Biomedical subjects
Publications and source records attributed to Y Shechter.
Until the discovery of insulin by Banting and Best in 1922, diabetes had a high mortality rate. Since then regular administration of the drug has brought it under control. Nevertheless it is not an ideal drug, in that it has to be injected and because of the incidence of insulin resistance. There is, therefore, a need for alternative forms of treatment and in recent years interest has centred on the possibilities of vanadium salts.
A case of carbamazepine-induced thrombocytopenia is reported. The patient, a 38-year-old female, developed thrombocytopenia 10 days after receiving 400 mg carbamazepine daily. The presence of an anti-IgG carbamazepine-dependent platelet-reactive antibody was documented. Using flow cytometry, the drug-induced antibody was detected at a drug concentration as low as 10(-6) M.
Previous studies have shown that the combination of vanadate and H2O2 generates peroxide(s) of vanadate (pervanadate) that is able to mimic insulin in stimulating lipogenesis or protein synthesis and inhibiting lipolysis in rat adipocytes. Here we report that pervanadate is a potent trigger of 3-O-methylglucose transport in rat adipocytes, with an effective concentration of 5 microM and a maximum at 20 microM. Moreover, pervanadate produced an additional activation of approximately 60% on glucose influx in cells treated with maximally activating concentrations of insulin. Vanadate was ineffective in potentiating insulin-stimulated glucose uptake. Quercetin, a bioflavonoid that inhibits insulin receptor tyrosine kinase, blunted this effect of pervanadate. Treatment of adipocytes with pervanadate inhibited protein phosphotyrosyl phosphatase activity of cell extracts in a dose-dependent manner, with an ID50 of 5 microM and complete inhibition at 80 microM. In contrast, vanadate (1-800 microM) did not appreciably inhibit cell phosphotyrosyl phosphatases. The inhibitory effect of pervanadate correlated with the increase in protein phosphotyrosine accumulation, as determined by Western blotting with antiphosphotyrosine antibodies. The most prominent phosphotyrosine-containing band detected in pervanadate-treated adipocytes was that of autophosphorylated insulin receptor, identified by immunoblotting or immunoprecipitation with antiinsulin receptor antibodies. The addition of insulin to pervanadate-treated adipocytes (20 microM) caused a further increase (approximately 70%) in receptor autophosphorylation. In a cell-free system using partially purified insulin receptor devoid of tyrosine phosphatase activity, pervanadate did not stimulate the receptor autophosphorylation or interfere with the stimulating effect of insulin. These results suggest that 1) pervanadate triggers glucose uptake by increasing autophosphorylation of insulin receptor, preventing its dephosphorylation; 2) under physiological conditions, cellular protein phosphotyrosyl phosphatase activity is high, thereby significantly opposing insulin-mediated hexose transport; and 3) pervanadate has the unique ability to markedly increase maximal cell responsiveness in stimulating glucose transport achieved at a saturating insulin concentration. These findings suggest a possible clinical application in the management of glucose uptake in pathological conditions of insulin resistance and hyperinsulinemia.
We describe 2 Arab patients, both offspring of unrelated consanguineous matings, with unusual facial appearance, severe mental retardation, microcephaly, cortical atrophy, seizures, hypotonia, dwarfism, and recurrent infections with neutrophilia. Neutrophil motility was markedly decreased but the opsonophagocytic activity was normal. Both patients lack the red blood cell (RBC) H antigen and manifest the Bombay (hh) phenotype. Familial endocardial fibroelastosis and familial tetralogy of Fallot segregated independently in one family. The occurrence of the same syndrome in 2 unrelated families suggests that the various aspects of the disorder are the pleiotropic effects of a single mutation. Homozygosity-by-descent for a deletion involving contiguous genes may explain the findings in this syndrome. Alternatively, a mutation which involves an ubiquitous GDP fucose donor rather than the enzyme (alpha 2-L-fucosyltransferase) or its substrate (glcNAc) may account for the pleiotropic manifestations in this syndrome.
