J Biol Chem, Vol. 274, Issue 37, 26617-26624, September 10, 1999

L-Glutamic Acid -Monohydroxamate
A POTENTIATOR OF VANADIUM-EVOKED GLUCOSE METABOLISM IN VITRO AND IN VIVO^*

Itzhak Goldwaser^§¶, Jinping Li, Eytan Gershonov^§, Michal Armoni, Eddy Karnieli, Mati Fridkin^§**, and Yoram Shechter

From the  Departments of Biological Chemistry and ^§ Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel and the  Institute of Endocrinology, Rambam Medical Center and the B. Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We report that the vanadium ligand L-Glu()HXM potentiates the capacity of free vanadium ions to activate glucose uptake and glucose metabolism in rat adipocytes in vitro (by 4-5-fold) and to lower blood glucose levels in hyperglycemic rats in vivo (by 5-7-fold). A molar ratio of two L-Glu()HXM molecules to one vanadium ion was most effective. Unlike other vanadium ligands that potentiate the insulinomimetic actions of vanadium, L-Glu()HXM partially activated lipogenesis in rat adipocytes in the absence of exogenous vanadium. This effect was not manifested by D-Glu()HXM. At 10-20 µM L-Glu()HXM, lipogenesis was activated 9-21%. This effect was approximately 9-fold higher (140 ± 15% of maximal insulin response) in adipocytes derived from rats that had been treated with vanadium for several days. Titration of vanadium(IV) with L-Glu()HXM led to a rapid decrease in the absorbance of vanadium(IV) at 765 nm, and ⁵¹V NMR spectroscopy revealed that the chemical shift of vanadium(IV) at 490 ppm disappeared with the appearance of a signal characteristic to vanadium(V) (530 ppm) upon adding one equivalent of L-Glu()HXM. In summary, L-Glu()HXM is highly active in potentiating vanadium-activated glucose metabolism in vitro and in vivo and facilitating glucose metabolism in rat adipocytes in the absence of exogenous vanadium probably through conversion of trace intracellular vanadium into an active insulinomimetic compound. We propose that the active species is either a 1:1 or 2:1 L-Glu()HXM vanadium complex in which the endogenous vanadium(IV) has been altered to vanadium(V). Finally we demonstrate that L-Glu()HXM- and L-Glu()HXM·vanadium-evoked lipogenesis is arrested by wortmannin and that activation of glucose uptake in rat adipocytes is because of enhanced translocation of GLUT4 from low density microsomes to the plasma membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intensive studies have been carried out during the last two decades on the insulinomimetic effects of vanadium (1-4). Vanadium salts mimic most of the effects of insulin on the main target tissues of the hormone in vitro and also induce normoglycemia and improve glucose homeostasis in insulin-deficient (5-7) and insulin-resistant diabetic rodents in vivo (5-8). On the basic research frontier, data continue to accumulate showing that vanadium salts manifest their insulin-like metabolic effects through alternative pathways not involving insulin receptor tyrosine kinase activation or phosphorylation of insulin receptor substrate 1 (9-19). The key events of this backup system appear to involve inhibition of protein-phosphotyrosine phosphatases and activation of nonreceptor protein-tyrosine kinases (20-23).

Vanadium salts are seriously considered as a possible treatment for diabetes, and several clinical studies have already been performed. In those studies, because of its toxicity, only low doses of vanadium (2 mg/kg/day) were used. Although ~20-fold lower than doses used in most animal studies, several beneficial effects were observed and documented (24-26). Any manipulation to elevate the insulinomimetic efficacy of vanadium without increasing its toxicity is of major clinical interest for the future care of diabetes (reviewed in Ref. 27).

Organically chelated vanadium compounds, such as vanadium·acetylacetonate and vanadium·RL-252,¹ are more potent than free vanadium in facilitating insulin-like effects in rat adipocytes (28, 29). Similarly, chelated vanadium compounds such as bis(maltolato)oxovanadium and bis(picolinato)oxovanadium are more effective than free vanadium in reducing circulating glucose levels in hyperglycemic streptozocin-treated rats (30-33).

