PubMed Health⌕ Search

Biomedical subjects

Y Shechter

Publications and source records attributed to Y Shechter.

At least 37 records · Page 2Linked to original sources
PubMed1997

A novel assay for evaluating glycogenolysis in rat adipocytes and the inability of insulin to antagonize glycogenolysis in this cell type.

We report here on a novel procedure for measuring glycogenolysis in rat adipocytes. In this procedure, cells are incubated for 30 min at 37 degrees C with insulin or vanadate, and with [U-14C]glucose to label the glycogen pool with radioactive glucose. The cells are washed and preincubated for an additional 1 h, before being assayed. The extent of glycogenolysis is determined by the decrease in radioactivity in precipitated glycogen, which was quite substantial under experimental conditions facilitating glycogenolysis. From the assay, we determined the following. (a) Glycogenolysis is activated in rat adipocytes in response to lipolytic hormones (i.e. catecholamines and adrenocorticotropic hormone). (b) Other agents and conditions elevating intracellular adenosine 3',5'-monophosphate levels (i.e. cholera toxin, dibutyryladenosine 3',5'-monophosphate, and isobutylmethylxanthine) also activate glycogenolysis. (c) Glycogenolysis (as opposed to lipolysis) is activated at concentrations of adrenocorticotropic hormone or isoproterenol 7-11-fold lower and at adenosine 3',5'-monophosphate concentrations 7-fold lower. (d) Calyculin A, a specific inhibitor of protein phosphatase 1, activates glycogenolysis as well. Calyculin A also activates lipolysis at an equimolar potency. (e) Insulin does not antagonize glycogenolysis in rat adipocytes. In conclusion, the assay allowed us to compare glycogenolysis to lipolysis within the same cell, and to find that the sensitivity to hormones and adenosine 3',5'-monophosphate was about 1 order of magnitude higher for glycogenolysis than for lipolysis. A more striking finding was the inability of insulin to antagonize glycogenolysis in the rat adipose cell, an effect which occurs readily in liver and muscle cells via protein phosphatase 1-activating machinery. This rules out a role for adipose protein phosphatase 1 activation in the mechanism by which insulin antagonizes lipolysis and supports the contention that the insulin effect in lowering adenosine 3',5'-monophosphate levels is the central mechanism by which insulin antagonizes lipolysis.

1-Methyl-3-isobutylxanthine↗
PubMed1997

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 (+4), zinc (Zn2+), tungstate (W), and molybdate (Mo) also had antilipolytic activity. The order of potency was vanadyl acetylacetonate > or = VOSO4 > or = NaVO3 > or = vanadyl-dipicolinate > Zn2+ >> W > Mo, and it correlated better with the inhibition of adipose membranal-PTPases in cell-free experiments. We have concluded that the antilipolytic effect of vanadate is 1) mechanistically distinct from that of insulin, 2) independent of phosphoinositol 3-kinase activation, and 3) independent of the lipolytic cascade. We also strongly suggest that the antilipolytic effect of vanadate emanates from inhibiting adipose membranal, rather than cytosolic PTPases, and present preliminary data showing distinct differences in catalysis between these two PTPase categories. Overall, the study indicates that antilipolysis can be manifested via alternative, insulin-independent, signal-transducing pathways.

Adipocytes↗
PubMed1997

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/l), 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 nonglycoprotein 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 micromol/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 micromol/l) and significantly reversed the antilipolytic effect of vanadate (IC50, 5.0 +/- 0.4 micromol/l). After vanadate treatment, a phosphorylated P55 protein is immunoprecipitated by antibodies to both phosphotyrosine and phosphatidylinositol (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.

Adipocytes↗
PubMed1996

Phenylarsine oxide and vanadate: apparent paradox of inhibition of protein phosphotyrosine phosphatases in rat adipocytes.

Vanadate mimics, whereas phenylarsine oxide (PAO) antagonizes, the effects of insulin in rat adipocytes. Both vanadate and PAO are documented inhibitors of protein-phosphotyrosine phosphatases. The relationship between the inhibition of 'inhibitory' PTPase and 'stimulatory' PTPase has been studied here in primary rat adipocytes. Low concentrations of PAO (IC50 = 0.6-2.0 microM) blocked the stimulating effects of insulin, vanadate and pervanadate on hexose uptake and glucose metabolism. Inhibition of isoproterenol-mediating lipolysis by vanadate and insulin was not blocked by PAO. The activating effects of okadaic acid on hexose uptake and glucose metabolism, which occur at points downstream to tyrosine phosphorylation, were also not blocked by PAO. Subsequent studies suggested that the PAO-sensitive PTPase comprises a minute fraction of the total adipocytic PTPase activity. To identify its location we applied procedures involving fractionations and activation of non-receptor adipocytic protein tyrosine kinase by PAO and vanadate in cell free assays. We found that the 'inhibitory' PTPase is exclusively associated with the membrane fraction whereas the 'stimulatory' PTPases are present in both the cytosolic and plasma membrane compartments. We next searched for markers, possibly associated with PAO-dependent desensitization and found that several proteins became phosphorylated on tyrosine moieties in the supernatant of PAO but not in vanadate pretreated adipocytes. In summary, we propose the presence of a minute, plasma membrane associated PTPase in primary rat adipocytes, inhibition of which arrests the activation of glucose metabolism. In contrast, inhibition of all the other cellular adipose PTPases, ultimately activates rather than inhibits these same bioeffects.

3-O-Methylglucose↗
PubMed1996

Evidence for the distinct vanadyl(+4)-dependent activating system for manifesting insulin-like effects.

Both exogenously added vanadate (oxidation state +5) and vanadyl (oxidation state +4) mimic the rapid responses of insulin through alternative signaling pathways, not involving insulin receptor activation [reviewed in Shechter et al. (1995) Mol. Cell. Biochem. 153, 39-47]. Vanadium exhibits complex chemistry, fluctuating between vanadate(+5) and vanadyl(+4), according to the prevailing conditions. Using several experimental approaches, we report here on a distinct vanadate(+5)-independent, vanadyl(+4)-dependent activating pathway. The key components of this pathway are membrane protein phosphotyrosine phosphatases (PTPases) and a cytosolic (nonreceptor) protein-tyrosine kinase (CytPTK). We further suggest that vanadate(+5) is not reduced rapidly to vanadyl(+4) inside the cell, and entered vanadyl sulfate(+4) is capable of undergoing spontaneous oxidation to vanadate(+5) in vivo. Finally, we show that the promotion and full expression of a downstream bioeffect such as lipogenesis requires both activation of CytPTK and prolonged stability of vanadyl(+4) against oxidation.

Adipocytes↗
PubMed1996

Familial relapsing thrombotic micro-angiopathy in two siblings.

UNLABELLED: Two siblings, a 12-year-old girl and a 7-year-old boy, had recurrent episodes of haemolytic-uraemic syndrome/thrombotic thrombocytopenic purpura, manifested mainly by thrombocytopenia and micro-angiopathic haemolytic anaemia. During 11 years of follow up the girl responded only to steroids, whereas many other therapeutic modalities were ineffective. Following treatment with low dose danazol relapses became fewer and of diminished severity and completely subsided after 6 months. The boy started his illness with signs of haemolytic uraemic syndrome and later developed neurological manifestations. During a 6 year follow up he responded only to plasma exchange. Although chronic thrombocytopenia persisted during the past 3 years, the boy's clinical condition improved. CONCLUSION: A family with two children with recurrent episodes of thrombotic thrombocytopenic purpura is described. In one child danazol could have had a beneficial effect.

Anemia, Hemolytic↗
PubMed1996

Vanadium salts as insulin substitutes: mechanisms of action, a scientific and therapeutic tool in diabetes mellitus research.

Vanadium and its compounds exhibit a wide variety of insulin-like effects. In this review, these effects are discussed with respect to the treatment of type I and type II diabetes in animal models, in vitro actions, antineoplastic role, treatment of IDDM and NIDDM patients, toxicity, and the possible mechanism(s) involved. Newly established CytPTK plays a major role in the bioresponses of vanadium. It has a molecular weight of approximately 53 kDa and is active in the presence of Co2+ rather than Mn2+. Among the protein-tyrosine kinase blockers, staurosporine is found to be a potent inhibitor of CytPTK but a poor inhibitor of InsRTK. Vanadium inhibits PTPase activity, and this in turn enhances the activity of protein tyrosine kinases. Our data show that inhibition of PTPase and protein tyrosine kinase activation has a major role in the therapeutic efficacy of vanadium in treating diabetes mellitus.

Animals↗
PubMed1995

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.

Alkaloids↗
PubMed1995

Permolybdate and pertungstate--potent stimulators of insulin effects in rat adipocytes: mechanism of action.

In previous studies, tungstate and molybdate were found to mimic the biological actions of insulin. It was suggested that these metallooxides initially inhibit vanadate-sensitive protein phosphotyrosine phosphatase (PTPase). This, in turn, stimulates a staurosporine-sensitive cytosolic protein tyrosine kinase (cytPTK), which activates several insulin bioeffects via insulin-independent pathways (Shisheva & Shechter, 1991, 1993; Elberg et al., 1994). Tungstate and molybdate, however, facilitate bioeffects in rat adipocytes only at high (millimolar) concentrations (Goto et al., 1992). We report here that incubations of tungstate or molybdate with hydrogen peroxide (H2O2) result in the formation of pertungstate (pW, peroxide of tungstate) or permolybdate (pMo, peroxide of molybdate). Pertungstate and permolybdate were found to stimulate all or most of the insulin bioeffects in rat adipocytes. Moreover, these permetallooxides are 80-180-fold more potent stimulators than the corresponding metallooxides. This shift in potency resembles that of pervanadate relative to vanadate in stimulating the same effect in rat adipocytes (Fantus et al., 1989). pW and pMo are also active in normalizing blood glucose levels in streptozotocin-induced diabetic rats. Further studies aimed at understanding the higher efficacy of this permetallooxide revealed the following: (a) All three permetallooxides (pV, pW, pMo) are oxidizing agents relative to reduced glutathione (GSH). They oxidize stoichiometric amounts of GSH to GSSG. (b) All three metallooxides do not oxidize GSH to GSSG. (c) Both metallooxides and permetallooxides inhibit rat adipocytic PTPase at micromolar quantities (IC50 = 3-10 microM). Permetallooxides, however, inhibited a larger PTPase fraction (80-100%) compared to metallooxides (40-70% of the total).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocytes↗
PubMed1995

Multiple sites of vanadate and peroxovanadate action in Xenopus oocytes.

In Xenopus laevis oocytes, the insulin mimics, vanadate and peroxovanadates (PV), stimulated the uptake of 3H-2-deoxyglucose and incorporation of 35S-methionine into protein. For both hexose transport and protein synthesis, peroxovanadates (produced by reacting vanadate and H2O2) were at least as potent as vanadate. Microinjection of peroxovanadates into the oocytes stimulated 2-deoxyglucose uptake. However, methionine incorporation was not stimulated by microinjection of peroxovanadate or vanadate solutions. Consistent with these results and with the possibility that vanadate and peroxovanadates enter the cell on a phosphate transporter, raising the medium phosphate concentration from 1 mM to 10 mM blocked vanadate-stimulated hexose transport and partially reduced peroxovanadates stimulation of hexose transport. Increased medium phosphate did not reduce stimulation of protein synthesis by either effector. Taken together, these data indicate that vanadate/peroxovanadates act at both intracellular and extracellular sites. Action at the former stimulates hexose uptake and action at the latter, protein synthesis.

Animals↗
PubMed1995

A Bombay individual lacking H and Le antigens but expressing normal levels of alpha-2- and alpha-4-fucosyltransferases.

BACKGROUND: The rare Bombay phenotype is usually due to a primary genetic defect in an alpha-2- or alpha-4-fucosyltransferase. The present study was done to investigate a patient with normal transferases, who exhibits the Bombay phenotype. CASE REPORT: Red cells of the patient, his parents, and siblings were phenotyped for A, B, and H antigens. The presence of B, H, and Le transferases in serum and saliva was measured. RESULTS: The parents and siblings were all group B, Le(a-b-). The propositus was typed as Oh, Le(a-b-). His serum contained anti-A, anti-B, and anti-H. Normal levels of B, H, and Le transferases were found in all family members including the patient. CONCLUSION: In an unusual case, a person has the Bombay phenotype, but normal levels of transferases in serum and saliva. A general defect in fucose metabolism seems to be the primary abnormality in this case.

ABO Blood-Group System↗
PubMed1995

Blood exchange [correction of exchance]-a rescue procedure for complicated falciparum malaria.

Falciparum malaria is the most hazardous form of malaria. Its high degree of parasitemia interferes with vital functions of most organs and is directly responsible for its high rate of mortality and morbidity. Quinine and other antimalarial drugs are relatively slow acting and not always effective due to the growing resistance developed by Plasmodium toward these drugs. Another emergency modality, which would remove the parasitic burden quickly and effectively, is thus much needed. We present a case of a 51-year-old sailor, who was admitted to the hospital because of complicated falciparum malaria. His situation deteriorated rapidly into a desparate stage, despite the various intensive treatments and quinine. He soon developed a systemic inflammatory response syndrome manifested as cerebral malaria, renal failure, acute respiratory distress syndrome and disseminated intravascular coagulation. An emergency blood exchange reversed the situation dramatically, and the patient recovered completely. It is recommended that any doctor, both in endemic and in non endemic areas, dealing with blood transfusions or infectious diseases, should be acquainted with this lifesaving modality, regardless of the controversy still surrounding this subject.

Emergencies↗
PubMed1994

A dynamic system for suppression and re-expression of insulin and pervanadate bioresponses in rat adipocytes. Treatment with okadaic acid and staurosporine.

In previous studies, we demonstrated that while okadaic acid stimulates glucose metabolism, it suppresses the bioresponses of insulin itself in rat adipocytes (Shisheva and Shechter, Endocrinology 129: 2279-2288, 1991). Both stimulation and suppression were attributed to okadaic acid-dependent inhibition of protein phosphatases 1 and 2A. We report here that exposure of adipocytes to staurosporine prior to okadaic acid restored insulin-stimulated actions on glucose metabolism. The effect was half-maximal at staurosporine concentrations as low as 70 nM and was fully expressed (80-87% of the control) at 400-500 nM. Similarly, the insulin-like effect of pervanadate, which was also suppressed by okadaic acid, was restored completely with staurosporine pretreatment. Staurosporine was less effective in restoring cell responses inhibited by high concentrations of okadaic acid, or when added to the cells after okadaic acid. Cell resensitization was unique to staurosporine and could not be produced by various agents that reduce cellular protein kinase A- or protein kinase C-dependent phosphorylation, such as phenylisopropyl adenosine (PIA), K-252a and GF 109203X. Staurosporine (400 nM) partially reversed lipolysis induced by okadaic acid but not that induced by beta-adrenergic stimulation. PIA, which antagonized okadaic acid-induced lipolysis to the same extent as staurosporine, was not capable of restoring insulin responses. Further studies aimed at elucidating this reversing effect revealed that staurosporine did not reactivate okadaic acid-inhibited protein phosphatases 1 and 2A in both cellular and cell-free systems. In summary, we report here a unique dynamic system in which insulin and pervanadate bioeffects can be fully suppressed and again re-expressed without reactivation of protein phosphatase 1 or 2A. The precise site for both effects, although still obscure, appears to be downstream from autophosphorylated insulin receptor.

Adipose Tissue↗
PubMed1994

Vanadium activates or inhibits receptor and non-receptor protein tyrosine kinases in cell-free experiments, depending on its oxidation state. Possible role of endogenous vanadium in controlling cellular protein tyrosine kinase activity.

We have shown that vanadium mimics several insulin effects in rat adipocytes, via a staurosporine sensitive cytosolic protein tyrosine kinase (CytPTK; Shisheva, A., and Shechter, Y. (1993) J. Biol. Chem. 268, 6463). Here we demonstrate that vanadium effects on protein tyrosine kinases are preserved after cell disintegration. Vanadium inhibits or activates protein tyrosine kinases depending on its oxidation state and the tyrosine kinase studied. Vanadyl (4+) but not vanadate (5+) inhibits receptor tyrosine kinases such as the insulin receptor (IC50 value = 23 +/- 4 microM) and the insulin-like growth factor-I receptor (IC50 = 19 +/- 3 microM). Inhibition is non-competitive with respect to ATP, Mn2+, or substrate concentrations. Preincubation of adipocytes with vanadyl (0.4 mM), and staurosporine (which arrests the cytosolic enzyme) substantially inhibited insulin-stimulated lipogenesis. Vanadyl is readily oxidized to vanadate by hydrogen peroxide. In contrast, CytPTKs were poorly inhibited by vanadyl, and vanadate stimulated several CytPTKs 2-6 fold. CytPTK derived from rat adipocytes, liver and brain were activated, and CytPTK from Nb2 lymphoma cells was not affected. CytPTK extracted from insulin-responsive tissues are more sensitive to vanadate activation (ED50 = 3 +/- 0.7 microM), whereas the brain enzyme is less sensitive (ED50 = 27 +/- 3 microM). Tungstate, molybdate, and phenylarsine oxide also stimulate CytPTK, suggesting that the vanadate effect is secondary to inhibiting protein phosphotyrosine phosphatases. This study supports a working hypothesis implicating the intracellular vanadyl pool in modulating CytPTK activity. Any physiological conditions converting vanadyl to vanadate (i.e. H2O2 production) will activate CytPTK and consequently CytPTK-dependent bioeffects.

Adipocytes↗
PubMed1994

The protein tyrosine phosphatase inhibitor, pervanadate, is a powerful antidiabetic agent in streptozotocin-treated diabetic rats.

The effect of pervanadate, a potent insulinomimetic agent that inhibits insulin receptor dephosphorylation in vitro, is now assessed in vivo. A single i.p. administration of pervanadate at concentrations as low as 700 micrograms vanadium/kg body wt markedly lowered blood glucose levels in streptozotocin-induced diabetic rats from 430 +/- 28 to 212 +/- 30 mg/100 ml within 3 h. A decrease was already observed half hour after treatment, continued in accelerating fashion to the 3rd h, and persisted for at least 24 h. The initial hyperglycemia reoccurred on the second day and remained thereafter. In comparable fashion, pervanadate decreased the blood glucose levels of control healthy rats, treated identically. Within this period body wt was not significantly altered in either group. This data indicate that rapid and efficient management of glucose homeostasis is achieved via inhibiting receptor dephosphorylation. This observation may lead to a new therapeutic approach of protein tyrosine phosphatase inhibition for future treatment of diabetes in general, and in insulin resistant states in particular.

Animals↗
PubMed1994

Group A beta-hemolytic streptococcal pharyngitis in children younger than 5 years.

We report on a prospective study of 152 children aged 3 months to 5 years, from a community pediatric clinic, who had signs of pharyngitis, temperature > or = 38 degrees C and were not treated by antibiotics during the preceeding week. Nose and throat cultures were taken from each child. Blood antistreptolysin (ASO) was examined. If the cultures were positive for group A beta hemolytic Streptococcus (GABHS), a second blood sample for ASO was obtained later. True streptococcal infection was defined in a case of a positive culture and an increase in the ASO titer of at least two tubes, while cases of positive cultures without significant changes in ASO titer were defined as carriers. Positive GABHS cultures were found in 23 cases. True group A Streptococcus infection was found only in patients > 2 years old. The carriers of GABHS increased gradually from 3% during the 1st year to 22% by the 5th year. This study demonstrated that in the population evaluated, the incidence of true GABHS infection in children < 2 years of age is low, as was observed in the past.

Age Distribution↗
PubMed1993

Role of cytosolic tyrosine kinase in mediating insulin-like actions of vanadate in rat adipocytes.

In previous studies we have identified a cytosolic protein tyrosine kinase (CytPTK) in rat adipocytes that is largely activated in vanadate-pretreated cells (Shisheva, A., and Shechter, Y. (1992) FEBS Lett. 300, 93-96). We report here that staurosporine and its analog K-252a are highly potent (ID50 = 3 and 100 nM, respectively) in inhibiting CytPTK activity of crude cell extract or partially purified enzyme preparations. Staurosporine and K-252a were less effective by more than 2 and 1 orders of magnitude, respectively, in inhibiting insulin receptor-catalyzed PolyGlu4Tyr phosphorylation in cell-free experiments. Preincubation of rat adipocytes with either staurosporine or K-252a selectively blocked the action of vanadate in activating glucose incorporation into lipids and its oxidation. Thus, staurosporine inhibited vanadate-stimulated lipogenesis and glucose oxidation (via glycolysis and the pentose phosphate pathway) in a concentration-dependent manner with ID50 of 75 and 300 nM, respectively. Insulin-stimulated bioeffects were not inhibited at this low range of staurosporine concentration. Staurosporine had no effect on vanadate-stimulated hexose uptake or on vanadate's antilipolytic action. Using staurosporine, we probed those insulinomimetic agents which facilitate their biological activity via the insulin receptor kinase (insulin, wheat germ agglutinin, concanavalin A, and pervanadate) or via CytPTK (vanadate and to a certain degree Mn2+ and Zn2+). These results suggest that (a) vanadate facilitates its insulin-like actions on glucose utilization via the cytosolic tyrosine kinase and (b) this enzyme does not participate in vanadate effects in stimulating hexose uptake and in inhibiting lipolysis. These findings explain further vanadate's post-insulin receptor actions and raise possible application in the management of glucose metabolism in insulin-independent fashion in pathological conditions.

Adipose Tissue↗