Gerard Elberg

Insulin-like actions of vanadate are mediated in an insulin-receptor-independent manner via non-receptor protein tyrosine kinases and protein phosphotyrosine phosphatases

Most or all mammalian cells contain vanadium at a concentration of 0.1–1.0 μM. The bulk of the vanadium in cells is probably in the reduced vanadyl (IV) form. Although this 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 years 1975–1980 the vanadate ion was shown to act as an efficient inhibitor of Na+,K+-ATPase and of other related phosphohydrolyzes as well. In 1980 it was observed that vanadate vanadyl, when added to intact rat adipocytes, mimics the biological actions of insulin in stimulating hexose uptake and glucose oxidation. This initiated a long, currently active, field of research among basic scientists and diabetologists. Several of the aspects studied are reviewed here.

Vanadate activates membranous nonreceptor protein tyrosine kinase in rat adipocytes.

The insulin-like effects of vanadate are independent of the insulin receptor and insulin receptor substrate 1 (IRS-1) phosphorylation. A cytosolic protein tyrosine kinase (CytPTK), sensitive to inhibition by nanomolar concentrations of staurosporine (concentration at which 50% inhibition occurs [IC50], 1–2 nmol/1), has been implicated in some (i.e., glucose oxidation, lipogenesis) but not all (i.e., hexose uptake, inhibition of lipolysis) of the insulin-like effects of vanadate. We report here the existence of another nonreceptor protein tyrosine kinase in rat adipocytes, located exclusively in the plasma membranes (MembPTK), which we suggest is associated with hexose uptake and the antilipolytic activity of vanadate. MembPTK is a nong-lycoprotein with an estimated molecular weight of 55–60 kDa. In a cell-free experiment, vanadate activates MembPTK seven- to ninefold (median effective dose, 17 ± 2 μmol/l). Vanadate-activated MembPTK is inhibited by staurosporine (IC50, 60 ± 5 nmol/l). In intact adipocytes, staurosporine antagonized vanadate-induced hexose uptake (IC50, 6.0 ± 0.3 μmol/l) and significantly reversed the antilipolytic effect of vanadate (IC50, 5.0 ± 0.4 μmol/l). After vanadate treatment, a phosphorylated P55 protein is immunoprecipitated by antibodies to both phosphotyrosine and phosphatidyli-nositol (PI) 3-kinase. In conclusion, rat adipocytes contain an additional vanadate-activatable nonreceptor membranous protein tyrosine kinase that may participate in the effects of vanadate not carried out by CytPTK. We also suggest that after treatment with vanadate, MembPTK is activated by autophosphorylation and interacts with PI 3-kinase. This may explain how vanadate activates PI 3-kinase without involving receptor activation and IRS-1 phosphorylation.

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 ...

Non-receptor cytosolic protein tyrosine kinases from various rat tissues

Adipocytic-cytosolic non-receptor protein tyrosine kinase (CytPTK) when activated can substitute for the insulin receptor tyrosine kinase (InsRTK), in manifesting several insulin effects in insulin-receptor independent fashion. Our aims here were to utilize PolyGlu4Tyr, a good experimental exogenous substrate for protein tyrosine kinases (PTKs) in general, for studying qualitative and quantitative parameters of CytPTKs extracted from different tissue cytosols. At the same time, we would search for a unique specific marker specifically characterizing CytPTKs. High speed supernatants of spleen, thymus, smooth muscle, lung and kidney were found to be rich in CytPTK activities. Their specific activities being 6- to 13-fold that of liver or adipose cytosols. Brain, testis and adrenal cytosols were an intermediate source of CytPTK activity, whereas CytPTK activity of heart and skeletal muscle was low. It was also evaluated that the capacity of the cytosol to phosphorylate PolyGlu4Tyr is 15-50% that of the non-stimulated Triton X-100 extractable plasma membrane PTKs. Fractionation of the cytosols on superose 12 column revealed several CytPTKs within the same tissue, their peaks ranging between 30 and 450 kDa. Immunoblotting analysis showed Fyn and Lyn were present in most tissue cytosols. Upon immunoprecipitation, however, with anti-Fyn or anti-Lyn, negligible amounts (< 2%) of the total cellular CytPTK were precipitated. Thus, these general markers of CytPTKs comprise only a minor proportion of the total intracellular PolyGlu4Tyr phosphorylating capacity. To see whether a specific marker for CytPTK could be detected, we also examined the requirement of CytPTKs for divalent ions, their preferred phosphate donor and their sensitivity to inhibition by known PTK inhibitors. We found that the order of reactivity with divalent cations was Co2+ > Mn2+ > Mg2+, while Zn2+ and Ca2+ did not support CytPTK activity. The best phosphate donor was ATP (ED50 = 5 microM), but other nucleoside 3-phosphates could substitute for ATP at high concentrations. With respect to these parameters, no basic difference exists between cytosolic and plasma-membrane PTKs. The PTK inhibitors, genestein and quercetin, inhibited both cytosolic and membranal PTKs at micromolar concentrations. In contrast, staurosporine was a potent inhibitor of CytPTKs (IC50 5-20 nM) and a poor inhibitor of membranal PTKs (IC50 10-40 microM). One of the conclusions we can draw from this study is that tissue cytosols contain PolyGlu4Tyr phosphorylating capacity in quantities greater than previously assumed and that the low level of phosphotyrosine found in cells is not the result of limited intracellular levels of CytPTKs.