In the wake of these findings, we have continued our search for more effective vanadium binding agents. Of special interest to us were vanadium chelators that synergize with vanadium both in vivo (i.e. in streptozocin rats) and in vitro (i.e. in isolated rat adipocytes) and therefore enable us to gain insight into the basic mechanism(s) by which such compounds potentiate the insulinomimetic activity of vanadium. Specifically, we have studied hydroxamic acid derivatives. These compounds are involved in the microbial transport of iron and are therefore applied therapeutically in conditions of iron deficiency (34). They are also inhibitors of urease activity and have been used in the treatment of hepatic coma. Monoamino acid hydroxamates are simple, nontoxic derivatives of amino acids. D-Aspartic acid -hydroxamate was shown to have antitumoral activity on murine leukemia L5178Y, both in vitro and in vivo, and is active against Friend leukemia cells in vitro as well (35). L-Glu()HXM is cytotoxic against L1210 cells in culture and remarkably antitumoral against L1210 leukemia and B16 melanoma in vivo (35, 36).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- D-[U-¹⁴C]glucose and 2-deoxy-D-[G-³H]glucose were purchased from NEN Life Science Products. Collagenase type I (134 units/mg) was obtained from Worthington. Porcine insulin was purchased from Eli Lilly Co. (Indianapolis, IN). Phloretin, 2-deoxyglucose, L-glutamic acid()-monohydroxamate, L-aspartic acid()monohydroxamate, glycine hydroxamate, L-isoleucine()hydroxamate, and L-tyrosine()hydroxamate were purchased from Sigma. RL-252 was prepared and characterized as described earlier (28).

Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) contained 110 mM NaCl, 25 mM NaHCO₃, 5 mM KCl, 1.2 mM KH₂PO₄, 1.3 mM CaCl₂, 1.3 mM MgSO₄. Krebs-Ringer bicarbonate HEPES (KRBH) buffer (pH 7.4) consisted of 117 mM NaCl, 10 mM NaHCO₃, 1 mM CaCl₂, 1 mM MgSO₄, 4 mM KH₂PO₄, 30 mM HEPES. All other chemicals and reagents used in this study were of analytical grade.

Streptozocin-treated Rats-- Diabetes was induced by a single intravenous injection of a freshly prepared solution of streptozocin (55 mg/kg body weight) in 0.1 M citrate buffer, pH 4.5 (9). The effect of the L-Glu()HXM·vanadium complex on blood glucose level was determined 8 days after induction of diabetes by streptozocin.

Cell Preparation and Bioassays-- Rat adipocytes were prepared from the fat pads of male Wistar rats (130-150 g) by collagenase digestion according to the method of Rodbell (37). Cell preparations showed more than 95% viability by Trypan blue exclusion at least 3 h after digestion. All bioassays were performed as described in figure legends. Glucose transport was carried out using 2-deoxy-D-[G-³H]glucose uptake (38), and lipogenesis (the incorporation of U-¹⁴C-labeled glucose into lipids) was performed according to Moody et al. (39). Briefly, freshly prepared rat adipocytes were suspended in KRBH, 0.7% BSA buffer and divided into about 50 plastic vials. Each vial contained 0.5 ml of adipocyte suspension (about 1.5 × 10⁵ cells). These were incubated for 2 h at 37 °C under an atmosphere of 95% O₂, 5% CO₂ with 0.16 mM [U-¹⁴C]glucose. Each assay contained vials with and without 17 nM insulin and the various test compounds. Lipogenesis was terminated by adding toluene-based scintillation fluid, and the extracted lipids were counted (39). Results are expressed as a percent of maximal insulin response. Only assays in which insulin activated lipogenesis 5-6-fold above basal (basal ~4000 cpm/1.5 × 10⁵ cells/2 h, V_insulin = 20,000-24,000 cpm/1.5 × 10⁵ cells/2 h) were taken into consideration. Insulin activated lipogenesis in this assay at an ED₅₀ value of 33 ± 3 pM. A concentration of 0.3 nM insulin and above already facilitated maximal (100%) response (i.e. Ref. 16). All assays were performed in duplicate or triplicate.

