Insulin cannot activate extracellular-signal-related kinase due to inability to generate reactive oxygen species in SK-N-BE(2) human neuroblastoma cells

The insulin-mediated Ras/mitogen-activated protein (MAP) kinase cascade was examined in SK-N-BE(2) and PC12 cells, which can and cannot produce reactive oxygen species (ROS), respectively. Tyrosine phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) was much lower in SK-N-BE(2) cells than in PC12 cells when the cells were treated with insulin. The insulin-mediated interaction of IRS-1 with Grb2 was observed in PC12 but not in SK-N-BE(2) cells. Moreover, the activity of extracellular-signal-related kinase (ERK) was much lower in SK-N-BE(2) than in PC12 cells when the cells were treated with insulin. Application of exogenous H2O2 caused increased tyrosine phosphorylation and Grb2 binding to IRS-1 in SK-N-BE(2) cells, while exposure to an H2O2 scavenger (N-acetylcysteine) or to a phophatidylinositol-3 kinase inhibitor (wortmannin), and expression of a dominant negative Rac1, decreased the activation of ERK in insulin-stimulated PC12 cells. These results indicate that the transient increase of ROS is needed to activate ERK in insulin-mediated signaling and that an inability to generate ROS is the reason for the insulin insensitivity of SK-N-BE(2) cells.     

Membrane localization of 3-phosphoinositide-dependent protein kinase-1 stimulates activities of Akt and atypical protein kinase C but does not stimulate glucose transport and glycogen synthesis in 3T3-L1 adipocytes

It is reported that 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is activated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner and phosphorylates Akt, p70S6 kinase, and atypical protein kinase C (PKC), but its function on insulin signaling is still unclear. We cloned a full-length pdk-1 cDNA from a human brain cDNA library, and the adenovirus to overexpress wild type PDK-1 (PDK-1WT) or membrane-targeted PDK-1 (PDK-1CAAX) was constructed. Overexpressed PDK-1WT existed mainly at cytosol, and PDK-1CAAX was located at the plasma membrane. In 3T3-L1 adipocytes, insulin induced mobility shift of PDK-1 protein, but overexpressed PDK-1WT and CAAX were shifted at the basal state. Insulin stimulated tyrosine phosphorylation of PDK-1WT, but PDK-1CAAX was already tyrosine-phosphorylated at the basal state. Overexpression of PDK-1WT led to a full activation of PKC zeta/lambda without insulin stimulation but showed only the minimum effects to stimulate phosphorylation of Akt and GSK-3. In contrast, the overexpression of PDK-1CAAX caused phosphorylation of Akt and GSK-3 more strongly without insulin stimulation. However, PDK-1CAAX did not affect 2-deoxyglucose uptake and inhibited glycogen synthesis, surprisingly. Finally, PDK-1CAAX expression inhibited insulin-induced ERK1/2 phosphorylation in a dose-dependent manner. Taken together, the translocation of PDK-1 from cytosol to the plasma membrane is critical for Akt and GSK-3 activation. On the other hand, only atypical PKC and Akt activation was insufficient for stimulation of glucose transport, and constitutive activation of Akt-GSK-3 pathway may inhibit glycogen synthesis and MAPK cascade in 3T3-L1 adipocytes.     

Human cancer, PTEN and the PI-3 kinase pathway

The PI-3 kinase pathway is a major driving force for human cancer. One common way of stimulating the PI-3 kinase pathway occurs through inactivation of the PTEN tumor suppressor. The mechanisms of PTEN inactivation include mutation, epigenetic silencing and post-translational modification. Improved insight into the regulation of PTEN is leading to a richer understanding of the contribution of PTEN and the PI-3 kinase pathway to human tumors. Understanding the pathology of PI-3 kinase signaling in tumors improves knowledge of cancer etiology and provides novel therapeutic targets.     

The tumor suppressor PTEN negatively regulates insulin signaling in 3T3-L1 adipocytes

