • Takagi Y.
      • Nishio Y.
      • Kikkawa R.
      • Shigeta Y.
      ,
      • Takayuki A.
      • Atsunori K.
      • Yoshihiko N.
      • Motoyoshi I.
      • Natsuki H.
      • Yasushi T.
      • Yoshihumi T.
      • Yukikazu S.
      • Ryuichi K.
      • Yukio S.
      ,
      • Ceriello A.
      • dello Russo P.
      • Amstad P.
      • Cerutti P.
      ,
      • Trocino R.A.
      • Akazawa S.
      • Ishibashi M.
      • Matsumoto K.
      • Matsuo H.
      • Yamamoto H.
      • Goto S.
      • Urata Y.
      • Kondo T.
      • Nagataki S.
      ).
      In the present studies, depletion of GSH with BSO reproduced the leftward shift in the vanadate dose-response curve seen in the insulin-resistant adipocytes. Furthermore, pretreatment of the adipocytes with the antioxidant and GSH precursor, NAC, blocked the enhanced sensitivity caused by the high G/I exposure. To determine whether these perturbations were associated with the predicted changes in vanadate to vanadyl conversion, EPR spectroscopy was performed. This revealed that enhanced vanadate sensitivity was associated with a decreased intensity of the vanadyl (+4) signal. Since total cell-associated vanadium, determined by atomic absorption, was similar under all these conditions, the EPR results indicate that, in the insulin-resistant and BSO-treated cells, there was a greater amount of vanadate (+5). In addition, these data imply that the insulin-resistant cells were under oxidative stress. Measurements of GSH showed that, as expected, BSO markedly depleted intracellular GSH. Exposure to high G/I also significantly decreased GSH, which was prevented by pretreatment with NAC. A decrease in cellular antioxidant concentration is indicative of oxidative stress, which was confirmed by the increased fluorescence intensity of DCF-loaded adipocytes exposed to high G/I. Taken together, these experiments indicate that vanadate (+5) is a more potent PTP inhibitor than vanadyl (+4) and that the increased intracellular vanadate (+5) in insulin-resistant adipocytes explains the paradoxical enhanced sensitivity to vanadate. This conclusion, based on results in intact cells, was confirmed by our in vitro findings, which demonstrated a greater potency of vanadate compared with vanadyl to inhibit recombinant PTP1B, an intracellular PTP shown recently to be a major regulator of IR Tyr dephosphorylation (
      • Kenner K.A.
      • Anyanwu E.
      • Olefsky J.M.
      • Kusari J.
      ,
      • Seely B.L.
      • Staubs P.A.
      • Reichart D.R.
      • Berhanu P.
      • Milarski K.L.
      • Saltiel A.R.
      • Kusari J.
      • Olefsky J.M.
      ,
      • Elchebly M.
      • Payette P.
      • Michaliszyn E.
      • Cromlish W.
      • Collins S.
      • Lee-Loy A.
      • Normandin D.
      • Cheng A.
      • Himms-Hagen J.
      • Chan C.-C.
      • Ramachandran C.
      • Gresser M.J.
      • Tremblay M.L.
      • Kennedy B.P.
      ). The greater efficacy of vanadate is also consistent with its trigonal bipyramidal structure, which resembles phosphate more closely than the tetrahedral geometry of vanadyl (
      • Crans D.C.
      • Mahroof-Tahir M.
      • Keramidas A.D.
      ,
      • Shaver A.
      • Ng J.B.
      • Hall D.A.
      • Posner B.I.
      ).
      In a recent study, Cuncic et al. (
      • Cuncic C.
      • Detich N.
      • Ethier D.
      • Tracey A.S.
      • Gresser M.J.
      • Ramachandran C.
      ) reported that cultured Jurkat T-lymphoma cells, which were unresponsive to vanadate, became sensitive to stimulation of protein tyrosine phosphorylation after GSH depletion. Pretreatment of cells with vanadate had no effect on PTP activity of solubilized cell membrane fractions, whereas after GSH depletion ∼50% inhibition by vanadate was observed. The authors suggested that the lack of reversibility of PTP inhibition in vitro in the presence of vanadium chelators such as EDTA was consistent with intracellular formation of pervanadate, which was previously demonstrated to cause irreversible inhibition by oxidizing the free PTP sulfhydryl to SO2 (
      • Huyer G.
      • Liu S.
      • Kelly J.
      • Moffat J.
      • Payette P.
