P85 Subunit Of Phosphoinositide 3-kinase Research Articles

Trans-10,cis-12, not cis-9,trans-11, conjugated linoleic acid decreases ErbB3 expression in HT-29 human colon cancer cells.

To examine whether trans-10,cis-12 CLA (t10c12) or cis-9,trans-11 CLA (c9t11) inhibits heregulin (HRG)-beta-stimulated cell growth and HRG-beta-ErbB3 signaling in HT-29 cells. We cultured HT-29 cells in the absence or presence of the CLA isomers and/or the ErbB3 ligand HRG-beta. MTT assay, [3H]thymidine incorporation, Annexin V staining, RT-PCR, Western blotting, immunoprecipitation, and in vitro kinase assay were performed. HRG-beta increased cell growth, but did not prevent t10c12-induced growth inhibition. T10c12 inhibited DNA synthesis and induced apoptosis of HT-29 cells, whereas c9t11 had no effect. T10c12 decreased the levels of ErbB1, ErbB2, and ErbB3 proteins and transcripts in a dose-dependent manner, whereas c9t11 had no effect. Immunoprecipitation/Western blot studies revealed that t10c12 inhibited HRG-beta-stimulated phosphorylation of ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) to ErbB3, ErbB3-associated PI3K activities, and phosphorylation of Akt. However, c9t11 had no effect on phospho Akt levels. Neither t10c12 nor c9t11 had any effect on HRG-beta-induced phosphorylation of ERK-1/2. These results indicate that the inhibition of HT-29 cell growth by t10c12 may be induced via its modulation of ErbB3 signaling leading to inhibition of Akt activation.

Protein-Protein Coupling/Uncoupling Enables Dopamine D2Receptor Regulation of AMPA Receptor-Mediated Excitotoxicity

here is considerable evidence that dopamine D2 receptors can modulate AMPA receptor-mediated neurotoxicity. However, the molecular mechanism underlying this process remains essentially unclear. Here we report that D2 receptors inhibit AMPA-mediated neurotoxicity through two pathways: the activation of phosphoinositide-3 kinase (PI-3K) and downregulation of AMPA receptor plasma membrane expression, both involving a series of protein-protein coupling/uncoupling events. Agonist stimulation of D2 receptors promotes the formation of the direct protein-protein interaction between the third intracellular loop of the D2 receptor and the ATPase N-ethylmaleimide-sensitive factor (NSF) while uncoupling the NSF interaction with the carboxyl tail (CT) of the glutamate receptor GluR2 subunit of AMPA receptors. Previous studies have shown that full-length NSF directly couples to the GluR2CT and facilitates AMPA receptor plasma membrane expression. Furthermore, the CT region of GluR2 subunit is also responsible for several other intracellular protein couplings, including p85 subunit of PI-3K. Therefore, the direct coupling of D2-NSF and concomitant decrease in the NSF-GluR2 interaction results in a decrease of AMPA receptor membrane expression and an increase in the interaction between GluR2 and the p85 and subsequent activation of PI-3K. Disruption of the D2-NSF interaction abolished the ability of D2 receptor to attenuate AMPA-mediated neurotoxicity by blocking the D2 activation-induced changes in PI-3K activity and AMPA receptor plasma membrane expression. Furthermore, the D2-NSF-GluR2-p85 interactions are also responsible for the D2 inhibition of ischemia-induced cell death. These data may provide a new avenue to identify specific targets for therapeutics to modulate glutamate receptor-governed diseases, such as stroke.