PTEN is a tumor suppressor with sequence homology to protein-tyrosine phosphatases and the cytoskeleton protein tensin. PTEN is capable of dephosphorylating phosphatidylinositol 3,4, 5-trisphosphate in vitro and down-regulating its levels in insulin-stimulated 293 cells. To study the role of PTEN in insulin signaling, we overexpressed PTEN in 3T3-L1 adipocytes approximately 30-fold above uninfected or control virus (green fluorescent protein)-infected cells, using an adenovirus gene transfer system. PTEN overexpression inhibited insulin-induced 2-deoxy-glucose uptake by 36%, GLUT4 translocation by 35%, and membrane ruffling by 50%, all of which are phosphatidylinositol 3-kinase-dependent processes, compared with uninfected cells or cells infected with control virus. Microinjection of an anti-PTEN antibody increased basal and insulin stimulated GLUT4 translocation, suggesting that inhibition of endogenous PTEN function led to an increase in intracellular phosphatidylinositol 3,4,5-trisphosphate levels, which stimulates GLUT4 translocation. Further, insulin-induced phosphorylation of downstream targets Akt and p70S6 kinase were also inhibited significantly by overexpression of PTEN, whereas tyrosine phosphorylation of the insulin receptor and IRS-1 or the phosphorylation of mitogen-activated protein kinase were not affected, suggesting that the Ras/mitogen-activated protein kinase pathway remains fully functional. Thus, we conclude that PTEN may regulate phosphatidylinositol 3-kinase-dependent insulin signaling pathways in 3T3-L1 adipocytes.     

Insulin inhibition of apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling cascade but does not involve internal ribosomal entry site (IRES) initiation

Although insulin normally activates global mRNA translation, it has a specific inhibitory effect on translation of apolipoprotein B (apoB) mRNA. This suggests that insulin induces a unique signaling cascade that leads to specific inhibition of apoB mRNA translation despite global translational stimulation. Recent studies have revealed that insulin functions to regulate apoB mRNA translation through a mechanism involving the apoB mRNA 5' untranslated region (5' UTR). Here, we further investigate the role of downstream insulin signaling molecules on apoB mRNA translation, and the mechanism of apoB mRNA translation itself. Transfection studies in HepG2 cells expressing deletion constructs of the apoB 5' UTR showed that the cis-acting region responding to insulin was localized within the first 64 nucleotides. Experiments using chimeric apoB UTR-luciferase constructs transfected into HepG2 cells followed by treatment with wortmannin, a PI-3K inhibitor, and rapamycin, an mTOR inhibitor, showed that signaling via PI-3K and mTOR pathways is necessary for insulin-mediated inhibition of chimeric 5' UTR-luciferase expression. In vitro translation of chimeric cRNA confirmed that the effects observed were translational in nature. Furthermore, using RNA-EMSA we found that wortmannin pretreatment blocked insulin-mediated inhibition of the binding of RNA-binding factor(s), migrating near the 110 kDa marker, to the 5' UTR. Radiolabeling studies in HepG2 cells also showed that insulin-mediated control of the synthesis of endogenously expressed full length apoB100 is mediated via the PI-3K and mTOR pathways. Finally, using dual-cistronic luciferase constructs we demonstrate that apoB 5' UTR may have weak internal ribosomal entry (IRES) translation which is not affected by insulin stimulation, and may function to stimulate basal levels of apoB mRNA translation.     

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN^fl/fl mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo.     

Insulin stimulates increased catalytic activity of phosphoinositide-dependent kinase-1 by a phosphorylation-dependent mechanism

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase downstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. Previous reports have suggested that PDK-1 has constitutive catalytic activity that is not regulated by stimulation of cells with growth factors. We now show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells may significantly increase the intrinsic catalytic activity of PDK-1. Insulin treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activity in an immune-complex kinase assay. Insulin stimulation of rat adipose cells also increased catalytic activity of endogenous PDK-1 immunoprecipitated from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosphatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not undergo phosphorylation or demonstrate increased kinase activity in response to insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulation. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cells and rat adipose cells can be significantly increased upon insulin stimulation.     

A novel positive feedback loop mediated by the docking protein Gab1 and phosphatidylinositol 3-kinase in epidermal growth factor receptor signaling

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4, 5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR.     

TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser⁴⁷³ in the hydrophobic motif, along with Thr³⁰⁸ in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr³⁰⁸, but the kinase(s) responsible for phosphorylating Akt at Ser⁴⁷³ (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser⁴⁷³ phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser⁴⁷³ in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.     

The protein kinase B/Akt signalling pathway in human malignancy

Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which in mammals comprises three highly homologous members known as PKBalpha (Akt1), PKBbeta (Akt2), and PKBgamma (Akt3). PKB/Akt is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components. The activation mechanism remains to be fully characterised but occurs downstream of phosphoinositide 3-kinase (PI-3K). PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), a lipid second messenger essential for the translocation of PKB/Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) and possibly other kinases. PKB/Akt phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer. Constitutive PKB/Akt activation can occur due to amplification of PKB/Akt genes or as a result of mutations in components of the signalling pathway that activates PKB/Akt. Although the mechanisms have not yet been fully characterised, constitutive PKB/Akt signalling is believed to promote proliferation and increased cell survival and thereby contributing to cancer progression. This review surveys recent developments in understanding the mechanisms and consequences of PKB/Akt activation in human malignancy.     