Insulin-like effects of tungstate and molybdate: mediation through insulin receptor independent pathways

The insulin-like effects of tungstate (W) and molybdate (Mo) were studied in rat adipocytes and compared to those of vanadate. Other than being less potent, W and Mo resembled vanadate in stimulating lipogenesis, in activating glucose oxidation, in enhancing rate of hexose uptake, and in inhibiting lipolysis. Tungstate and molybdate did not activate the insulinreceptor tyrosine kinase (InsRTK). Quercetin which blocks InsRTK activity and insulin stimulation of glucose metabolism, failed to inhibit when these bioeffects were stimulated by W or Mo. The metalooxide, however, activated a staurosporine sensitive non receptor, cytosolic protein tyrosine kinase (CytPTK), and staurosporine blocked W or Mo dependent lipogenesis in rat adipocytes. Staurosporine did not prevent Mo and W either from activating hexose transport, or from inhibiting lipolysis. Tungstate and molybdate were less effective than vanadate in inhibiting adipose PTPases in cell free systems. Membranal PTPases were more sensitive to W and Mo inhibition than cytosolic PTPases. While the presence of a nucleophile such as hydroxylamine reversed inhibition of PTPase by vanadate it did not affect inhibition by W or Mo. In summary, the insulinomimetic effects of W and Mo appear to resemble qualitatively that of vanadate in all respects. Both act in an insulin receptor-independent-fashion, activate CytPTK and trigger additional effects that are not mediated by the InsRTK or by CytPTK. The quantitative differences may be attributed to reduced capacity of W and Mo relative to vanadate to inhibit the relevant PTPases in intact cells.

Lactogenic hormones rapidly activate p21( ras )/mitogen-activated protein kinase in Nb2-11C rat lymphoma cells.

Lactogenic hormone-dependent Nb2-11C cells proliferate in response to prolactin (PRL) or human growth hormone (hGH). We have investigated the activation of p21ras and mitogen-activated protein kinase (MAP-kinase) by hGH in lactogen-dependent Nb2-11C and in autonomous hormone-independent Nb2-SP rat lymphoma cells. Exposure of Nb2-11C cells to hGH resulted in a dose-dependent activation of p21ras and of MAP-kinase. Activation occurs at physiological hGH concentration and with a rapid onset (∼1 min) reaching maximal level at 10–20 min. In contrast, in Nb2-SP autonomous lactogen-independent cells, p21ras and MAP-kinase are constitutively activated and a challenge with lactogenic hormone had a modest additional activating effect. TPA, an activator of protein kinase C, enhanced p21ras and MAP-kinase activity in Nb2-11C cells but failed to induce proliferation. The mechanism of activation of p21ras in Nb2-11C cells by lactogenic hormones involves both an increased binding of guanine nucleotides to p21ras as well as an increase in GTP/GDP+GTP ratio. In summary, we have demonstrated here that activation of the p21ras/MAP-kinase pathway follows PRL receptor activation but is not sufficient for the lactogenic hormone-dependent mitogenesis.