Western Immunoblot Analysis of GLUT4 in Subcellular Membranes Following Stimulation of Rat Adipocytes-- Adipocytes prepared from 6-week-old rats were incubated with and without insulin and with L-Glu()HXM alone and complexed with vanadate as specified in the figure. Cells were then homogenized and fractionated to low density microsomal membrane (LDM) and plasma membrane (PM) fractions by differential ultracentrifugation according to Ref. 40. Membrane proteins were then solubilized in sample buffer for 30 min at 25 °C, resolved on 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and immunoblotted with anti-GLUT4 antisera (41). Visualization was performed by phosphoimaging. The relative intensity of bands corresponding to GLUT4 was quantitated using MacBas 1000.

⁵¹V NMR Spectroscopy-- The ⁵¹V NMR spectra were recorded on a 200-MHz Bruker WPS4 (4.7T) spectrometer. Spectrum width of 16,000 H₃, a 90^o pulse angle, and an accumulation time of 0.28 were used. The chemical shifts are reported relative to the external reference standard VOCl₂ (490 ppm).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

L-Glutamic Acid()Monohydroxamate Potentiates Vanadium-evoked Lipogenesis in Rat Adipocytes-- In this set of experiments, rat adipocytes were incubated for 10-20 min with submaximal concentrations of vanadate (10-30 µM), L-Glu()HXM (10-30 µM), or an equimolar combination of them. The capacity to activate lipogenesis relative to insulin was then determined. As shown in Fig. 1, the combination was highly synergistic. For example, at 10 µM vanadate or L-Glu()HXM, lipogenesis was 17 ± 3 and 9 ± 2%, respectively, whereas the combination produced a marked incredible 93 ± 4% activation of maximal insulin response. At 20 µM, the extent of lipogenesis was 37 ± 3, 20 ± 3, and 121 ± 7%, and at 30 µM, it was 42 ± 4, 23 ± 4, and 143 ± 7% of maximal. Wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase, fully blocked the activating effects of vanadate, L-Glu()HXM, and its combination with vanadate (Fig. 1, right columns). Thus L-Glu()HXM potentiated vanadate-evoked lipogenesis about 3.5-5-fold; the higher concentrations reached a level that is about 140% of that achieved by saturating concentrations of insulin or vanadate. A finding of significant interest to us was the ability of L-Glu()HXM to partially activate lipogenesis even in the absence of exogenous vanadium (Fig. 1). This finding is examined in great detail in connection with Fig. 6.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Increase in the lipogenic capacity of vanadium(V) following the addition of L-glutamic acid()monohydroxamate. Freshly prepared rat adipocytes (3 × 105 cells/ml) suspended in KRB buffer, pH 7.4, containing 0.7% BSA were preincubated for 10 min with the indicated concentrations of NaVO3, free Glu()HXM, and a 1:1 complex of L-Glu()HXM·NaVO3. The cells were then supplemented with [U-14C]glucose, and lipogenesis was performed for 2 h at 37 °C. Radioactivity incorporated into extracted lipids was then determined. Maximal response (100%) is that obtained in the presence of 17 nM insulin.

In Fig. 2, lipogenesis in rat adipocytes was evaluated at a fixed, low concentration of vanadate (5 µM) with increasing concentrations of L-Glu()HXM. Lipogenesis was negligible at 5 µM vanadate or L-Glu()HXM alone (4-6% of maximal insulin effect) but is augmented to 27.0 ± 3% when they were given in combination (at a molar stoichiometry of 1:1). At 2:1 and 3:1 Glu()HXM·vanadium molar stoichiometry, lipogenesis expanded to 43 and 57%, respectively, of maximal response. Thus a substantial synergistic effect is obtained at a 1:1 molar ratio and is increased further at a 2:1 molar stoichiometry and even higher, though much less pronounced, at a 3:1 molar ratio (Fig. 2).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Stimulation of lipogenesis at varying molar ratios of L-Glu()HXM to vanadium(V). Freshly prepared rat adipocytes (3 × 105 cells/ml) suspended in KRB buffer, pH 7.4, containing 0.7% BSA were preincubated for 10 min with the indicated concentrations of 1:1 to 3:1 molar stoichiometry of L-Glu()HXM to NaVO3 or with free NaVO3(V) and free L-Glu()HXM. The cells were then supplemented with [U-14C]glucose, and lipogenesis was performed for 2 h at 37 °C. Radioactivity incorporated into extracted lipids was then determined. Maximal response (100%) is that obtained in the presence of 17 nM insulin.