β1 Integrins Are Receptors for Thrombospondin 1

Thrombospondin 1 (TSP1) is an endogenous antiangiogenic protein, and it is known that some of these effects are mediated by binding of the type-1 repeat (TSR) domains to the cell surface receptor CD36. However, Short et al. show that TSP1 and a TSR-containing peptide inhibited migration of human umbilical vein endothelial cells (HUVEC), which do not express CD36, in response to vascular endothelial growth factor (VEGF). Fluorescence-activated cell sorting (FACS) indicated that β1 integrins were abundant on HUVEC, and antibodies against β1 integrins that activated β1 integrins signaling inhibited cell migration in a dose-dependent manner. Concentrations of activating antibodies against β1 integrins that were too low to inhibit migration directly interfered with the inhibitory activity of the TSR peptide, but antibodies against β3 integrins and nonactivating antibodies against β1 integrins did not. Small interfering RNAs (siRNAs) were used to decrease β1 or β3 integrins, and individually these did not block VEGF-induced migration; however, siRNA treatment to reduce β1 integrin did block the inhibitory effect of the TSR peptide. Immunoblotting of HUVEC proteins bound to beads coated with TSR peptide indicated a direct interaction between TSR and α5 β1 integrins. Antibodies against α5 or α3 blocked the TSR peptide inhibition of VEGF-induced migration. Both antagonists of phospholipase C-γ (PLC-γ) and of phosphoinositide 3-kinase (PI3K) inhibited migration. TSR and the PLC-γ antagonist had additive effects, whereas TSR and the PI3K inhibitor did not. The p85 subunit of PI3K was found in a complex with β1 integrin; thus, PI3K appears to be involved in the inhibitory effects of β1 signaling on migration. Finally, the involvement of the β1 integrins in mediating TSP1 inhibitory effects was present in cells that do express the CD36 receptor for TSR. In cells that do express CD36, antibodies to CD36 blocked inhibition of migration by the TSR peptide; however, migration in response to full-length TSP1 was not blocked by the antibody to CD36 but was blocked by the β1 integrin antibody. Thus, inhibition of migration by TSP1 appears to involve both CD36 and β1 integrins and can be mediated solely by β1 integrins in cells lacking CD36. Understanding the mechanisms of inhibitors of angiogenesis may aid in designing such inhibitors for cancer therapy. S. M. Short, A. Derrien, R. P. Narsimhan, J. Lawler, D. E. Inger, B. R. Zetter, Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by β1 integrins. J. Cell Biol. 168 , 643-653 (2005). [Abstract] [Full Text]

Adenoviral Overexpression of the Glutamylcysteine Ligase Catalytic Subunit Protects Pancreatic Islets against Oxidative Stress

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.

Association of the Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) with the p85 subunit of phosphoinositide 3-kinase

To investigate additional functions of the T cell adaptor, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD (SLP-76), we performed a yeast two-hybrid assay using the N-terminal region of SLP-76 fused with the kinase domain of Syk. By screening a human leukemia cDNA library, we identified the p85 subunit of phosphoinositide 3-kinase (PI3K) as one of the interacting molecules. Unlike the SH2 domain of Vav or Nck, tyrosine phosphorylation of SLP-76 at position 113 or 128 was sufficient for it to associate with the N-terminal SH2 of p85. Collectively, these data suggest that SLP-76 may play a role in PI3K signaling pathways.

Interaction of the retinal insulin receptor beta-subunit with the p85 subunit of phosphoinositide 3-kinase.

Recently, we have shown that phosphoinositide 3-kinase (PI3K) in retina is regulated in vivo through light activation of the insulin receptor beta-subunit. In this study, we have cloned the 41 kDa cytoplasmic region of the retinal insulin receptor (IRbeta) and used the two-hybrid assay of protein-protein interaction in the yeast Saccharomyces cerevisiae to demonstrate the interaction between the p85 subunit of PI3K and the cytoplasmic region of IRbeta. Under conditions where IRbeta autophosphorylates, substitution of Y1322F and M1325P in IRbeta resulted in the abolition of p85 binding to the IRbeta, confirming that the p85 subunit of PI3K binds to Y1322. The binding site for p85 on IRbeta was also confirmed in the yeast three-hybrid system. Using the C-terminal region of IRbeta (amino acids 1293-1343 encompassing the YHTM motif) as bait and supplying an exogenous tyrosine kinase gene to yeast cells, we determined that the IRbeta-pYTHM motif interacts with p85. We also used retinal organ cultures to demonstrate insulin activation of the insulin receptor and subsequent binding of p85, measured through GST pull-down assays with p85 fusion proteins. Further, the Y960F mutant insulin receptor, which does not bind IRS-1, is capable of bringing down PI3K activity from retina lysates. On the other hand, in response to insulin, IRS-2 is able to interact with the p85 subunit of PI3K in the retina. These results suggest that multiple signaling pathways could regulate the PI3K activity and subsequent activation of Akt in the retina.

Insulin and Hypoxia Share Common Target Genes but Not the Hypoxia-inducible Factor-1α

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.

Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway.

