Immunocytochemical Detection of Phosphatidylinositol 3-kinase Activation by Insulin and Leptin

Intracellular signaling mediated by phosphatidylinositol 3-kinase (PI3K) is important for a number of cellular processes and is stimulated by a variety of hormones, including insulin and leptin. A histochemical method for assessment of PI3K signaling would be an important advance in identifying specific cells in histologically complex organs that are regulated by growth factors and peptide hormones. However, current methods for detecting PI3K activity require either homogenization of the tissue or cells or the ability to transfect probes that bind to phosphatidylinositol 3,4,5 trisphosphate (PIP3), the reaction product of PI3K catalysis. Here we report the validation of an immunocytochemical method to detect changes in PI3K activity, using a recently developed monoclonal antibody to PIP3, in paraformaldehyde-fixed bovine aortic endothelial cells (BAECs) in culture and in hepatocytes of intact rat liver. Treatment with either insulin or leptin increased BAEC PIP3 immunoreactivity, and these effects were blocked by pretreatment with PI3K inhibitors. Furthermore, infusion of insulin into the hepatic portal vein of fasted rats caused an increase of PIP3 immunostaining in hepatocytes that was associated with increased serine phosphorylation of the downstream signaling molecule protein kinase B/Akt (PKB/Akt). We conclude that immunocytochemical PIP3 staining can detect changes in PI3K activation induced by insulin and leptin in cell culture and intact liver.

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Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.17.1.190 ◽

1997 ◽

Vol 17 (1) ◽

pp. 190-198 ◽

Cited By ~ 116

Author(s):

E U Frevert ◽

B B Kahn

Keyword(s):

Dna Synthesis ◽

Glucose Transport ◽

Recombinant Adenovirus ◽

Glycogen Synthase ◽

Mitogen Activated Protein Kinase ◽

Phosphatidylinositol 3 Kinase ◽

Pi3k Activity ◽

Mitogen Activated Protein ◽

Pi3k Activation ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.

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Protein kinase A and protein kinase Cα/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm

Reproduction ◽

10.1530/rep-09-0314 ◽

2010 ◽

Vol 140 (1) ◽

pp. 43-56 ◽

Cited By ~ 16

Author(s):

T Rotman ◽

N Etkovitz ◽

A Spiegel ◽

S Rubinstein ◽

H Breitbart

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Reproductive Tract ◽

Female Reproductive Tract ◽

Phosphatidylinositol 3 Kinase ◽

Highly Active ◽

Protein Kinase Cα ◽

Pi3k Activation ◽

Kinase A ◽

Phosphatidylinositol 3

In order to acquire fertilization competence, spermatozoa have to undergo biochemical changes in the female reproductive tract, known as capacitation. Signaling pathways that take place during the capacitation process are much investigated issue. However, the role and regulation of phosphatidylinositol 3-kinase (PI3K) in this process are still not clear. Previously, we reported that short-time activation of protein kinase A (PRKA, PKA) leads to PI3K activation and protein kinase Cα (PRKCA, PKCα) inhibition. In the present study, we found that during the capacitation PI3K phosphorylation/activation increases. PI3K activation was PRKA dependent, and down-regulated by PRKCA. PRKCA is found to be highly active at the beginning of the capacitation, conditions in which PI3K is not active. Moreover, inhibition of PRKCA causes significant activation of PI3K. Similar activation of PI3K is seen when the phosphatase PPP1 is blocked suggesting that PPP1 regulates PI3K activity. We found that during the capacitation PRKCA and PPP1CC2 (PP1γ2) form a complex, and the two enzymes were degraded during the capacitation, suggesting that this degradation enables the activation of PI3K. This degradation is mediated by PRKA, indicating that in addition to the direct activation of PI3K by PRKA, this kinase can enhance PI3K phosphorylation indirectly by enhancing the degradation and inactivation of PRKCA and PPP1CC2.

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Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.279.2.e266 ◽

2000 ◽

Vol 279 (2) ◽

pp. E266-E274 ◽

Cited By ~ 9

Author(s):

Paul G. Drake ◽

Alejandro Balbis ◽

Jiong Wu ◽

John J. M. Bergeron ◽

Barry I. Posner

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Insulin Receptor ◽

Rat Liver ◽

Intracellular Signaling ◽

Kinase Activity ◽

Glycogen Synthase ◽

Metabolic Effects ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

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Phosphatidylinositol 3-Kinase and Prostaglandylinositol Cyclic Phosphate (Cyclic PIP), a Mediator of Insulin Action, in the Signal Transduction of Insulin

Biological Chemistry ◽

10.1515/bc.2000.140 ◽

2000 ◽

Vol 381 (11) ◽

pp. 1139-1141 ◽

Cited By ~ 1

Author(s):

A. Gypakis ◽

H.K. Wasner

Keyword(s):