We report here that quercetin, a naturally occurring bioflavonoid, is an effective blocker of insulin receptor tyrosine kinase-catalyzed phosphorylation of exogenous substrate. The ID50 was estimated to be 2 +/- 0.2 microM in cell-free experiments, using a partially purified insulin receptor and a random copolymer of glutamic acid and tyrosine as a substrate. Insulin-stimulated autophosphorylation of the receptor itself was not blocked by quercetin (up to 500 microM). In intact rat adipocytes, quercetin inhibited insulin-stimulating effects on glucose transport, oxidation, and its incorporation into lipids. Inhibition of lipogenesis (50%) occurred at 47 +/- 4 microM, whereas full inhibition was evident at 110 +/- 10 microM quercetin. In contrast, the effect of insulin in inhibiting lipolysis remained unaltered in quercetin-treated adipocytes. The inhibitor was devoid of general adverse cell affects. Basal activities and the ability of lipolytic agents to stimulate lipolysis were not affected. Inhibition by quercetin enabled us to evaluate which insulinomimetic agents are dependent on tyrosine phosphorylation of endogenous substrates for stimulating glucose metabolism. Quercetin blocked lipogenesis mediated by insulin, wheat germ agglutinin, and concanavalin A. The lipogenic effect of Zn2+ and Mn2+ was partially blocked, whereas that of vanadate was not affected at all.(ABSTRACT TRUNCATED AT 250 WORDS)
Previous studies suggested that insulin receptor tyrosine kinase (IRTK) is the sole tyrosine kinase in rat adipocytes. We now report that this cell type also contains a cytosolic soluble protein tyrosine kinase (CytPTK) which is not related to IRTK. The enzyme phosphorylated PolyGlu4Tyr with high efficiency at a rate of 20 +/- 2 pmol PTyr/20 micrograms PolyGlu4Tyr/20 min/micrograms cytosolic protein. Upon gel filtration chromatography the enzyme activity was eluted as a single peak corresponding to a molecular mass of 53 +/- 3 kDa. Unlike IRTK, CytPTK activity was supported by Co2+ rather than by Mn2+, and it was not inactivated by N-ethylmaleimide. The enzyme was extremely sensitive to inhibition by staurosporine (ID50 = 3 nM) as opposed to IRTK (ID50 = 8 microM). In addition, CytPTK (but not IRTK) was largely activated by vanadate ions. Agents which affect the serine/threonine phosphorylation state of cell proteins did not alter CytPTK activity when subjected to intact adepocytes. In a cell-free system CytPTK activity was largely reduced by pretreatment with immobilized alkaline phosphatase at physiological pH. The possibility that CytPTK participates in insulin-independent regulation of glucose metabolism is suggested.
A novel family of vanadyl ion (VO2+, oxidation state +4) carriers is introduced. These carriers possess C2 symmetry, utilize two hydroxamate groups as ion binding sites, and optionally possess asymmetric carbons. Binding efficiencies and hydrophobicities are regulated by the use of a modular assembly. When applied to rat adipocytes, these carriers augment the potency of vanadyl ions to stimulate glucose metabolism. The complexes shift the dose-response curve to the left. Also, the maximal effect of vanadyl ions which is in the order of 20-30% of that of insulin is shifted toward maximal (100-115%) stimulation. Among several chelators studied, the order of synergistic potency was RL-252 greater than or equal to RL-262 greater than 1367. RL-239, RL-280, and RL-261 had smaller effects, whereas RL-282 had a negligible effect. The synergistic action of RL-252 (and other chelators as well) on VO2+ was already observed at a molar ratio of 1:0.01 of VO2+ to RL-252, respectively, and maximal augmentation occurred at a molar ratio of 1:0.1. The superiority of the hydrophobic chelators relative to the hydrophilic ones, together with the low molar ratio of chelator to VO2+ to achieve maximal effect, strongly suggests that these chelators act as vanadyl ionophores. This notion was confirmed by carrier-facilitated extraction of VO2+ from water into CHCl3 with the following order of decreasing efficacy: RL-262 greater than RL-252 greater than 1367 greater than RL-261. The chelators' potentiating effect may therefore be related to facilitated transport of VO2+ ions into the cells' interiors.(ABSTRACT TRUNCATED AT 250 WORDS)
In this study we have searched for sulfhydryl reagents which can be radiolabeled and detect minute quantities of SH-proteins. Iodoacetamidotyramine reacts with sulfhydryls at a low rate, having a pseudo-first order rate constant, kappa obs = 3 +/- 0.2 M-1 s-1, at neutral pH. In contrast, N-ethylmaleimide-containing reagents, such as tyrosine-MIB and tyramine-MIB were three orders of magnitude more reactive in alkylating sulfhydryls. Pseudo-first order rate constants, kappa obs, were in the range of 5200-5700 M-1 s-1. Therefore, a simple and convenient procedure was designed for the synthesis and the radioactive labeling of tyramine-MIB. Simplification was attained by virtue of the specific-'affinity' adsorption of [125I]tyramine-MIB (and not the other intermediates) to small Sephadex G-10 column and its elution with ethanol. [125I]Tyramine-MIB was stable for weeks in dried form and for hours in acidic to neutral aqueous solutions. The reagent, when radiolabeled to high specific activity (0.5 Ci/mumol), detected sulfhydryl proteins at concentrations as low as 1-10 pM. The applicability of the reagent in studying biological systems was demonstrated by adding it to intact adipocytes and the consequent labeling of a single protein with an apparent Mr = 32,000, which is most likely an externally oriented surface plasma membrane SH-protein. [125I]Tyramine-MIB reactivity and sensitivity exceeds that of protein-tyrosyl radioiodination by the chloramine-T procedure and is expected to assist in studying minute quantities of SH-proteins.