Eosinophil major basic protein (MBP) is an effective stimulus for neutrophil superoxide (O(2)(-)) production, degranulation, and IL-8 production. In this study we evaluated the participation of phosphoinositide 3-kinase (PI3K) and PI3K-associated signaling events in neutrophil activation by MBP. Inhibition of PI3K activity blocked MBP-stimulated O(2)(-) production, but not degranulation or IL-8 production. Measurement of Akt phosphorylation at Ser(473) and Thr(308) confirmed that MBP stimulated PI3K activity and also demonstrated indirectly activation of phosphoinositide-dependent kinase-1 by MBP. Genistein and the Src kinase family inhibitor, 4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, inhibited MBP-stimulated phosphorylation of Akt. 4-Amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also inhibited MBP-stimulated O(2)(-) production. MBP stimulated phosphorylation and translocation of the p85 subunit of class I(A) PI3K, but not translocation of the p110gamma subunit of class I(B) PI3K, to the neutrophil membrane. Inhibition of protein kinase Czeta (PKCzeta) inhibited MBP-stimulated O(2)(-) production. Measurement of phosphorylated PKCzeta (Thr(410)) and PKCdelta (Thr(505)) confirmed that PKCzeta, but not PKCdelta, is activated in MBP-stimulated neutrophils. The time courses for phosphorylation and translocation of the p85 subunit of class I(A) PI3K, activation of Akt, and activation of PKCzeta were similar. Moreover, inhibition of PI3K activity inhibited MBP-induced activation of PKCzeta. We conclude that MBP stimulates a Src kinase-dependent activation of class I(A) PI3K and, in turn, activation of PKCzeta in neutrophils, which contributes to the activation of NADPH oxidase and the resultant O(2)(-) production in response to MBP stimulation.

Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line.

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-alpha led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-alpha-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.

Interleukin-2 receptors and interleukin-2-mediated signaling in myelin: activation of diacylglycerol kinase and phosphatidylinositol 3-kinase

Myelin was previously shown to possess neurotransmitter and cytokine receptors that trigger well-defined signaling mechanisms within the multilamellar structure. The present study reveals the presence of an interleukin-2 (IL-2) receptor in isolated mouse CNS myelin that responds to recombinant mouse IL-2 by activating diacylglycerol kinase (DAGK) and phosphoinositide 3-kinase (PI3K); additional evidence suggests participation by protein tyrosine kinase. Activation of myelin DAGK by IL-2 occurred in brain stem tissue mince and was blocked by chelerythrin chloride, indicating an essential role for myelin-localized protein kinase C. Two inhibitors of PI3K, wortmannin and LY294002, blocked endogenous PI3K as well as that enhanced by IL-2. Activation of PI3K by IL-2 was also blocked by tyrphostin A25, a selective inhibitor of PTK, suggesting activation of the latter by IL-2 is upstream to PI3K activation. This reaction resulted in tyrosine phosphorylation of a protein tentatively identified as the p85 subunit of PI3K. Developmental changes were noted in that receptor density and signaling activity were robust during the period of rapid myelination and declined rapidly thereafter.

Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes.

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr(612) and Tyr(941) (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr(896) (a Grb2 binding site); and (iii) Tyr(1229) (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr(1158) and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (>or=20 min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.

Association of the X-linked Lymphoproliferative Disease Gene Product SAP/SH2D1A with 2B4, a Natural Killer Cell-activating Molecule, Is Dependent on Phosphoinositide 3-Kinase

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.

Pi 3-kinase and its up- and down-stream modulators as potential targets for the treatment of type II diabetes.

Type 2 diabetes is caused by a combination of impaired insulin secretion and, to a greater extent, resistance of target tissues to insulin action. Phosphoinositide 3-kinase (PI3K) plays a key role in insulin signaling and has been shown to be blunted in tissues of type 2 diabetes subjects. There is emerging biochemical and, particularly, genetic evidence suggesting that insulin resistance can potentially be treated via modulation of PI3K by targeting PI3K itself or its up and down-stream modulators. These potential targets include Src homology 2 domain containing inositol 5-phosphatase 2 (SHIP2), phosphatase and tensin homolog deleted on chromosome ten (PTEN), kappaB kinase beta (IKKbeta), PKC isoforms, and the PI3K p85 subunit. There is evidence suggesting that their inhibition affects PI3K activity and improves insulin sensitivity in vivo. In the current review, we will discuss the role of these molecules in insulin-mediated activation of PI3K, the rational for targeting these molecules for diabetes treatment, and some critical issues in terms of drug development.

PI 3-kinase and its up- and down-stream modulators as potential targets for the treatment of type II diabetes

Type 2 diabetes is caused by a combination of impaired insulin secretion and, to a greater extent, resistance of target tissues to insulin action. Phosphoinositide 3-kinase (PI3K) plays a key role in insulin signaling and has been shown to be blunted in tissues of type 2 diabetes subjects. There is emerging biochemical and, particularly, genetic evidence suggesting that insulin resistance can potentially be treated via modulation of PI3K by targeting PI3K itself or its up and down-stream modulators. These potential targets include Src homology 2 domain containing inositol 5-phosphatase 2 (SHIP2), phosphatase and tensin homolog deleted on chromosome ten (PTEN), kappaB kinase beta (IKKbeta), PKC isoforms, and the PI3K p85 subunit. There is evidence suggesting that their inhibition affects PI3K activity and improves insulin sensitivity in vivo. In the current review, we will discuss the role of these molecules in insulin-mediated activation of PI3K, the rational for targeting these molecules for diabetes treatment, and some critical issues in terms of drug development.

