Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI 3-kinase activity and a subsequent rise in sodium transport (Markadieu N, Crutzen R, Blero D, Erneux C, Beauwens R. Am J Physiol Renal Physiol 288: F1201–F1212, 2005). We therefore questioned whether insulin would produce an intracellular burst of H2O2 leading to PI 3-kinase activation and subsequent increase in sodium transport. An acute production of reactive oxygen species (ROS) in A6 cells incubated with the oxidation-sensitive fluorescent probe 5,6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate was already detected after 2 min of insulin stimulation. This fluorescent signal and the increase in sodium transport were completely inhibited in monolayers incubated with peggylated catalase, indicating that H2O2 is the main intracellular ROS produced upon insulin stimulation. Similarly, preincubation of monolayers with different chelators of either superoxide (O2•−; nitro blue tetrazolium, 100 μM) or H2O2 (50 μM ebselen), or blockers of NADPH oxidase (Nox) enzymes (diphenyleneiodonium, 5 μM; phenylarsine oxide, 1 μM and plumbagin, 30 μM) prevented both insulin-stimulated H2O2 production and insulin-stimulated sodium transport. Furthermore, diphenyleneiodonium pretreatment inhibited the recruitment of the p85 PI 3-kinase regulatory subunit in an anti-phosphotyrosine immunoprecipitate in insulin-stimulated cells. In contrast, PI-103, an inhibitor of class IA PI 3-kinase, inhibited insulin-stimulated sodium transport but did not significantly reduce insulin-stimulated H2O2 production. Taken together, our data suggest that insulin induces an acute burst of H2O2 production which participates in an increase in phosphatidylinositol 3,4,5- trisphosphate production and subsequently stimulation of sodium transport.

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Phosphatidylinositol 3,4,5-trisphosphate: an early mediator of insulin-stimulated sodium transport in A6 cells

AJP Renal Physiology ◽

10.1152/ajprenal.00314.2003 ◽

2004 ◽

Vol 287 (2) ◽

pp. F319-F328 ◽

Cited By ~ 15

Author(s):

Nicolas Markadieu ◽

Daniel Blero ◽

Alain Boom ◽

Christophe Erneux ◽

Renaud Beauwens

Keyword(s):

Sodium Transport ◽

Cell Monolayer ◽

Sodium Reabsorption ◽

Na Channel ◽

Insulin Stimulation ◽

Cell Monolayers ◽

A6 Cells ◽

Cell Extracts ◽

Pkb Phosphorylation ◽

Stimulation Of

Insulin stimulates sodium transport across A6 epithelial cell monolayers. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) was suggested as an early step in the insulin-stimulated sodium reabsorption (Ref. 35). To establish that the stimulation of the PI 3-kinase signaling cascade is causing stimulation of apical epithelial Na channel, we added permeant forms of phosphatidylinositol (PI) phosphate (P) derivatives complexed with a histone carrier to A6 epithelium. Only PIP3 and PI( 3 , 4 )P2 but not PI( 4 , 5 )P2 stimulated sodium transport, although each of them penetrated into A6 cell monolayers as assessed using fluorescent permeant phosphoinositides derivatives. By Western blot analysis of A6 cell extracts, the inositol 3-phosphatase PTEN and the protein kinase B PKB were both detected. To further establish that the stimulation of sodium transport induced by insulin is related to PIP3 levels, we transfected A6 cells with human PTEN cDNA and observed a 30% decrease in the natriferic effect of insulin. Similarly, the increase in sodium transport observed by addition of permeant PIP3 was also reduced by 30% in PTEN-overexpressing cells. PKB, a main downstream effector of PI 3-kinase, was phosphorylated at both Thr 308 and Ser 473 residues upon insulin stimulation of the A6 cell monolayer. PKB phosphorylation in response to insulin stimulation was reduced in PTEN-overexpressing cells. Permeant PIP3 also increased PKB phosphorylation. Taken together, the present results establish that the d-3-phosphorylated phosphoinositides PIP3 and PI( 3 , 4 )P2 mediate the effect of insulin on sodium transport across A6 cell monolayers.

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Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers

AJP Renal Physiology ◽

10.1152/ajprenal.00383.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. F1201-F1212 ◽

Cited By ~ 22

Author(s):

Nicolas Markadieu ◽

Raphaël Crutzen ◽

Daniel Blero ◽

Christophe Erneux ◽

Renaud Beauwens

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Sodium Transport ◽

Kinase Activity ◽

Mapk Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Cell Monolayers ◽

Epidermal Growth ◽

Kinase Pathway ◽

Stimulation Of

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin stimulation of sodium transport in A6 cell monolayers. In this study, we investigate whether stimulation of the PI 3-kinase by other agents also provoked an increase in sodium transport. Both epidermal growth factor (EGF) and H2O2 provoked a rise in sodium transport that was inhibited by LY-294002, an inhibitor of PI 3-kinase activity. PI 3-kinase activity was estimated in extracts from A6 cell monolayers directly by performance of a PI 3-kinase assay. We also estimated the relative importance of the PI 3-kinase pathway by two different methods: 1) coprecipitation of the p85 regulatory subunit with anti-phosphotyrosine antibodies and 2) phosphorylation of PKB on both Ser 473 and Thr 308 residues observed by Western blotting. Since the mitogen-activated protein kinase (MAPK) pathway has also been implicated in the regulation of sodium transport, we also investigated whether this pathway is turned on by insulin, H2O2, or EGF. Phosphorylation of ERK1/2 was increased only transiently by insulin and H2O2 but quite sustainedly by EGF. Inhibitors of this pathway (U-0126 and PD-98059) failed to affect the insulin and H2O2 stimulation of sodium transport but increased substantially the stimulation induced by EGF. The latter effect was associated with an increase in PKB phosphorylation, thus suggesting that the stimulation of the MAPK pathway prevents, in part, the stimulation of the PI 3-kinase pathway in the transport of sodium stimulated by EGF.

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PI 3-Kinase Activation Is Required for Insulin Stimulation of Glucose Transport into L6 Myotubes

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1994.2703 ◽

1994 ◽

Vol 205 (1) ◽

pp. 570-576 ◽

Cited By ~ 27

Author(s):

J. Berger ◽

N. Hayes ◽

D.M. Szalkowski ◽

B. Zhang

Keyword(s):

Glucose Transport ◽

Insulin Stimulation ◽

Kinase Activation ◽

L6 Myotubes ◽

Stimulation Of

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The relationship between insulin binding, insulin activation of insulin-receptor tyrosine kinase, and insulin stimulation of glucose uptake in isolated rat adipocytes. Effects of isoprenaline

Biochemical Journal ◽

10.1042/bj2740787 ◽

1991 ◽

Vol 274 (3) ◽

pp. 787-792 ◽

Cited By ~ 19

Author(s):

H H Klein ◽

S Matthaei ◽

M Drenkhan ◽

W Ries ◽

P C Scriba

Keyword(s):

Glucose Uptake ◽

Kinase Activity ◽

Coupling Efficiency ◽

Insulin Binding ◽

Receptor Kinase ◽

Insulin Stimulation ◽

Insulin Effect ◽

Kinase Activation ◽

The Relationship ◽

Stimulation Of

We have studied the relationship between insulin activation of insulin-receptor kinase and insulin stimulation of glucose uptake in isolated rat adipocytes. Glucose uptake was half-maximally or maximally stimulated, respectively, when only 4% or 14% of the maximal kinase activity had been reached. To investigate this relationship also under conditions where the insulin effect on activation of receptor kinase was decreased, the adipocytes were exposed to 10 microM-isoprenaline alone or with 5 micrograms of adenosine deaminase/ml. An approx. 30% (isoprenaline) or approx. 50% (isoprenaline + adenosine deaminase) decrease in the insulin effect on receptor kinase activity was found at insulin concentrations between 0.4 and 20 ng/ml, and this could not be explained by decreased insulin binding. The decreased insulin-effect on kinase activity was closely correlated with a loss of insulin-sensitivity of glucose uptake. Moreover, our data indicate that the relation between receptor kinase activity and glucose uptake (expressed as percentage of maximal uptake) remained unchanged. The following conclusions were drawn. (1) If activation of receptor kinase stimulates glucose uptake, only 14% of the maximal kinase activity is sufficient for maximal stimulation. (2) Isoprenaline decreases the coupling efficiency between insulin binding and receptor-kinase activation, this being accompanied by a corresponding decrease in sensitivity of glucose uptake. (3) Our data indicate that the signalling for glucose uptake is closely related to receptor-kinase activity, even when the coupling efficiency between insulin binding and kinase activation is altered. They thus support the hypothesis that receptor-kinase activity reflects the signal which originates from the receptor and which is transduced to the glucose-transport system.

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Intracellular Na+ and K+ activities during insulin stimulation of rat soleus muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1982.242.3.e193 ◽

1982 ◽

Vol 242 (3) ◽

pp. E193-E200

Author(s):

R. J. Stark ◽

J. O'Doherty

Keyword(s):

Membrane Potential ◽

Soleus Muscle ◽

Intracellular Sodium ◽

Resting Membrane Potential ◽

Insulin Stimulation ◽

Subsequent Increase ◽

Extracellular Glucose ◽

Rat Soleus Muscle ◽

Sodium And Potassium ◽

Stimulation Of

The action of insulin on the resting membrane potential (Em) and intracellular sodium and potassium activities (aNa, aK) of rat soleus muscle fibers was determined by direct intracellular measurements of aNa, aK, and Em using Na-selective, K-selective, and conventional microelectrodes. The use of these microelectrodes allowed us to continuously monitor these parameters in the same fiber. Although we were able to accurately measure aNa and aK and continuously monitor their levels throughout periods of insulin stimulation of up to 20 min duration, we were unable to detect any significant change in Em, aNa, or aK. Varying the concentration of insulin or extracellular glucose failed to alter our observations. These results indicate that the action of insulin on the sarcolemma and subsequent increase in glucose transport must result from some mechanism independent of a change in membrane potential or intracellular sodium or potassium activity.

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Role of the Src Homology 2 (SH2) Domain and C-Terminus Tyrosine Phosphorylation Sites of SH2-Containing Inositol Phosphatase (SHIP) in the Regulation of Insulin-Induced Mitogenesis1

Endocrinology ◽

10.1210/endo.140.10.7028 ◽

1999 ◽

Vol 140 (10) ◽

pp. 4585-4594 ◽

Cited By ~ 10

Author(s):

Tsutomu Wada ◽

Toshiyasu Sasaoka ◽

Manabu Ishiki ◽

Hiroyuki Hori ◽

Tetsuro Haruta ◽

...

Keyword(s):