1. Bovine insulin dissolved in 0.05 M deoxycholic acid was absorbed through oral intubation in fish weighing 200-300 g. 2. The peak of absorption appeared in all fish, 30-45 min after intubation and was followed by a gradual decrease. The extent of absorption was extremely variable, with up to a 20-fold difference between individuals. No detectable insulin was found in fish intubated with vehicle. 3. The absorbed hormone retained 17-88% of its lipogenic bioactivity in vitro. The absorbed insulin lowered the levels of several amino acids in the intubated fish, indirectly indicating that its bioactivity was also retained in vivo.
The effects of Zn2+ in mimicking insulin in vivo and in vitro are further characterized. Like insulin, Zn2+ stimulated the conversion of [U-14C]-, [1-14C]-, and [6-14C]glucose to lipids in rat adipocytes. Maximum stimulation of lipogenesis was 55-80% of maximum insulin response after preincubation (30 min at 37 degrees C) of adipocytes with ZnCl2 (0.4 mM). Under these conditions, the half-maximum effect was achieved at 0.17 +/- 0.02 mM of ZnCl2. Similarly, an insulinlike effect of Zn2+ was observed on the oxidation of glucose by both pathways, glycolytic and hexose monophosphate shunt. In contrast, unlike insulin, Zn2+ did not inhibit lipolysis but rather exhibited a slight lipolytic activity. Also, the effect of Zn2+ on hexose influx did not exceed 14 +/- 3% that of insulin. The stimulatory effects of Zn2+ were not related to generation of H2O2. Catalase (100 micrograms/ml) did not inhibit Zn(2+)-stimulated glucose oxidation and its incorporation into lipids. Zn2+ had an additive effect on either insulin- or vanadate-stimulated conversion of [1-14C]glucose to fat, and together, the effect was approximately 140% of the maximum rate of lipogenesis. Chelation of intracellular Zn2+ by the cell-permeable chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine did not significantly affect the ability of insulin to stimulate lipogenesis. Adipocytes derived from STZ rats were largely refractory to the modulating action of insulin. In contrast, the effect of Zn2+ on lipogenesis in these cells was more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)
Modification of human growth hormone (hGH) with a low equimolar concentration of fluorescein isothiocyanate (FITC) yielded a derivative containing 1 mol of fluorescein/mol of protein. The site of modification was identified as lysine-70. Lysine-70 of hGH is about 3-fold more reactive than a "normal" lysine in a protein, having pseudo-first-order kinetics Kobs = 110 +/- 7 M-1 min-1 at pH 10.5. The pKa of the lysine was estimated to be 10.7, within the normal range of normal epsilon-lysine moieties in proteins. This higher chemical reactivity seems to favor selective labeling of this moiety at low FITC concentrations. To obtain monomodified derivatives, hGH was derivatized with 0.6 equiv of FITC, and the modified derivatives were separated from unreacted hormone by means of HPLC using a Mono Q column. Its biological activity, determined by Nb2 bioassay, decreased to 40%, and its affinity toward lactogen receptors in Nb2 cells and toward somatogen receptors in bovine liver decreased respectively to 30% and 20%. The present study indicates that out of the seven amino groups of human growth hormone, the epsilon-amino group of lysine-70 is excessively reactive toward FITC. Second, this particular amino group contributes to receptor binding and receptor activation. Lysine-70 is located in the loop between the first and second helix and close to the carboxy-terminal end of the first helix. This contribution is most likely the result of the formation of an electrostatic interaction between the hormone and the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)
A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.
The ortho, meta, and para forms of hydroxyphenyl acetate were found to be inhibitory in the order of ortho greater than para greater than meta in three distinct biological assays: (a) insulin-dependent assimilation of glucose into lipids in intact adipocytes, (b) growth and proliferation of Nb2 rat lymphoma cells, and (c) tyrosine phosphorylation of copolymer (Glu4Tyr) under cell-free conditions. Although relatively high concentrations of o-hydroxyphenyl acetate (OHPA) were required to inhibit these processes, the inhibitor exhibited a low index of cytotoxicity and high specificity toward inhibiting tyrosine- (but not serine-) specific kinases. Cell cycle analysis of the DNA histograms in Nb2 cells revealed that exposure to OHPA did not change the initiation of the G0/G1----S transition but drastically reduced its rate and a subsequent cell proliferation. Kinetic experiments in which the inhibitor was added or withdrawn through different phases of cell cycle confirmed this conclusion. OHPA inhibition of cell growth appears to be limited to eukaryotic cells as the growth of either gram-positive or gram-negative bacteria was unaffected by the presence of the inhibitor. The study supports the following conclusions: (a) Events that are dependent on tyrosine phosphorylation are indeed essential for mammalian cell growth and proliferation. (b) Neither the initial nor intermediate events of the proliferative cascade that occur in the Nb2 cells prior to DNA synthesis are dependent on the activity of protein tyrosine kinase(s) that are inhibited by OHPA. (c) Cell growth of prokaryotic cells and yeast may lack protein tyrosine kinase activity or be less dependent on events requiring tyrosine phosphorylation. (d) Inhibition of the insulin-dependent lipogenesis is subsequent to the inhibition of insulin receptor tyrosine kinase activity.
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We have studied the effects of oral administration of vanadate, an insulinometic agent and a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) in vitro, on blood glucose and PTPase action, in two hyperinsulinemic rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Oral administration of vanadate (0.25 mg/ml in the drinking water) to ob/ob mice for 3 wk lowered blood glucose level from 236 +/- 4 to 143 +/- 2 mg/dl without effect on body weight. Administration of vanadate to db/db mice produced a similar effect. Electron microscopic examination revealed no signs of hepatotoxicity after 47 d of treatment. There was a slight reduction in insulin receptor autophosphorylation when tested by immunoblotting with antiphosphotyrosine antibody after in vivo stimulation, and the phosphorylation of the endogenous substrate of the insulin receptor, pp185, was markedly decreased in the ob/ob mice. Both cytosolic and particulate PTPase activities in liver of ob/ob mice measured by dephosphorylation of a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation were decreased by approximately 50% (P less than 0.01). In db/db diabetic mice, PTPase activity in the cytosolic fraction was decreased to 53% of control values (P less than 0.02) with no significant difference in the particulate PTPase activity. Treatment with vanadate did not alter hepatic PTPase activity as assayed in vitro, or receptor and substrate phosphorylation as assayed in vivo, in ob/ob mice despite its substantial effect on blood glucose. These data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase.
The insulin-like effects of okadaic acid (OKA) in rat adipocytes were further characterized. Okadaic acid did not alter insulin receptor function. This includes undisturbed insulin binding and receptor-mediated ligand internalization in OKA-treated cells. Also, the tyrosine kinase activity of the insulin receptor was not modified in a cell-free system. The stimulating effects of OKA were significantly increased by preincubating (40 min) the cells at 37 C. At lower temperatures (i.e. 26-30 C), OKA did not mimic insulin. Maximal stimulation of lipogenesis occurred at 0.5 microM and then declined at higher concentrations. The insulin-like effects of OKA on lipogenesis did not persist after removal of the agent by washing at 37 C. Okadaic acid maximally stimulated the incorporation of [1-14C]glucose into lipids and the oxidation of [6-14C]glucose into 14CO2, but unlike insulin, it had little if any effect of oxidizing [1-14C]glucose to 14CO2 or incorporating [6-14C]glucose into lipids. Okadaic acid was equivalent to insulin in stimulating 3-O-methyl-glucose uptake. Since the insulin-like effects of OKA did not persist after preincubation and washing, the effects of insulin in OKA-treated cells could be evaluated. The adipocytes were found to be fully refractory to the modulating actions of insulin. Thus, insulin did not stimulate glucose transport, its oxidation, or its incorporation into lipids, and failed to reverse lipolysis. Unresponsiveness was fully developed after 40-min preincubation at 37 C with 3 microM OKA and was half-maximal at 0.13 microM OKA. It persisted at least over a period of 150 min. The effect of OKA was restricted to the stimulating actions of insulin and vanadate. Basal activities were not altered, nor was the ability of the desensitized cells to respond to isoproterenol. The lack of an insulin-like effect of OKA on some metabolic pathways enabled us to demonstrate that OKA (0.25 microM) also rendered adipocytes fully unresponsive to insulin in the continuous presence of the agent. Western blotting of the 40,000 x g pellets with antibodies to phosphotyrosine revealed the appearance of a protein with an apparent mol wt of 43,000 in OKA-desensitized cells. In summary, OKA mimics some of insulin bioeffects, but concomitantly renders the cells tolerant to the modulating action of the hormone itself.
Previous studies have demonstrated that vanadate ions mimic many of the actions of insulin in in vitro systems. Also, vanadate administered to diabetic hyperglycemic rats lowers their blood glucose levels to normal values. In this study we demonstrate that vanadate inhibits glucose output in the isolated perfused rat liver. Glucose production was suppressed maximally (about 50% to 60%), on addition of extremely low vanadate ion concentrations (0.5 to 1 mumol/L). This concentration is about two log units lower than the vanadate ion concentrations that are required to activate hexose uptake and glucose metabolism in vitro and is within the range of endogenous intracellular vanadium concentration. Insulin had little or no effect in inhibiting hepatic glucose output in this experimental system. The effect of vanadate ions is rapid in onset and is not accompanied by any signs of liver toxicity as assessed by various criteria. In conclusion, the study indicates that (a) vanadate ions inhibits hepatic glucose output, maximally and at extremely low, nontoxic concentrations (ID50 = 0.7 +/- 0.1 mumol/L). (b) The modulation action of the ion is fast and probably occurs at point(s) distal to the insulin receptor itself. (c) The liver participates in the process of maintaining euglycemia in diabetic rats receiving optimal doses of vanadate orally.
Previous studies have shown that reduced carbamoylmethylated lysozyme (RCAM-lysozyme, MW approximately 14.5K) is a substrate and inhibitor (Ki approximately 0.6 microM) of insulin receptor kinase (InsRK) autophosphorylation (Kohanski & Lane, 1986; Lane & Kohanski, 1986). In this study we have prepared a family of defined modified derivatives of RCAM-lysozyme and used them to probe the nature of the substrate and inhibitory sites of InsRK. All open-chain derivatives of lysozyme in which either the tryptophanyl, methionyl, cysteinyl, arginyl, or histidyl side chains were modified served as substrates and were potent inhibitors of InsRK autophosphorylation. This was true whether the substitutions were either hydrophilic or hydrophobic, although the hydrophilic derivatives had a higher inhibitory potency. Tryptic peptides derived from RCAM-lysozyme, however, were inactive as inhibitors, and a mixture of the three cyanogen bromide fragments (containing 12, 24, and 93 amino acids, respectively) was found to be less potent in inhibiting the receptor kinase. Derivatization of either tyrosyl or carboxyl side chains produced derivatives that were neither substrates nor capable of inhibiting receptor autophosphorylation. Derivatives with modified amino groups were substrates for InsRK but were not able to inhibit InsRK autophosphorylation. The present study suggests that (a) unphosphorylated InsRK has a large hydrophilic substrate binding domain and is effectively inhibited by long-chain polypeptides but not by short sequences, (b) some of the amino, carboxyl, and hydroxyphenyl side chains are essential to the inhibitory nature of these polypeptides, and (c) derivatives that fail to inhibit autophosphorylation can still be recognized and phosphorylated by active InsRK.(ABSTRACT TRUNCATED AT 250 WORDS)