L-Glu()HXM Potentiates Vanadate-evoked Glucose Uptake-- Fig. 3 shows activation of 2-deoxyglucose uptake by low concentrations of vanadate (20 µM), L-Glu()HXM (40 µM), and by the 2:1 molar combination, of them. 2-Deoxyglucose undergoes insulin- or vanadate-evoked influx into the cell via the same transporters as glucose and is phosphorylated in situ to 2-deoxyglucose 6-phosphate with no further metabolism (42, 43). Therefore, this measurement reflects an effect on glucose entry into the cell in a manner largely independent of the metabolism of the endogenous saccharide. Vanadate (20 µM) and L-Glu()HXM (40 µM) affected 2-deoxyglucose uptake of 7 ± 0.7 and 31 ± 4% of maximal insulin effect, respectively. Together they caused 2-deoxyglucose uptake 117 ± 9% of maximal insulin response (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Potentiation of hexose uptake by L-Glu()HXM·vanadium (2:1). Adipocytes (2 × 106 cells/ml) suspended in KRBH buffer containing 1% BSA were preincubated in the presence and absence of insulin (17 nM), sodium metavanadate (20 µM), L-Glu()HXM (40 µM), and their combination (at 1:2 molar stoichiometry). Aliquots (70 µl) were transferred into tubes containing 2-deoxy-D-[6-3H]glucose (0.1 mM final concentration). Phloretin (0.1 mM) was added after 3 min for transport termination. This was followed by centrifugation of aliquots through a silicone layer.

L-Glu()HXM Alone and L-Glu()HXM·Vanadate Lead to Translocation of GLUT4 from LDM to PM Fractions in Rat Adipocytes-- Incubation of rat adipocytes with L-Glu()HXM and L-Glu()HXM·vanadate led to a decrease in the content of GLUT4 in the LDM fraction and an increase in the PM fraction (Fig. 4). The decrease in GLUT4 content in the low density lipoprotein fraction amounted to 32 ± 3, 3 ± 1, and 68 ± 5% of maximal insulin response upon incubating the cells with L-Glu()HXM (40 µM), vanadate (20 µM, not shown), and the combination, respectively (calculated from Fig. 4). Under similar experimental conditions, L-Glu()HXM, vanadate, and the combination activated 2-deoxyglucose uptake to an extent of 31 ± 4, 7 ± 0.7, and 117 ± 9% of maximal insulin response (Fig. 3), suggesting a contributing effect of the complex to glucose influx in addition to its effect in recruiting GLUT4 transporters from the low density lipoprotein to the PM fraction.²

View larger version (49K): [in this window] [in a new window]   Fig. 4.   L-Glu()HXM alone or complexed with vanadate induces translocation of GLUT4 from LDM to PM fraction in rat adipocytes. Rat adipocytes were incubated for 30 min at 37 °C in the presence and the absence of insulin (17 nM) and the indicated concentrations of L-Glu()HXM or L-Glu()HXM·vanadate. Cells were then homogenized and fractionated to PM and LDM by differential ultracentrifugation, and GLUT4 protein was identified by Western immunoblot analysis ("Experimental Procedures"). Immunoreactive GLUT4 proteins were visualized by phosphoimaging (top panels) and were quantitated using MacBas 1000 software (histograms, bottom panels).