Map Kinase ◽

Tyrosine Phosphorylation ◽

Insulin Receptors ◽

Sh2 Domain ◽

Regulatory Function ◽

Phosphorylation Sites ◽

Insulin Stimulation ◽

Kinase Activation ◽

Stimulation Of

Abstract To examine the role of SHIP in insulin-induced mitogenic signaling, we used a truncated SHIP lacking the SH2 domain (ΔSH2-SHIP) and a Y917/1020F-SHIP (2F-SHIP) in which two tyrosines contributing to Shc binding were mutated to phenylalanine. Wild-type (WT)-, ΔSH2-, and 2F-SHIP were transiently transfected into Rat1 fibroblasts overexpressing insulin receptors (HIRc). Insulin-stimulated tyrosine phosphorylation of WT-SHIP and ΔSH2-SHIP, whereas tyrosine phosphorylation of 2F-SHIP was not detectable, indicating that 917/1020-Tyr are key phosphorylation sites on SHIP. Although SHIP can bind via its 917/1020-Tyr residues and SH2 domain to Shc PTB domain and 317-Tyr residue, respectively, insulin-induced SHIP association with Shc was more greatly decreased in 2F-SHIP cells than that inΔ SH2-SHIP cells. Insulin stimulation of Shc association with Grb2, which is important for p21ras-MAP kinase activation, was decreased by overexpression of WT- and 2F-SHIP. Importantly, insulin-induced Shc·Grb2 association was not detectably reduced in ΔSH2-SHIP cells. In accordance with the extent of Shc association with Grb2, insulin-induced MAP kinase activation was relatively decreased in both WT-SHIP and 2F-SHIP cells, but not in ΔSH2-SHIP cells. To examine the functional role of SHIP in insulin’s biological action, insulin-induced mitogenesis was compared among these transfected cells. Insulin stimulation of thymidine incorporation and bromodeoxyuridine incorporation was decreased in WT-SHIP cells compared with that of control HIRc cells. Expression of 2F-SHIP also significantly reduced insulin-induced mitogenesis, whereas it was only slightly affected by overexpression of ΔSH2-SHIP. Furthermore, the reduction of insulin-induced mitogenesis in WT-SHIP cells was partly compensated by coexpression of Shc. These results indicate that SHIP plays a negative regulatory role in insulin-induced mitogenesis and that the SH2 domain of SHIP is important for its negative regulatory function.

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Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4902-4911.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4902-4911

Author(s):

B Cheatham ◽

C J Vlahos ◽

L Cheatham ◽

L Wang ◽

J Blenis ◽

...

Keyword(s):

Protein Kinase ◽

Dna Synthesis ◽

Mitogen Activated Protein Kinase ◽

Phosphatidylinositol 3 Kinase ◽

Insulin Stimulation ◽

S6 Kinase ◽

Kinase Activation ◽

Mitogen Activated Protein ◽

Stimulation Of ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

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TNF-α impairs insulin signaling and insulin stimulation of glucose uptake in C2C12muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.276.5.e849 ◽

1999 ◽

Vol 276 (5) ◽

pp. E849-E855 ◽

Cited By ~ 47

Author(s):

Luis F. del Aguila ◽

Kevin P. Claffey ◽

John P. Kirwan

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Tyrosine Phosphorylation ◽

Insulin Signaling ◽

Insulin Stimulation ◽

Kinase Activation ◽

Tnf Α ◽

Impaired Insulin ◽

Stimulation Of

Physiological stressors such as sepsis and tissue damage initiate an acute immune response and cause transient systemic insulin resistance. This study was conducted to determine whether tumor necrosis factor-α (TNF-α), a cytokine produced by immune cells during skeletal muscle damage, decreases insulin responsiveness at the cellular level. To examine the molecular mechanisms associated with TNF-α and insulin action, we measured insulin receptor substrate (IRS)-1- and IRS-2-mediated phosphatidylinositol 3-kinase (PI 3-kinase) activation, IRS-1-PI 3-kinase binding, IRS-1 tyrosine phosphorylation, and the phosphorylation of two mitogen-activated protein kinases (MAPK, known as p42MAPK and p44MAPK) in cultured C2C12myotubes. Furthermore, we determined the effects of TNF-α on insulin-stimulated 2-deoxyglucose (2-DG) uptake. We observed that TNF-α impaired insulin stimulation of IRS-1- and IRS-2-mediated PI 3-kinase activation by 54 and 55% ( P< 0.05), respectively. In addition, TNF-α decreased insulin-stimulated IRS-1 tyrosine phosphorylation by 40% ( P < 0.05). Furthermore, TNF-α repressed insulin-induced p42MAPKand p44MAPK tyrosine phosphorylation by 81% ( P < 0.01). TNF-α impairment of insulin signaling activation was accompanied by a decrease ( P < 0.05) in 2-DG uptake in the muscle cells (60 ± 4 vs. 44 ± 6 pmol ⋅ min−1 ⋅ mg−1). These data suggest that increases in TNF-α may cause insulin resistance in skeletal muscle by inhibiting IRS-1- and IRS-2-mediated PI 3-kinase activation as well as p42MAPK and p44MAPK tyrosine phosphorylation, leading to impaired insulin-stimulated glucose uptake.

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