Insulin Receptor ◽

Glucose Transport ◽

Functional Role ◽

Specific Inhibitor ◽

Insulin Receptor Substrate ◽

Phosphatidylinositol 3 Kinase ◽

Downstream Signaling ◽

Cyclic Phosphate ◽

Phosphatidylinositol 3

Abstract It has been suggested that downstream signaling from the insulin receptor to the level of the protein kinases and protein phosphatases is accomplished by prostaglandylinositol cyclic phosphate (cyclic PIP), a proposed second messenger of insulin. However, evidence points also to both phosphatidylinositol 3-kinase, which binds to the tyrosine phosphorylated insulin receptor substrate-1, and the Ras complex in insulin's downstream signaling. We have examined whether a correlation exists between these various observations. It was found that wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, prevented insulin-induced, as well as cyclic PIP-induced activation of glucose transport, indicating that PI 3-kinase action on glucose transport involves downstream signaling of both insulin and cyclic PIP. Wortmannin has no effect on cyclic PIP synthase activity nor on the substrate production for cyclic PIP synthesis either, indicating that the functional role of PI 3-kinase is exclusively downstream of cyclic PIP.

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Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase

Blood ◽

10.1182/blood.v93.8.2578 ◽

1999 ◽

Vol 93 (8) ◽

pp. 2578-2585 ◽

Cited By ~ 80

Author(s):

Carinne Lecoq-Lafon ◽

Frédérique Verdier ◽

Serge Fichelson ◽

Stany Chrétien ◽

Sylvie Gisselbrecht ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Intracellular Signaling ◽

Direct Consequence ◽

Receptor Activation ◽

Phosphatidylinositol 3 Kinase ◽

Erythroid Progenitors ◽

Epo Receptor ◽

Gm Csf ◽

Macrophage Colony ◽

Phosphatidylinositol 3

Abstract Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.

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Structural studies of the phosphatidylinositol 3-kinase (PI3K) SH3 domain in complex with a peptide ligand: role of the anchor residue in ligand binding

Biological Chemistry ◽

10.1515/bc.2010.003 ◽

2010 ◽

Vol 391 (1) ◽

Cited By ~ 14

Author(s):

Renu Batra-Safferling ◽

Joachim Granzin ◽

Susanne Mödder ◽

Silke Hoffmann ◽

Dieter Willbold

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Protein Interactions ◽

Intracellular Signaling ◽

Sh3 Domain ◽

Peptide Ligand ◽

Type I ◽

Phosphatidylinositol 3 Kinase ◽

Anchor Residue ◽

Phosphatidylinositol 3

Abstract Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P-3 is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.

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Hypothalamic Phosphatidylinositol 3-Kinase Pathway of Leptin Signaling Is Impaired during the Development of Diet-Induced Obesity in FVB/N Mice

Endocrinology ◽

10.1210/en.2007-1307 ◽

2007 ◽

Vol 149 (3) ◽

pp. 1121-1128 ◽

Cited By ~ 55

Author(s):

Anantha S. Metlakunta ◽

Maitrayee Sahu ◽

Abhiram Sahu

Keyword(s):

Energy Homeostasis ◽

Early Period ◽

Pi3k Pathway ◽

Leptin Resistance ◽

Phosphatidylinositol 3 Kinase ◽

Leptin Signaling ◽

Diet Induced Obesity ◽

Pi3k Activity ◽

Central Leptin Resistance ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI3K) pathway of leptin signaling plays an important role in transducing leptin action in the hypothalamus. Obesity is usually associated with resistance to the effect of leptin on food intake and energy homeostasis. Although central leptin resistance is thought to be involved in the development of diet-induced obesity (DIO), the mechanism behind this phenomenon is not clearly understood. To determine whether DIO impairs the effect of leptin on hypothalamic PI3K signaling, we fed 4-wk-old FVB/N mice a high-fat diet (HFD) or low-fat diet (LFD) for 19 wk. HFD-fed mice developed DIO in association with hyperleptinemia, hyperinsulinemia, and impaired glucose and insulin tolerance. Leptin (ip) significantly increased hypothalamic PI3K activity and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) levels in LFD-fed mice but not in DIO mice. Immunocytochemical study confirmed impaired p-STAT3 activation in various hypothalamic areas, including the arcuate nucleus. We next tested whether both PI3K and STAT3 pathways of leptin signaling were impaired during the early period of DIO. Leptin failed to increase PI3K activity in DIO mice that were on a HFD for 4 wk. However, leptin-induced p-STAT3 activation in the hypothalamus measured by Western blotting and immunocytochemistry remained comparable between LFD- and HFD-fed mice. These results suggest that the PI3K pathway but not the STAT3 pathway of leptin signaling is impaired during the development of DIO in FVB/N mice. Thus, a defective PI3K pathway of leptin signaling in the hypothalamus may be one of the mechanisms of central leptin resistance and DIO.

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Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00150.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. E889-E897 ◽

Cited By ~ 79

Author(s):

Yasuki Higaki ◽

Toshio Mikami ◽

Nobuharu Fujii ◽

Michael F. Hirshman ◽

Katsuhiro Koyama ◽

...