      • Kennedy B.
      • Tsaprailis G.
      • Gresser M.J.
      • Ramachandran C.
      ). Since pervanadate, similar to vanadate, is not paramagnetic and not seen by EPR spectroscopy, we cannot rule out a contribution of de novogenerated peroxovanadium to the increased sensitivity to vanadate observed in the adipocytes treated with high glucose. However, given that adipocytes are 500–1000-fold more sensitive to pervanadate than vanadate (
      • Fantus I.G.
      • Kadota S.
      • Deragon G.
      • Foster B.
      • Posner B.I.
      ,
      • Posner B.I.
      • Faure R.
      • Burgess J.W.
      • Bevan A.P.
      • Lachance D.
      • Zhang-Sun G.
      • Fantus I.G.
      • Ng J.B.
      • Hall D.A.
      • Lum B.S.
      • Shaver A.
      ) and considering the magnitude of the differences in sensitivity of glucose uptake and PTP inhibition observed, it is not necessary to invoke a contribution of generated pervanadate to explain our results.
      In summary, the greater intrinsic potency of vanadate (+5) compared with vanadyl (+4), especially in the nanomolar range as seen in the absence of DTT, along with the higher ratio of vanadate (+5)/vanadyl (+4) concentrations in the insulin-resistant cells appear adequate to explain the shifts in sensitivity of the biological responses seen in the intact cells.
      The use of vanadium compounds to treat diabetes has been successful in rodents (
      • Shechter Y.
      ,
      • Orvig C.
      • Thompson K.H.
      • Battell M.
      • McNeill J.H.
      ,
      • Brichard S.M.
      • Henquin J.-C.
      ,
      • Tsiani E.
      • Fantus I.G.,.
      ). In people with diabetes and overt hyperglycemia, oral administration of vanadium compounds did result in modest improvement in glucose concentrations (
      • Cohen N.
      • Halberstam M.
      • Shlimovich P.
      • Chang C.J.
      • Shamoon H.
      • Rossetti L.
      ,
      • Goldfine A.B.
      • Simonson D.C.
      • Folli F.
      • Patti M.E.
      • Kahn C.R.
      ). However, when administered to subjects with insulin resistance who did not manifest hyperglycemia, vanadate had no significant effect (
      • Halberstam M.
      • Cohen N.
      • Shlimovich P.
      • Rossetti L.
      • Shamoon H.
      ). These clinical observations are consistent with a more potent effect of vanadium in the presence of elevated glucose. It is possible that the oxidative stress induced by high glucose may increase sensitivity to vanadate in vivo as we found in isolated cells.
      Despite the difficulty in achieving effective circulating concentrations of vanadium in humans, interest in the inhibition of PTP1B and concomitant activation of the IR Tyr kinase remains high, particularly since enhanced insulin sensitivity and resistance to high fat diet-induced insulin resistance was reported in the PTP1B−/− mouse (
      • Elchebly M.
      • Payette P.
      • Michaliszyn E.
      • Cromlish W.
      • Collins S.
      • Lee-Loy A.
      • Normandin D.
      • Cheng A.
      • Himms-Hagen J.
      • Chan C.-C.
      • Ramachandran C.
      • Gresser M.J.
      • Tremblay M.L.
      • Kennedy B.P.
      ). The specificity of vanadium compounds for different PTPs may be amenable to modification by alteration of ligands (
      • Posner B.I.
      • Faure R.
      • Burgess J.W.
      • Bevan A.P.
      • Lachance D.
      • Zhang-Sun G.
      • Fantus I.G.
      • Ng J.B.
      • Hall D.A.
      • Lum B.S.
      • Shaver A.
      ,
      • McNeill J.H.
      • Yuen V.G.
      • Dai S.
      • Orvig C.
      ). This study now demonstrates that the relative potency of vanadium may be regulated by intracellular redox state such that increased efficacy may be achieved in tissues under oxidative stress. This property could be utilized as a method to target PTP inhibition to specific tissues.

      Acknowledgments

      We thank Dr. D. Templeton for help with the atomic absorption spectroscopy, Dr. J. Boggs for advice on EPR, G. Deragon for technical assistance, B. Baubinas for secretarial support, and Drs. C. Yip, S. Grinstein, and D. Crans for helpful discussion.

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