The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

The p85 subunit of phosphoinositide 3-kinase is associated with β-catenin in the cadherin-based adhesion complex

Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell–cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via β-catenin. We also show that the interaction of p85 and β-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

Internalin B Activates Nuclear Factor-κB via Ras, Phosphoinositide 3-Kinase, and Akt

Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB. Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB. Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation. This process could be involved in host defense or the inhibition of apoptosis during infection.

Zinc Has an Insulin-Like Effect on Glucose Transport Mediated by Phosphoinositol-3-Kinase and Akt in 3T3-L1 Fibroblasts and Adipocytes

Zinc has insulin-like effects on cells, including promotion of both lipogenesis and glucose transport. The relationship between zinc and the stimulation of glucose transport is unclear. We hypothesize that zinc affects the insulin-signaling pathway. In this study, the effect of zinc on glucose transport and insulin signaling was examined in 3T3-L1-preadipocytes and -adipocytes. Treatment of cells with up to 200 micromol/L zinc significantly increased glucose transport (P < 0.05). The effect of zinc on adipocytes was greater than on preadipocytes, and the effect of zinc plus insulin was greater than that of either insulin or zinc alone. Cytochalasin D, which disrupts actin filaments, attenuated the increase of glucose transport induced by zinc or insulin (P < 0.05). At 100 nmol/L, wortmannin, the phosphoinositide (PI) 3-kinase inhibitor, decreased basal glucose transport and blocked zinc-stimulated glucose transport in both cell types (P < 0.05). H7, an inhibitor of protein kinase C, did not reduce basal glucose transport but decreased zinc-induced glucose transport (P < 0.05). Zinc increased tyrosine phosphorylation of the insulin receptor beta subunit of both preadipocytes and adipocytes after 5-10 min of treatment (P < 0.05). Zinc at 200 micromol/L did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)-1 or -2; further, there was no effect of zinc on the association of the p85 subunit of PI 3-kinase and IRS-1. Zinc significantly increased serine-473 phosphorylation of Akt in both preadipocytes and adipocytes (P < 0.05). The PI 3-kinase inhibitor, wortmannin, totally blocked the effect of zinc on Akt activation. Hence, it appears that zinc can induce an increase in glucose transport into cells and potentiate insulin-induced glucose transport, likely acting through the insulin-signaling pathway.

GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.

GLUT4 vesicle trafficking in rat adipocytes after ethanol feeding: regulation by heterotrimeric G-proteins.

Long-term ethanol consumption decreases insulin-stimulated glucose uptake in isolated rat adipocytes. Here we investigate the mechanisms for this decrease. Male Wistar rats were fed for 4 weeks with a liquid diet containing 35% of the calories from ethanol and compared with pair-fed controls. Stimulation of 3-O-methylglucose transport in isolated adipocytes by insulin was decreased by 70% after ethanol feeding. However, stimulation by insulin of the tyrosine phosphorylation of the p85 subunit of phosphoinositide 3-kinase and the phosphorylation of Akt were not affected by ethanol feeding. GLUT4 was mobilized from intracellular light microsomes in response to insulin in both pair-fed and ethanol-fed rats, resulting in 4.3-fold and 3.3-fold increases in GLUT4 associated with plasma membrane in pair-fed and ethanol-fed rats respectively. Surface-accessible GLUT4, assessed by a trypsin cleavage assay or cell-surface labelling with bis-mannose photolabel, was increased 2.3-fold and 1.6-fold respectively, in pair-fed rats after treatment with insulin. In contrast, insulin did not increase surface-accessible GLUT4 in ethanol-fed rats. Treatment of adipocytes with R-phenylisopropyladenosine, an adenosine A1 receptor agonist, increased the transport of 3-O-methylglucose and trypsin-accessible GLUT4, in adipocytes from both pair-fed and ethanol-fed rats. These results demonstrate that whereas the insulin-mediated signalling and translocation of GLUT4 to the plasma membrane is maintained after ethanol feeding, the final fusion of GLUT4 vesicles to the plasma membrane is disrupted, preventing the stimulation of glucose uptake by insulin. Fusion of GLUT4 with the plasma membrane can be stimulated by the activation of adenosine A1 receptors.