Keyword(s):

Oxidative Stress ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Intracellular Signaling ◽

Phosphatidylinositol 3 Kinase ◽

Maximal Increase ◽

Skeletal Muscle Glucose Uptake ◽

Dose Dependent Increase ◽

Dose Dependent ◽

Phosphatidylinositol 3

We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 μmol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor N G-monomethyl-l-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 μmol/l had no effect on ATP concentrations and did not increase the activities of either the α1 or α2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism.

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Aquaporins and Fetal Membranes From Diabetic Parturient Women: Expression Abnormalities and Regulation by Insulin

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2015-2057 ◽

2015 ◽

Vol 100 (10) ◽

pp. E1270-E1279 ◽

Cited By ~ 8

Author(s):

Damien Bouvier ◽

Marion Rouzaire ◽

Geoffroy Marceau ◽

Cécile Prat ◽

Bruno Pereira ◽

...

Keyword(s):

Intracellular Signaling ◽

Kinase Inhibitor ◽

Fetal Membranes ◽

Phosphatidylinositol 3 Kinase ◽

Intracellular Signaling Pathway ◽

The Impact ◽

Phosphatidylinositol 3

Context: During pregnancy, aquaporins (AQPs) expressed in fetal membranes are essential for controlling the homeostasis of the amniotic volume, but their regulation by insulin was never explored in diabetic women. Objective: The aim of our study was to investigate the involvement of AQPs 1, 3, 8, and 9 expressed in fetal membranes in diabetic parturient women and the control of their expression by insulin. Design and Participants: From 129 fetal membranes in four populations (controls, type 1, type 2 [T2D], and gestational diabetes [GD]), we established an expression AQP profile. In a second step, the amnion was used to study the control of the expression and functions of AQPs 3 and 9 by insulin. Main Outcomes and Measures: The expression of transcripts and proteins of AQPs was studied by quantitative RT-PCR and ELISA. We analyzed the regulation by insulin of the expression of AQPs 3 and 9 in the amnion. A tritiated glycerol test enabled us to measure the impact of insulin on the functional characteristics. Using an inhibitor of phosphatidylinositol 3-kinase, we analyzed the insulin intracellular signaling pathway. Results: The expression of AQP3 protein was significantly weaker in groups T2D and GD. In nondiabetic fetal membranes, we showed for the amnion (but not for the chorion) a significant repression by insulin of the transcriptional expression of AQPs 3 and 9, which was blocked by a phosphatidylinositol 3-kinase inhibitor. Conclusion: In fetal membranes, the repression of AQP3 protein expression and functions observed in vivo is allowed by the hyperinsulinism described in pregnant women with T2D or GD.

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Parallel Phosphatidylinositol-3 Kinase and p42/44 Mitogen-Activated Protein Kinase Signaling Pathways Subserve the Mitogenic and Antiapoptotic Actions of Insulin-Like Growth Factor I in Osteoblastic Cells

Endocrinology ◽

10.1210/en.2003-0350 ◽

2003 ◽

Vol 144 (11) ◽

pp. 4886-4893 ◽

Cited By ~ 77

Author(s):

Andrew Grey ◽

Qi Chen ◽

Xin Xu ◽

Karen Callon ◽

Jill Cornish

Keyword(s):

Signaling Pathways ◽

Intracellular Signaling ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Osteoblastic Cells ◽

Phosphatidylinositol 3 Kinase ◽

P70s6 Kinase ◽

Igf I ◽

Convergence Point ◽

Phosphatidylinositol 3

Abstract IGF-I is an endocrine and paracrine regulator of skeletal homeostasis, principally by virtue of its anabolic effects on osteoblastic cells. In the current study, we examined the intracellular signaling pathways by which IGF-I promotes proliferation and survival in SaOS-2 human osteoblastic cells. Inhibition of each of the phosphatidylinositol-3 kinase (PI-3 kinase), p42/44 MAPK, and p70s6 kinase pathways partially inhibited the ability of IGF-I to stimulate osteoblast proliferation and survival. Because activation of p70s6 kinase is downstream of both PI-3 kinase and p42/44 MAPK activation in osteoblasts treated with IGF-I, this ribosomal kinase represents a convergence point for IGF-I-induced PI-3 kinase and p42/44 MAPK signaling in osteoblastic cells. In addition, abrogation of PI-3 kinase-dependent Akt signaling, which does not inhibit IGF-I-induced p70s6 kinase phosphorylation, also inhibited the antiapoptotic effects of IGF-I in osteoblasts. Finally, interruption of Gβγ signaling partially abrogated the ability of IGF-I to promote osteoblast survival, without inhibiting signaling through PI-3 kinase/Akt, p42/44 MAPKs, or p70s6 kinase. These data suggest that IGF-I signals osteoblast mitogenesis and survival through parallel, partly overlapping intracellular pathways involving PI-3 kinase, p42/44 MAPKs, and Gβγ subunits.

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