L-Glu()HXM·Vanadate Normalizes Blood Glucose Levels in Streptozocin-treated Diabetic Rats-- In the experiments summarized in Fig. 5, streptozocin-treated rats received intraperitoneally sodium metavanadate (0.05 mmol/kg body weight), L-Glu()HXM (0.1 mmol/kg body weight), or a combination of the two compounds 8 days after the induction of diabetes. As shown in the figure, vanadate and L-Glu()HXM, at these concentrations, had a rather minor effect in reducing the high circulating glucose levels characterizing these hyperglycemic rats. The combination, however, was highly efficient at normalizing blood glucose levels. Normoglycemia was evident 1 day after the first administration and remained so following two more administrations. The glucose levels then remained close to normal for the next 3 days (Fig. 5).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effect of L-Glu()HXM·vanadate administration on blood glucose levels of streptozocin-treated rats. Male Wistar rats, 8 days after induction of diabetes (circulating glucose levels 310-340 mg/dalton), were divided into several groups. At the time points indicated by the arrows (intraperitoneal, at 11:00 a.m.), groups of diabetic rats received either vanadate (0.05 mmol/kg body weight ), L-Glu()HXM (0.1 mmol/kg body weight ), L-Glu()HXM (0.1 mmol/kg) and vanadate (0.05 mmol/kg, ), or none (). Circulating glucose levels were determined daily (at 8.00 a.m.). Each point in the figure represents the arithmetic mean of plasma glucose for 5 rats. The dashed line indicates the arithmetic mean of plasma glucose of control healthy male Wistar rats.

Activation of Lipogenesis in Rat Adipocytes by L-Glu()HXM in the Absence of Exogeneous Vanadium-- L-glutamic acid()HXM also activated lipogenesis in the absence of added vanadium, and this effect was studied in detail (Fig. 6). The dose-response curve (Fig. 6A) indicates that activation is already evident at 5 µM L-Glu()HXM and that higher concentrations reach a level of 40 ± 7% of maximal insulin response (median effective dose = 35 ± 4 µM). Other amino acid hydroxamates such as L-Tyr()HXM, Gly()HXM, and L-Ile()HXM also activated lipogenesis, but they were considerably less potent (ED₅₀ = 250 ± 30 µM, 40 ± 5% of maximal insulin effect). L-Aspartic acid -monohydroxamate showed higher lipogenic activity compared with the -amino acid hydroxamates and was slightly less potent than L-Glu()HXM (ED₅₀ = 45 ± 7 µM, Fig. 6B). N-acetyl-L-Glu()HXM and L-Glu()HXM--methyl ester were virtually ineffective, indicating the need for a free -amino and, to a somewhat lesser extent, a free -carboxyl moiety for the activation of lipogenesis by L-Glu()HXM in the rat adipose cell (Fig. 6C). Stereospecificity appears crucial as well, because the D-isomer of Glu()HXM was ineffective. All these findings indicate that activation of lipogenesis by L-Glu()HXM depends on a specific entry of this L-amino acid analog into the adipose cell. Further investigation has led us to suggest that L-Glu()HXM enters the adipose cell primarily through the non-Na⁺-dependent glutamine transport system.²

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Activation of lipogenesis by L-Glu()HXM in the absence of exogenous vanadium. Comparison to other amino acid()hydroxamates and ineffectiveness of the D-isomer and of chemically modified L-Glu()HXM derivatives. Freshly prepared adipocytes (3 × 105 cells/ml) suspended in KRB buffer, pH 7.4, containing 0.7% BSA were preincubated for 10 min with the indicated concentrations of the various test compounds. The cells were then supplemented with [U-14C]glucose (final concentration 0.16 mM), and lipogenesis was performed for 2 h at 37 °C. Radioactivity incorporated into extracted lipids was then determined. Maximal response (100%) is that obtained in the presence of 17 nM insulin.

Several organic chelators, which potentiate the insulinomimetic activity of vanadium either in vitro or in vivo, have been documented. These include acetylacetonate (29), maltol (30, 31), picolinate (32, 33), and RL-252 (28). In Fig. 6D, we have examined whether they are capable of activating lipogenesis in the absence of exogenous vanadium. Unlike L-Glu()HXM, none of these agents were able to activate lipogenesis in the rat adipose cell at concentrations of 100 µM (Fig. 6D) or lower (not shown).