Dependence of insulin-stimulated glucose transporter 4 translocation on 3-phosphoinositide-dependent protein kinase-1 and its target threonine-410 in the activation loop of protein kinase C-zeta.

Previous studies have suggested that 1) atypical protein kinase C (PKC) isoforms are required for insulin stimulation of glucose transport, and 2) 3-phosphoinositide-dependent protein kinase-1 (PDK-1) is required for activation of atypical PKCs. Presently, we evaluated the role of PDK-1, both in the activation of PKC-zeta, and the translocation of epitope-tagged glucose transporter 4 (GLUT4) to the plasma membrane, during insulin action in transiently transfected rat adipocytes. Overexpression of wild-type PDK-1 provoked increases in the activity of cotransfected hemagglutinin (HA)-tagged PKC-zeta and concomitantly enhanced HA-tagged GLUT4 translocation. Expression of both kinase-inactive PDK-1 and an activation-resistant form of PKC-zeta that is mutated at Thr-410, the immediate target of PDK-1 in the activation loop of PKC-zeta, inhibited insulin-induced increases in both HA-PKC-zeta activity and HA-GLUT4 translocation to the same extent as kinase-inactive PKC-zeta. Moreover, the inhibitory effects of kinase-inactive PDK-1 were fully reversed by cotransfection of wild-type PDK-1 and partly reversed by wild-type PKC-zeta, but not by wild-type PKB. In contrast to the T410A PKC-zeta mutant, an analogous double mutant of PKB (T308A/S473A) that is resistant to PDK-1 activation had only a small effect on insulin-stimulated HA-GLUT4 translocation and did not inhibit HA-GLUT4 translocation induced by overexpression of wild-type PDK-1. Our findings suggest that both PDK-1 and its downstream target, Thr-410 in the activation loop of PKC-zeta, are required for insulin-stimulated glucose transport.     

Requirement for 3-phosphoinositide-kependent dinase-1 (PDK-1) in insulin-induced glucose uptake in immortalized brown adipocytes

To provide insight into the physiological importance of 3-phosphoinositide-dependent kinase-1 (PDK-1) in the metabolic actions of insulin, we have generated mice that harbor a PDK-1 gene containing LoxP sites (PDK-1(lox/lox) mice) and established immortalized brown preadipocyte cell lines both from these animals and from wild-type mice. Exposure to appropriate hormonal inducers resulted in the differentiation of >80% of the immortalized brown preadipocytes derived from both types of mice into mature adipocytes. Introduction of the Cre recombinase with the use of adenovirus-mediated gene transfer induced a dose-dependent decrease in the abundance of PDK-1 in PDK-1(lox/lox) adipocytes but not in the wild-type cells. In Cre-expressing PDK-1(lox/lox) adipocytes in which the abundance of PDK-1 was reduced by approximately 85%, the insulin-induced phosphorylation both of Akt on threonine 308 and of p70 S6 kinase on threonine-389 was markedly inhibited. The phosphorylation both of Akt on serine 473 and of p42 and p44 isoforms of mitogen-activated protein kinase induced by insulin was not affected by Cre expression, indicating that the latter specifically inhibits PDK-1-dependent signaling. Both glucose uptake and the translocation of glucose transporter 4 to the plasma membrane induced by insulin as well as glucose uptake induced by a constitutively active form of phosphoinositide 3-kinase were also greatly inhibited by Cre expression in PDK-1(lox/lox) adipocytes. Phosphorylation of AMP-activated protein kinase and glucose uptake induced by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) were not affected by Cre expression in PDK-1(lox/lox) adipocytes. These results indicate that PDK-1 is essential for insulin-induced glucose uptake in adipocytes.     

Insulin activates phospholipase C-gamma1 via a PI-3 kinase dependent mechanism in 3T3-L1 adipocytes

Previously we have shown that the insulin receptor and phospholipase C-gamma1 physically interact in the 3T3-L1 adipocyte cell line. In this study, we investigated the ability of insulin and PDGF to stimulate PLC-gamma1 enzyme activity as measured by PI-(4,5)P(2) hydrolysis. Both insulin and PDGF caused a rapid (<1 min) increase in PLC activity associated with the respective receptor. PDGF treatment resulted in a higher and more sustained stimulation of PLC-gamma1 activity compared to insulin (0.95 pmol/min/mg vs 0.68 pmol/min/mg). Furthermore, insulin and PDGF promoted increases in total cellular DAG, one of the products of PI-(4,5)P(2) hydrolysis. Insulin-stimulated PLC activity appears to be downstream of PI-3Kinase as the DAG increase was partially blocked by Wortmannin and addition of PI-(3,4,5)P(3) activated PLC-gamma1 in vitro. Inhibition of PLC using U73122 or an inhibitory peptide caused a decrease in insulin-stimulated 2-deoxyglucose transport and GLUT4 translocation that was rescued by the addition of OAG, a cell-permeable synthetic DAG.     

Association of PI-3 kinase with PAK1 leads to actin phosphorylation and cytoskeletal reorganization

The family of p21-activated kinases (PAKs) have been implicated in the rearrangement of actin cytoskeleton by acting downstream of the small GTPases Rac and Cdc42. Here we report that even though Cdc42/Rac1 or Akt are not activated, phosphatidylinositol-3 (PI-3) kinase activation induces PAK1 kinase activity. Indeed, we demonstrate that PI-3 kinase associates with the N-terminal regulatory domain of PAK1 (amino acids 67-150) leading to PAK1 activation. The association of the PI-3 kinase with the Cdc42/Rac1 binding-deficient PAK1(H83,86L) confirms that the small GTPases are not involved in the PI-3 kinase-PAK1 interaction. Furthermore, PAK1 was activated in cells expressing the dominant-negative forms of Cdc42 or Rac1. Additionally, we show that PAK1 phosphorylates actin, resulting in the dissolution of stress fibers and redistribution of microfilaments. The phosphorylation of actin was inhibited by the kinase-dead PAK1(K299R) or the PAK1 autoinhibitory domain (PAK1(83-149)), indicating that PAK1 was responsible for actin phosphorylation. We conclude that the association of PI-3 kinase with PAK1 regulates PAK1 kinase activity through a Cdc42/Rac1-independent mechanism leading to actin phosphorylation and cytoskeletal reorganization.     

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.     

The effects of intracellular calcium depletion on insulin signaling in 3T3-L1 adipocytes

We have examined the requirement for intracellular calcium (Ca(2+)) in insulin signal transduction in 3T3-L1 adipocytes. Using the Ca(2+) chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), we find both augmentation and inhibition of insulin signaling phenomena. Pretreatment of cells with 50 microM BAPTA-AM did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 or insulin receptor (IR)beta. The decreased mobility of IRS1 normally observed after chronic stimulation with insulin, due to serine phosphorylation, was completely eliminated by Ca(2+) chelation. Correlating with decreased insulin-induced serine phosphorylation of IRS1, phosphotyrosine-mediated protein-protein interactions involving p85, IRS1, IRbeta, and phosphotyrosine-specific antibody were greatly enhanced by pretreatment of cells with BAPTA-AM. As a result, insulin-mediated, phosphotyrosine-associated PI3K activity was also enhanced. BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca(2+) depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca(2+) in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca(2+) is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca(2+) blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

PTEN, but not SHIP2, suppresses insulin signaling through the phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 adipocytes

Glucose homeostasis is controlled by insulin in part through the stimulation of glucose transport in muscle and fat cells. This insulin signaling pathway requires phosphatidylinositol (PI) 3-kinase-mediated 3'-polyphosphoinositide generation and activation of Akt/protein kinase B. Previous experiments using dominant negative constructs and gene ablation in mice suggested that two phosphoinositide phosphatases, SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) negatively regulate this insulin signaling pathway. Here we directly tested this hypothesis by selectively inhibiting the expression of SHIP2 or PTEN in intact cultured 3T3-L1 adipocytes through the use of short interfering RNA (siRNA). Attenuation of PTEN expression by RNAi markedly enhanced insulin-stimulated Akt and glycogen synthase kinase 3alpha (GSK-3alpha) phosphorylation, as well as deoxyglucose transport in 3T3-L1 adipocytes. In contrast, depletion of SHIP2 protein by about 90% surprisingly failed to modulate these insulin-regulated events under identical assay conditions. In control studies, no diminution of insulin signaling to the mitogen-activated protein kinases Erk1 and Erk2 was observed when either PTEN or SHIP2 were depleted. Taken together, these results demonstrate that endogenous PTEN functions as a suppressor of insulin signaling to glucose transport through the PI 3-kinase pathway in cultured 3T3-L1 adipocytes.     

Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.