Enhanced sensitivity of insulin-resistant adipocytes to vanadate is associated with oxidative stress and decreased reduction of vanadate (+5) to vanadyl (+4).

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(-7) m), sensitivity to insulin-stimulated glucose transport was decreased. In contrast, there was a paradoxical enhanced sensitivity to vanadate of the insulin-resistant cells (EC(50) for control, 325 +/- 7.5 microm; EC(50) for insulin-resistant, 171 +/- 32 microm; 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 with N-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 PTP1B in vitro was more sensitive to vanadate (+5) than vanadyl (+4). In conclusion, the paradoxical 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.

Tyrosine phosphatase inhibitors, vanadate and pervanadate, stimulate glucose transport and GLUT translocation in muscle cells by a mechanism independent of phosphatidylinositol 3-kinase and protein kinase C.

Vanadate and pervanadate (pV) are protein tyrosine phosphatase (PTP) inhibitors that mimic insulin to stimulate glucose transport. To determine whether phosphatidylinositol (PI) 3-kinase is required for vanadate and pV, as it is for insulin, cultured L6 myotubes were treated with vanadate and pV. The two compounds stimulated glucose transport to levels similar to those stimulated by insulin; however, while PI 3-kinase activity and the increase in the lipid products PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were inhibited by wortmannin after stimulation by all three agents--insulin, vanadate, and pV--wortmannin blocked glucose transport stimulated by insulin but not vanadate or pV. Vanadate and pV stimulated the translocation of GLUTs from an intracellular compartment to the plasma membrane; this stimulation was not blocked by wortmannin, but insulin-induced GLUT translocation was inhibited. Similar results were obtained in cultured H9c2 cardiac muscle cells in which wortmannin did not inhibit glucose transport or the vanadate-induced translocation of GLUT4 in c-myc-GLUT4 transfected cells. The ser/thr kinase PKB (Akt/PKB/RAC-PK) is activated by insulin, lies downstream of PI 3-kinase, and has been implicated in signaling of glucose transport. Insulin and pV stimulated PKB activity, and both were inhibited by wortmannin. In contrast, vanadate, at concentrations that maximally stimulated glucose transport, did not significantly increase PKB activity. To determine the potential role of protein kinase C (PKC), L6 cells were incubated chronically with phorbol myristate acetate (PMA) or acutely with the PKC inhibitors calphostin C and bisindolylmaleimide. There was no inhibition of glucose transport stimulation by insulin, vanadate, or pV, and a combination of wortmannin and PKC inhibitors also failed to block the effect of vanadate and pV. In contrast, disassembly of the actin network with cytochalasin D blocked the stimulation of glucose transport by all three agents. In conclusion, vanadate and pV are able to stimulate glucose transport and GLUT translocation by a mechanism independent of PI 3-kinase and PKC. Similar to that by insulin, glucose transport stimulation by vanadate and pV requires the presence of an intact actin network.

Dexamethasone suppresses in vivo levels of bone collagen synthesis in neonatal mice.

The objective of this study was to determine the acute effects of glucocorticoids on in vivo levels of bone collagen synthesis in neonatal mice. Mice were injected with vehicle or dexamethasone at the start of the experiment. At 22 h, mice were given a 10 microCi injection of [3H]proline. At 24 h, the mice were sacrificed and the incorporation of [3H]proline into collagenase-digestible CDP labeling) and noncollagen (NCP labeling) protein in calvariae were determined by digestion with bacterial collagenase. Calvarial RNA was analyzed for COL 1A1 and osteocalcin mRNA levels by Northern blotting. After 24 h, vehicle-treated mice showed a 9.8 +/- 1.0% weight gain while dexamethasone-treated mice (1 mg/kg) had a 7.4 +/- 0.8% weight loss. Dexamethasone (1 mg/kg) decreased CDP and NCP labeling in calvariae by 51 +/- 4% and 17 +/- 4%, respectively (13 experiments). The inhibitory effect on protein labeling was selective for collagen since dexamethasone decreased the percent collagen synthesis from 25.4 +/- 1.6% to 16.6 +/- 1.0% (13 experiments). Dexamethasone at 3 mg/kg also decreased CDP labeling and the percent collagen synthesis in calvariae. There was a 30% reduction in COL1A1 mRNA levels and a 67% decrease in osteocalcin mRNA levels. To determine the reversibility of the inhibition of collagen synthesis, mice were given a single injection of dexamethasone (1 mg/kg) and then injected with [3H]proline 2 h prior to sacrifice at 24, 48, or 72 h. The reduction in CDP labeling observed at 24 h was fully reversed by 48-72 h. Moreover, by 72 h, the-rate of weight gain by dexamethasone-treated mice was similar to vehicle-treated controls. These data show that administration of dexamethasone to neonatal mice leads to a selective decrease in bone collagen synthesis within 24 h that is accompanied by down-regulation of osteocalcin and COL1A1 mRNA levels. This model will be useful in determining mechanisms by which high dose glucocorticoids inhibit bone formation in vivo.