Extensive Potentiation of L-Glu()HXM-evoked Lipogenesis in Rat Adipocytes in Vitro Following Enrichment with Vanadium in Vivo-- The findings presented in Figs. 1-4 have taught us that L-Glu()HXM potentiates the insulinomimetic potency of vanadium and that activation of lipogenesis by L-Glu()HXM alone never exceeds 40 ± 7% of maximal insulin effect (Fig. 6). To examine whether L-Glu()HXM-evoked lipogenesis can be affected by the level of intracellular vanadium, a group of male Wistar rats received daily subcutaneous administrations of vanadate (0.1 mmol/kg/day) over a period of 5 days to raise the level of endogenous vanadium. Rats were then sacrificed 7 h after the last administration. Adipocytes were prepared, and the effect of L-Glu()HXM on lipogenesis was compared with that in nontreated freshly prepared adipocytes. As shown in Fig. 7, vanadium-enriched adipocytes became dramatically sensitive to L-Glu()HXM-evoked lipogenesis. This was valid both in terms of a leftward shift in the dose-response curve to L-Glu()HXM (ED₅₀ = 6.4 ± 0.3 µM versus ED₅₀ = 35 ± 4 µM in control adipocytes) and in terms of the degree of lipogenesis (145 ± 15 versus 40 ± 7% of maximal insulin response, i.e. Fig. 6). At 10 µM, L-Glu()HXM already stimulated lipogenesis and amounted to 120% of maximal insulin effect in the vanadium-enriched adipose cells (as opposed to only 8.0 ± 1.5% in control adipocytes) (Fig. 7).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Activation of lipogenesis by L-Glu()HXM. Comparison between normal adipocytes and vanadium-enriched adipocytes. Male Wistar rats received daily subcutaneously injected NaVO3 (0.1mmol/kg/day) for 5 days (called enriched vanadium rats). The rats were then sacrificed (7 h after the last administration). Lipogenesis was performed comparing the freshly prepared rat adipocytes (3 × 105 cells/ml) from nonenriched vanadium rats with the enriched ones suspended in KRB buffer, pH 7.4, containing 0.7% BSA. The cells were preincubated for 10 min with the indicated concentrations of L-Glu()HXM. The cells were then supplemented with [U-14C]glucose, and lipogenesis was performed for 2 h at 37 °C. Radioactivity incorporated into extracted lipids was then determined. Maximal response (100%) is that obtained in the presence of 17 nM insulin.

Spectroscopic Studies-- Previously we found in cell-free experiments that vanadium(IV), at neutral pH values, undergoes slow spontaneous oxidation to vanadium(V). This occurs similarly in the presence of 10 mM reduced glutathione, an ineffectual reductant of vanadium(V), at neutral pH values with a t_1/2 value of 1 ± 0.1 h at 25 °C (29). The results summarized in Fig. 8 show the V⁵¹ NMR spectra of vanadium dichloride(IV) at pH 7.0 prior to and after the addition of L-Glu()HXM. Vanadium dichloride(IV) appeared as a single peak with a chemical shift of 490 ppm in its ⁵¹V spectrum, indicating one main species present at >95% purity. Upon the addition of L-Glu()HXM (1 equivalent), the chemical shift of vanadium(IV) at 490 ppm disappeared within minutes and the principal chemical shift characterizing vanadium(V) at 530 ppm appeared (Fig. 8).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   51V NMR spectra of vanadium(V), vanadium(IV), and a mixture of vanadium(IV) with L-Glu()monohydroxamate. A, 51V NMR spectrum of sodium metavanadate (20 mM, pH 7.1); B, 51V NMR spectrum of vanadium dichloride(IV) (20 mM, pH 6.8); C, 51V NMR spectrum of a mixture (1:1 molar ratio) of VOCl2(IV) and L-Glu()HXM (20 mM, pH 7.0). Spectra were monitored with fresh solutions. In C, the spectrum was monitored 5 min after the addition of L-Glu()HXM to VOCl2.

