Organic vanadium chelators potentiate vanadium-evoked glucose metabolism in vitro and in vivo: establishing criteria for optimal chelators.

Several ligands, when complexed with vanadium, potentiate its insulinomimetic activity both in vivo and in vitro. We have recently found that L-Glu-gamma-monohydroxamate (HXM) and L-Asp(beta)HXM were especially potent in this regard. In the present study, we used vanadium-enriched adipose cells and cell-free experimental systems to determine the features of L-Glu(gamma)HXM and L-Asp(beta)HXM that turn these ligands into optimal-synergizing vanadium chelators. We found that L-Glu(gamma)HXM and L-Asp(beta)(HXM) possess the following characteristics: 1) They associate with vanadium(+5) at pH 7.2 within a narrow range of an apparent formation constant of 1.3 to 1.9 x 10(2) M(-1); 2) they have nearly the same binding affinity for the vanadyl(+4) cation and the vanadate(+5) anion at physiological pH values; and 3) they form intense ultraviolet absorbing complexes upon associating with vanadium(+4) at 1 and 3 M stoichiometry, respectively, at pH 3.0. Vanadium ligands lacking any of these three defined criteria synergize less effectively with vanadium to activate glucose metabolism.

Insulin-like effects of vanadium: basic and clinical implications.

Most mammalian cells contain vanadium at a concentration of about 20 nM, the bulk of which is probably in the reduced vanadyl (+4) form. Although this trace element is essential and should be present in the diet in minute quantities, no known physiological role for vanadium has been found thus far. In the late 1970s the vanadate ion was shown to act as an efficient inhibitor of Na+,K+-ATPase as well as of other related phosphohydrolases. In 1980 vanadium was reported to mimic the metabolic effects of insulin in rat adipocytes. During the last decade, vanadium has been found to act in an insulin-like manner in all three main target tissues of the hormone, namely skeletal muscles, adipose, and liver. Subsequent studies revealed that the action of vanadium salts is mediated through insulin-receptor independent alternative pathway(s). The investigation of the antidiabetic potency of vanadium soon ensued. Vanadium therapy was shown to normalize blood glucose levels in STZ-rats and to cure many hyperglycemia-related deficiencies. Therapeutic effects of vanadium were then demonstrated in type II diabetic rodents, which do not respond to exogenously administered insulin. Finally, clinical studies indicated encouraging beneficial effects. A major obstacle, however, is overcoming vanadium toxicity. Recently, several organically chelated vanadium compounds were found more potent and less toxic than vanadium salts in vivo. Such a newly discovered organic chelator of vanadium is described in this review.

A novel approach for a water-soluble long-acting insulin prodrug: design, preparation, and analysis of [(2-sulfo)-9-fluorenylmethoxycarbonyl](3)-insulin.

In this study we designed, prepared, and analyzed a water-soluble, long-acting insulin derivative whose protracted action in vivo is based on a new principle rather than on slower absorption rates of suspended insulin formulations. To this end, we have prepared (9-fluorenylmethoxycarbonyl-SO(3)H)(3)-insulin ((FMS)(3)-insulin), a derivative having three 9-fluorenylmethoxycarbonyl-SO(3)H (FMS) moieties covalently linked to the three amino side chains of insulin. (FMS)(3)-insulin is soluble in aqueous buffers at neutral pH, at a concentration range of 0.15-0.60 mM, and has about 1% of both the biological potency and the receptor-binding affinity of the native hormone. Upon incubation at pH 7.4 and 37 degrees C, it undergoes a slow hydrolysis with linear regeneration of insulin possessing full biological potency. A single subcutaneous administration of (FMS)(3)-insulin to streptozocin-treated rats lowered circulating glucose levels for a prolonged period (t(1/2) = 30 h). Similarly, intraperitoneal administration of (FMS)(3)-insulin to healthy rats had a prolonged glucose-lowering effect. In this experimental system, recovery from hypoglycemia proceeded with a t(1/2) value of 14 +/- 1 h, as compared with t(1/2) = 8.0 +/- 1 h for native insulin and t(1/2) = 10.0 +/- 1 h for NPH-insulin. (FMS)(3)-insulin is more resistant to proteolysis and appears to be nonimmunogenic. On the whole, (FMS)(3)(-)insulin represents a prototype version of a water-soluble, long-acting preparation of insulin. It is nearly inactive at the time of administration, and therefore can be administered, at high dose, with no concern for hypoglycemia. Because of its decreased receptor-binding affinity, (FMS)(3)-insulin evades receptor-mediated endocytosis and degradation and, hence, persists for a long period in the circulation. The insulin constituent of the (FMS)(3)-insulin conjugate undergoes a slow, spontaneous activation in the circulatory system, manifesting a prolonged glucose-lowering action in vivo. According to the data presented here, (FMS)(3)-insulin represents a typical prodrug: a compound which by itself shows only marginal activity but over time it is chemically hydrolyzed to the fully active hormone.

Vanadate restores glucose 6-phosphate in diabetic rats: a mechanism to enhance glucose metabolism.

Vanadate mimics the metabolic actions of insulin. In diabetic rodents, vanadate also sensitizes peripheral tissues to insulin. We have analyzed whether this latter effect is brought about by a mechanism other than the known insulinomimetic actions of vanadium in vitro. We report that the levels of glucose 6-phosphate (G-6-P) in adipose, liver, and muscle of streptozotocin-treated (STZ)-hyperglycemic rats are 77, 50, and 58% of those in healthy control rats, respectively. Normoglycemia was induced by vanadium or insulin therapy or by phlorizin. Vanadate fully restored G-6-P in all three insulin-responsive peripheral tissues. Insulin did not restore G-6-P in muscle, and phlorizin was ineffective in adipose and muscle. Incubation of diabetic adipose explants with glucose and vanadate in vitro increased lipogenic capacity three- to fourfold (half-maximally effective dose = 11 +/- 1 microM vanadate). Lipogenic capacity was elevated when a threshold level of approximately 7.5 +/- 0.3 nmol G-6-P/g tissue was reached. In summary, 1) chronic hyperglycemia largely reduces intracellular G-6-P in all three insulin-responsive tissues; 2) vanadate therapy restores this deficiency, but insulin therapy does not restore G-6-P in muscle tissue; 3) induction of normoglycemia per se (i.e., by phlorizin) restores G-6-P in liver only; and 4) glucose and vanadate together elevate G-6-P in adipose explants in vitro and significantly restore lipogenic capacity above the threshold of G-6-P level. We propose that hyperglycemia-associated decrease in peripheral G-6-P is a major factor responsible for peripheral resistance to insulin. The mechanism by which vanadate increases peripheral tissue capacity to metabolize glucose and to respond to the hormone involves elevation of this hexose phosphate metabolite and the cellular consequences of this elevated level of G-6-P.

L-Glutamic acid gamma-monohydroxamate. A potentiator of vanadium-evoked glucose metabolism in vitro and in vivo.

We report that the vanadium ligand L-Glu(gamma)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(gamma)HXM molecules to one vanadium ion was most effective. Unlike other vanadium ligands that potentiate the insulinomimetic actions of vanadium, L-Glu(gamma)HXM partially activated lipogenesis in rat adipocytes in the absence of exogenous vanadium. This effect was not manifested by D-Glu(gamma)HXM. At 10-20 microM L-Glu(gamma)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(gamma)HXM led to a rapid decrease in the absorbance of vanadium(IV) at 765 nm, and (51)V 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(gamma)HXM. In summary, L-Glu(gamma)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(gamma)HXM vanadium complex in which the endogenous vanadium(IV) has been altered to vanadium(V). Finally we demonstrate that L-Glu(gamma)HXM- and L-Glu(gamma)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.