Vanadium(IV) (i.e. vanadyl sulphate or VOCl₂) has a characteristic "blue" absorbance with _{765 nm} = 14 ± 0.3, whereas vanadium(V) does not absorb at all at this wavelength (29). The addition of 2-3 equivalents of L-Glu()HXM to VOCl₂(IV) (50 mM at pH 7.5) led rapidly to a near total decrease in vanadium(IV) absorbance at 765 nm (Fig. 9). Fig. 8B depicts complex formation as a function of the pH in the range of pH 2-9. Decrease is minimal at pH 4.0, quite significant at pH 5.0, half-maximal at pH 5.7, and reaches a stable plateau at pH range 7-9 (Fig. 9B).

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Decrease in absorbance of vanadium(IV) at 765 nm upon addition of L-Glu()HXM. Effect of pH: A, left column, absorbance of VOCl2 alone (50 mM in H2O); right column, absorbance of VOCl2 (50 mM) and L-Glu()HXM (150 mM) titrated with NaHCO3 to pH 7.4. B, samples of VOCl2 (50 mM) and L-Glu()HXM (100 mM) in H2O were titrated either with HCl or with NaHCO3 before absorbance at 765 nm and were monitored to obtain the pH values indicated in the figure. L-Glu()HXM alone does not absorb at 765 nm. Vanadium dichloride alone, which tends to precipitate at neutral pH values, remains completely soluble at all pH values in the presence of two or more equivalents of L-Glu()HXM.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been consistently observed that chelated vanadium compounds are more potent than the free metaloxide in facilitating the metabolic actions of insulin. This was demonstrated in vitro with systems like rat adipocytes, as well as in diabetic rodents such as streptozocin-treated hyperglycemic rats (28-33, 44). Because of the variations in the experimental models used, the oxidation state of vanadium applied, and the different administration modes, the basis for the higher insulinomimetic potencies of complexed vanadium remained rather speculative. Because this topic has immediate therapeutic relevance, we looked for new vanadium chelators characterized by: (a) higher synergistic potencies than previously documented for vanadium chelators with respect to vanadium-evoked glucose uptake and glucose metabolism both in vitro and in diabetic rats in vivo, (b) low indices of toxicity, and (c) reasonable solubility in aqueous, neutral media after complexation with vanadium.

In this study, we have introduced the L-isomer of glutamic acid()monohydroxamate as it satisfactorily fulfilled the above criteria. It potentiated vanadium-activated hexose uptake, glucose metabolism, and recruitment of GLUT4 transporters from LDM to PM fractions (Figs. 1-4). In vivo it potentiated the efficacy of vanadium to lower blood glucose levels in streptozocin rats (Fig. 5). This amino acid analog has negligible toxicity in mammals.² Both L-Glu()HXM alone and its complexes with vanadium are fairly soluble in aqueous media at neutral pH values. An important finding was that L-Glu()HXM alone, in the absence of exogenous vanadium, showed a reasonable amount of insulinomimetic activity in that it activated glucose uptake and glucose metabolism in the rat adipose cell (Figs. 1-3). Further investigation revealed that this activating effect is unique to the L-isomer of Glu()HXM but is not facilitated by the D-isomer. Nonmodified -amino and -carboxyl moieties appear essential. This intrinsic activity is exclusive to L-Glu()HXM not being shared by any of the other vanadium chelators that potentiate the actions of vanadium in vivo or in vitro (Fig. 6, A-D, and Refs. 28-33). Our assumption that L-Glu()HXM permeates into the cell interior and transforms the "dormant" intracellular vanadium pool into an insulinomimetic-activated species gains credence from the dramatic sensitization of vanadium-enriched adipocytes to L-Glu()HXM-evoked lipogenesis (Fig. 7).

It should be mentioned at this point that because of the extreme complexity of aqueous vanadium chemistry (reviewed in Refs. 46-49), the intracellular milieu of the mammalian cell is still "a black box" with respect to the state and the form of entered vanadium. With the endogenously present vanadium pool, experiments have shown that it exists mostly as vanadium(IV), though some researchers may wonder even about this experimental finding because vanadium in its IV oxidation state is only stable at acidic pH values (pH < 3.0) and readily oxidizes to vanadium(V) at neutral pH even in the presence of high glutathione concentrations (28, 46). The intracellular vanadium pool, however, can be preserved in its IV oxidation form at neutral pH values if it is chelated by ascorbic acid (not shown) or to endogenous proteins (50, 51). At the low physiological level of intracellular vanadium, the cell should have the capacity to chelate all the endogenous vanadium.

Our experimental findings that L-Glu()HXM alone enhances glucose uptake and glucose metabolism (Figs. 1 and 2) together with the apparent rapid conversion of vanadium(IV) to vanadium(V) upon complexation (Figs. 8 and 9) strongly support the contention that vanadium(V) rather than vanadium(IV), and in a chelated form, is the active insulinomimetic species that facilitates the activation of glucose uptake and its metabolism in rat adipocytes. Although most of our previous cell-free experiments support this conclusion, we were not fully convinced prior to the completion of this study. This is because protein phosphotyrosine phosphatases (with p-nitrophenylphosphate as a substrate) are inhibited by both vanadium(IV) and vanadium(V), free or chelated, at nearly the same concentrations (see Ref. 52). On the other hand, adipose nonreceptor protein-tyrosine kinases, whether cytosolic or membranal, are with one exception activated by vanadium(V) but not at all by vanadium(IV) (22, 23). We have only observed vanadium(IV)-evoked activation of nonreceptor protein-tyrosine kinases when membranal protein phosphotyrosine phosphatases were extracted with Triton X-100 and added to the cytosolic protein-tyrosine kinase fraction (29). These experimental conditions, however, are not likely to occur in the intact cell system. For example, broken plasma membrane fragments (or deoxycholate-treated membranal fragments) did not support activation of cytosolic protein-tyrosine kinases in the presence of vanadium(IV) (29).

In summary, L-Glu()HXM appears superior to previously documented organic chelators of vanadium in potentiating its activation of glucose uptake and glucose metabolism in vitro and in vivo. Taken together with earlier studies, this may be attributed to one or more of the following: (a) increased efficiency of this specific combination to permeate into cells or tissues; (b) a favorable 5-coordinated, rather than octahedral topography of this complex in an aqueous, neutral environment (Ref. 50);² and/or (c) higher intracellular stability of the L-Glu()HXM-vanadium complex. Finally, we have recently observed that vanadate does not inhibit alkaline phosphatase in the presence of L-Glu()HXM.² This inhibitory effect of vanadate (53) is undesirable from our point of view as it may contribute to vanadium toxicity in mammals, but not to the efficacy of vanadium to manifest the metabolic actions of insulin (reviewed in Ref. 54). This and other basic and diabetological aspects raised here are being further investigated.

	ACKNOWLEDGEMENTS

We thank Elana Friedman for typing the manuscript, Dr. Sandra Moshonov and Dov P. Grossman for editing it, and Dr. Sun Qian for technical assistance.

	FOOTNOTES

^* This work was supported by grants from the Minerva Foundation, Munich, Germany, The Israel Academy of Science Foundation, the Israeli Ministry of Health, and The Lapid Pharmaceutical Company.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.

^¶ Partial fulfillment of the requirements for the Ph.D. degree.

^** Lester Pearson Professor of Protein Chemistry.

The incumbent of the C. H. Hollenberg Chair in Metabolic and Diabetes Research established by the friends and associates of Dr. C.H. Hollenberg of Toronto, Canada. To whom correspondence should be addressed. Tel.: 972-8-9343698; Fax: 972-8-9344118.

² I. Goldwaser, J. Li, E. Gershonov, M. Armoni, E. Karnieli, M. Fridkin, and Y. Shechter, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: RL-252, [(CH₂)₂-C-{CH₂O-(CH₂)₂-CO-NHCH(iBu)CONOHCH₂}₂]; L-Glu()HXM, L-glutamic acid -monohydroxamate; GLUT4, glucose transporter 4; PM, plasma membranes; LDM, low density microsomes; BSA, bovine serum albumin; VOCl₂, vanadyl dichloride; NaVO₃, sodium metavanadate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES