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PubMed2003

Gab2 tyrosine phosphorylation by a pleckstrin homology domain-independent mechanism: role in epidermal growth factor-induced mitogenesis.

In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (DeltaPHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Cytosolic action of thyroid hormone leads to induction of hypoxia-inducible factor-1alpha and glycolytic genes.

Thyroid hormone (TH) effects are mediated through T(3), which regulates gene expression by binding to the nuclear TH receptors, TRalpha and TRbeta. Using microarrays and real-time PCR we found mRNAs of the following genes increased in response to T(3) in a TRbeta-specific manner: the transcription factor hypoxia-inducible factor (HIF)-1alpha, its target genes glucose transporter (GLUT)1 and platelet-type phosphofructokinase (PFKP), and the monocarboxylate transporter (MCT)4. The products of these genes have important roles in cellular glucose metabolism. HIF-1alpha expression and activity can be regulated through phosphatidylinositol-OH-3-kinase (PI3K) and MAPK signaling; thus the possibility of alternative, nonnuclear pathways of TH action was raised. We examined the involvement of these pathways in mediating TH effects by treating human skin fibroblasts with 2 nm T(3) in the absence or presence of either the PI3K inhibitor LY294002 or the MAPK inhibitor PD98059. T(3) induced HIF-1alpha mRNA by 2.7-fold (+/-0.4; P < 0.013). This increase was completely abrogated by LY294002 (1.1 +/- 0.1; nonsignificant = 0.57), but preserved in the presence of PD98059 (2.2 +/- 0.2; P < 0.009). Western blotting confirmed these results at the protein level, indicating dependency on the PI3K pathway. The same pattern of response was observed for GLUT1, PFKP, and MCT4 expression. To examine whether HIF-1alpha is directly induced, we used the translation inhibitor cycloheximide (CHX). T(3) induction of HIF-1alpha mRNA was not affected by CHX, whereas T(3) effect on GLUT1, PFKP, and MCT4 mRNA was completely abrogated by CHX. These results demonstrate that cytosolic activation of the PI3K signaling pathway has a role in TH-mediated direct (HIF-1alpha) and indirect (GLUT1, PFKP, MCT4) gene expression, and possibly provides a link between TH and cellular glucose metabolism in human fibroblasts.

Chromones↗

PubMed2006

Vav3 oncogene is overexpressed and regulates cell growth and androgen receptor activity in human prostate cancer.

The purpose of this research was to investigate the role of Vav3 oncogene in human prostate cancer. We found that expression of Vav3 was significantly elevated in androgen-independent LNCaP-AI cells in comparison with that in their androgen-dependent counterparts, LNCaP cells. Vav3 expression was also detected in other human prostate cancer cell lines (PC-3, DU145, and 22Rv1) and, by immunohistochemistry analysis, was detected in 32% (26 of 82) of surgical specimens of human prostate cancer. Knockdown expression of Vav3 by small interfering RNA inhibited growth of both androgen-dependent LNCaP and androgen-independent LNCaP-AI cells. In contrast, overexpression of Vav3 promoted androgen-independent growth of LNCaP cells induced by epidermal growth factor. Overexpression of Vav3 enhanced androgen receptor (AR) activity regardless of the presence or absence of androgen and stimulated the promoters of AR target genes. These effects of Vav3 could be attenuated by either phosphatidylinositol 3-kinase (PI3K) inhibitors or dominant-negative Akt and were enhanced by cotransfection of PI3K. Moreover, phosphorylation of Akt was elevated in LNCaP cells overexpressing Vav3, which could be blocked by PI3K inhibitors. Finally, we ascertained that the DH domain of Vav3 was responsible for activation of AR. Taken together, our data show that overexpression of Vav3, through the PI3K-Akt pathway, inappropriately activates AR signaling axis and stimulates cell growth in prostate cancer cells. These findings suggest that Vav3 overexpression may be involved in prostate cancer development and progression.

Blotting, Western↗

PubMed2004

Signaling pathways controlling primordial germ cell migration in zebrafish.

During their migration, zebrafish primordial germ cells (PGCs) rely on directional cues provided by the chemokine SDF-1a, whose receptor is CXCR4b. The molecular mechanisms whereby CXCR4b activation is interpreted intracellularly into directional migration are not known. Here we investigate the role of two important biochemical pathways -- G-protein-dependent and phosphoinositide 3-kinase (PI3K)-dependent signaling -- in directing PGC migration in zebrafish. We show that G proteins of the Gi family are essential for directional migration but not for PGC motility. Inhibition of PI3K signaling in PGCs slows down their migration and leads to abnormal cell morphology as well as to reduced stability of filopodia. Invariably, during directed PGC migration, the distribution of the products of PI3K activity - phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and/or phosphatidylinositol (3,4)bisphosphate [PtdIns(3,4)P(2)] -- is not polarized, and reducing the level of these 3-phosphoinositides does not affect the ability of PGCs to migrate directionally. We therefore conclude that Gi-dependent signaling is essential for directional migration, whereas the PI3K pathway is important for the actual motility of PGCs.

Animals↗

PubMed2007

Regulation of the phosphatidylinositol 3-kinase-Akt and the mitogen-activated protein kinase pathways by ursolic acid in human endometrial cancer cells.

The phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the mitogen activated protein kinase (MAPK) pathway are important in the development and proliferation of various human cancers. It has been found recently that ursolic acid treatment affects growth and apoptosis in cancer cells. We sought to determine whether prominent signaling pathways, including the PI3K-Akt pathway and the MAPK (JNK, P38, and P44/42) pathway mediate these effects. Endometrial cancer cells often have high levels of phosphorylated Akt seen in conjunction with a PTEN mutation or deletion. Elevation in Akt protects the cancer cell from apoptosis. Ursolic acid treatment moderately decreased PI3K levels in SNG-II cells. Treatment also decreased phospho-Akt and phospho-P44/42 in a dose- and time-dependent fashion, dramatically in SNG-II cells and moderately in HEC108 cells. This effect was most pronounced following treatment with 50 mum ursolic acid for 72 h. Our study found inhibition of both the PI3K-Akt pathway and the MAPK pathway in two endometrial cancer cell lines, SNG-II and the poorly differentiated HEC108 cell line.

Cell Line, Tumor↗

PubMed2003

Insulin-like growth factor-1 rescues the mutated FGF receptor 3 (G380R) expressing ATDC5 cells from apoptosis through phosphatidylinositol 3-kinase and MAPK.

UNLABELLED: An activated mutation in the FGFR3 gene causes ACH. To examine the effects of IGF-1, which is an important mediator of GH, on apoptosis, we analyzed a chondrogenic cell line expressing the FGFR3 mutants. Our findings that IGF-1 prevented the apoptosis through PI3K and MAPK pathways may explain how GH treatment improves the disturbed bone growth in ACH. INTRODUCTION: Achondroplasia (ACH), which is caused by a point mutation of the fibroblast growth factor receptor 3 (FGFR3) gene in the transmembrane domain (G380R), is one of the most common genetic forms of dwarfism. Recently, using a chondrogenic cell line, ATDC5, we have showed that the constitutively active FGFR3 mutants induced an apoptosis of chondrocytes. We have also reported that growth hormone (GH) treatment increased the growth rate in achondroplasia in parallel with the increment of serum levels of insulin-like growth factor (IGF)-1, suggesting an important role of IGF-1 in skeletal development. In this study, to clarify the mechanism by which GH treatment improved the phenotype of ACH patients, we examined the possible effects of IGF-1 on an apoptosis induced by FGFR3 mutant in ATDC5. MATERIALS AND METHODS: Using adenovirus vector, wildtype or mutant FGFR3 (G380R) was introduced into ATDC5. Analysis of apoptosis was estimated by TUNEL assay. Expression levels of apoptosis-related genes and activation of signaling molecules were analyzed by immunoblot. RESULTS: MTT assay showed that the cell number was reduced in ATDC5 cells expressing the mutant FGFR3 (G380R; ATDC5-mtR3 cells), suggesting that ATDC5-mtR3 cells might fall into apoptosis. IGF-1, which is an important mediator of GH, restored cell proliferation and reduced apoptosis in ATDC5-mtR3 cells. IGF-1 also decreased the ratio of Bax/Bcl-2 in the cells. To investigate which signaling cascade is responsible for antiapoptotic effects of IGF-1, we examined the role of phosphatidylinositol 3-kinase (PI3K) and MAPK in ATDC5-mtR3 cells. Specific inhibitors of PI3K or MAPK blocked the antiapoptotic effects of IGF-1 in ATDC5-mtR3 cells. CONCLUSIONS: Our findings, showing IGF-1 prevents the apoptosis induced by FGFR3 mutation through the PI3K pathway and MAPK pathway, explain the mechanisms by which GH treatment improves the disturbed bone growth in ACH.

Animals↗

PubMed2000

Different signaling pathways are involved in CCK(B) receptor-mediated MAP kinase activation in COS-7 cells.

Recently, the involvement of the MAP kinase ERK in mitogenic signaling of cholecystokininB (CCK(B)) receptors has been shown. However, the intracellular effector systems involved in this signaling pathway are poorly defined. In this study, we used COS-7 cells transiently transfected with the human CCK(B) receptor to investigate cholecystokinin-induced MAP kinase activation. CCK-8 induced activation of ERK2 which is associated with its phosphorylation and localization in the nucleus. The CCK-8-dependent ERK stimulation is sensitive to wortmannin an inhibitor of phosphoinositide 3-kinases (PI3Ks) indicating the involvement of PI3K activity. To identify the PI3K species involved in mitogenic signaling of the CCK(B) receptor several dominant-negative mutants of PI3K regulatory and catalytic subunits were transiently expressed. Surprisingly, different catalytically inactive mutants of the G protein-sensitive PI3Kgamma did not affect ERK stimulation induced by CCK, whereas a dominant-negative mutant of the regulatory p85 subunit induced significant inhibition of CCK-dependent ERK activity. These results indicate an involvement of PI3K class 1A species alpha, beta or/and delta in signal transduction via CCK(B) receptors. In addition, protein kinase C (PKC)-dependent signaling pathways contribute to CCK(B)-mediated MAP kinase signaling as shown by inhibition of CCK-8-induced ERK activation by the PKC inhibitor bisindolylmaleimide.

Animals↗

PubMed2001

Phosphoinositide 3-kinase inhibition in cancer treatment.

Over the past ten years, our knowledge of the integral role that the phospho-inositide 3-kinases (PI3Ks) and their 3'-phosphorylated lipid products (3'-phosphorylated phosphoinositides; 3P-PIs) play in the mediation of signal transduction, cytoskeletal rearrangements and membrane trafficking has expanded considerably. They are now known to be involved in the regulation of cell growth, differentiation, mobility, proliferation and survival and hence they have become a potential target for the control of the growth and spread of cancer cells. More recently, the correlation of the multiplicity of isomers (both catalytic and regulatory) within the different classes of the PI3Ks with their functional relevance has become possible. This, combined with our further understanding of the protein recognition patterns for their different 3P-PIs and the newly-described pathways in the control of the levels of these by dephosphorylation, has provided new aspects and areas for interference in these multiple PI3K signalling pathways. However, in the search for effective, non-toxic, drugs for use in the treatment of cancers, these individual targets for PI3K inhibition need to be further correlated with the specific in vivo effects on cell survival, invasivity and metastatic potential. Here, the range of PI3K inhibition targets are discussed in the light of recent experimental findings, with a view to the exploitation of their specificities in new approaches to effective cancer treatments based on PI3K activity inhibition.

Androstadienes↗

PubMed2004

Activation of the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin signaling pathway is required for metabotropic glutamate receptor-dependent long-term depression.

Hippocampal long-term depression (LTD) is a long-lasting decrease in synaptic strength that is most commonly studied at glutamatergic inputs to pyramidal cells in hippocampal area CA1. Activation of G-protein-coupled group I (including types 1 and 5) metabotropic glutamate receptors (mGluRs) by the pharmacological agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) elicits LTD in area CA1 of the hippocampus. Recent reports have shown that de novo protein synthesis is necessary for DHPG-induced LTD. However, relatively little is known about the signaling pathways that couple mGluRs to translation initiation. In this study, we investigated whether the activation of the phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which has been shown to regulate translation initiation, is necessary for mGluR-LTD induced by DHPG. We found that brief incubations of mouse hippocampal slices with DHPG resulted in increased phosphorylation of Akt and mTOR in hippocampal area CA1. Two structurally unrelated PI3K inhibitors, LY294002 and wortmannin, blocked the DHPG-induced increases in phosphorylation of Akt and mTOR. Biochemical fractionation studies showed that the DHPG-induced increase in the phosphorylation of Akt and mTOR could be detected in synaptoneurosome preparations, and immunohistochemical analysis revealed that similar increases could be detected in both stratum pyramidale and stratum radiatum in area CA1. Finally, we observed that both PI3K inhibitors and rapamycin, an mTOR inhibitor, prevented mGluR-LTD induced by DHPG. Together, our findings indicate that activation of the PI3K-Akt-mTOR signaling cascade is required for mGluR-LTD and suggest that this pathway may couple group I mGluRs to translation initiation in hippocampal area CA1.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2007

Phosphatidylinositol 3-kinase and Akt nonautonomously promote perineurial glial growth in Drosophila peripheral nerves.

Drosophila peripheral nerves, structured similarly to their mammalian counterparts, comprise a layer of motor and sensory axons wrapped by an inner peripheral glia (analogous to the mammalian Schwann cell) and an outer perineurial glia (analogous to the mammalian perineurium). Growth and proliferation within mammalian peripheral nerves are increased by Ras pathway activation: loss-of-function mutations in Nf1, which encodes the Ras inhibitor neurofibromin, cause the human genetic disorder neurofibromatosis, which is characterized by formation of neurofibromas (tumors of peripheral nerves). However, the signaling pathways that control nerve growth downstream of Ras remain incompletely characterized. Here we show that expression specifically within the Drosophila peripheral glia of the constitutively active Ras(V12) increases perineurial glial thickness. Using chromosomal loss-of-function mutations and transgenes encoding dominant-negative and constitutively active proteins, we show that this nonautonomous effect of Ras(V12) is mediated by the Ras effector phosphatidylinositol 3-kinase (PI3K) and its downstream kinase Akt. We also show that the nonautonomous, growth-promoting effects of activated PI3K are suppressed by coexpression within the peripheral glia of FOXO+ (forkhead box O) a transcription factor inhibited by Akt-dependent phosphorylation. We suggest that Ras-PI3K-Akt activity in the peripheral glia promotes growth of the perineurial glia by inhibiting FOXO. In mammalian peripheral nerves, the Schwann cell releases several growth factors that affect the proliferative properties of neighbors. Some of these factors are oversecreted in Nf1 mutants. Our results raise the possibility that neurofibroma formation in individuals with neurofibromatosis might result in part from a Ras-PI3K-Akt-dependent inhibition of FOXO within Schwann cells.

Animals↗

PubMed2006

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro.

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.

Animals↗

PubMed2007

Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed.

Human ESCs (hESCs) respond to signals that determine their pluripotency, proliferation, survival, and differentiation status. In this report, we demonstrate that phosphatidylinositol 3-kinase (PI3K) antagonizes the ability of hESCs to differentiate in response to transforming growth factor beta family members such as Activin A and Nodal. Inhibition of PI3K signaling efficiently promotes differentiation of hESCs into mesendoderm and then definitive endoderm (DE) by allowing them to be specified by Activin/Nodal signals present in hESC cultures. Under conditions where hESCs are grown in mouse embryo fibroblast-conditioned medium under feeder-free conditions, approximately 70%-80% are converted into DE following 5 days of treatment with inhibitors of the PI3K pathway, such as LY 294002 and AKT1-II. Microarray and quantitative polymerase chain reaction-based gene expression profiling demonstrates that definitive endoderm formation under these conditions closely parallels that following specification with elevated Activin A and low fetal calf serum (FCS)/knockout serum replacement (KSR). Reduced insulin/insulin-like growth factor (IGF) signaling was found to be critical for cell fate commitment into DE. Levels of insulin/IGF present in FCS/KSR, normally used to promote self-renewal of hESCs, antagonized differentiation. In summary, we show that generation of hESC-DE requires two conditions: signaling by Activin/Nodal family members and release from inhibitory signals generated by PI3K through insulin/IGF. These findings have important implications for our understanding of hESC self-renewal and early cell fate decisions.

Activins↗

PubMed2002

Inhibitors of phosphatidylinositol 3-kinase amplify insulin release from islets of lean but not obese mice.

We examined the effects of phosphatidylinositol 3-kinase (PI3K) inhibition by wortmannin or LY294002 on glucose-induced secretion from mouse islets. Islets were collagenase isolated and perifused or subjected to Western blot analyses and probed for insulin receptor-signaling components. In agreement with previous studies, mouse islets, when compared with rat islets, were minimally responsive to 10 mM glucose stimulation. The inclusion of 50 nM wortmannin or 10 microM LY294002 significantly amplified 10 mM glucose-induced release from mouse islets. The effect of wortmannin was abolished by the calcium channel antagonist nitrendipine or by lowering the glucose level to 3 mM. Wortmannin had no effect on 10 mM alpha-ketoisocaproate-induced secretion. In contrast to its potentiating effect on islets from CD-1 mice, wortmannin had no effect on 10 mM glucose-induced release from ob/ob mouse islets. Western blot analyses revealed the presence of the insulin receptor, insulin receptor substrate proteins 1 and 2 and PI3K in CD-1 islets. These results support the concept that a PI3K-dependent signaling pathway exists in beta-cells and that it may function to restrain glucose-induced insulin secretion from beta-cells. They also suggest that, as insulin resistance develops in peripheral tissues, a potential result of impaired PI3K activation, the same biochemical anomaly in beta-cells promotes a linked increase in insulin secretion to maintain glucose homeostasis.

Analysis of Variance↗

PubMed2006

Akt and Mammalian target of rapamycin regulate separate systems of proteolysis in renal tubular cells.

EGF suppresses proteolysis via class 1 phosphatidylinositol 3-kinase (PI3K) in renal tubular cells. EGF also increases the abundance of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and transcription factors (e.g., pax2) that are degraded by the lysosomal pathway of chaperone-mediated autophagy. To determine if EGF regulates chaperone-mediated autophagy through PI3K signaling, this study examined the effect of inhibiting PI3K and its downstream mediators Akt and the mammalian target of rapamycin (mTOR). Inhibition of PI3K with LY294002 prevented EGF-induced increases in GAPDH and pax2 abundance in NRK-52E renal tubular cells. Similar results were seen with an adenovirus encoding a dominant negative Akt (DN Akt). Expression of a constitutively active Akt increased GAPDH and pax2 abundance. An mTOR inhibitor, rapamycin, did not prevent EGF-induced increases in these proteins. Neither DN Akt nor rapamycin alone had an effect on total cell protein degradation, but both partially reversed EGF-induced suppression of proteolysis. DN Akt no longer affected proteolysis after treatment with a lysosomal inhibitor, methylamine. In contrast, methylamine or the inhibitor of macroautophagy, 3-methyladenine, did not prevent rapamycin from partially reversing the effect of EGF on proteolysis. Notably, rapamycin did not increase autophagasomes detected by monodansylcadaverine staining. Blocking the proteasomal pathway with either MG132 or lactacystin prevented rapamycin from partially reversing the effect of EGF on proteolysis. It is concluded that EGF regulates pax2 and GAPDH abundance and proteolysis through a PI3K/Akt-sensitive pathway that does not involve mTOR. Rapamycin has a novel effect of regulating proteasomal proteolysis in cells that are stimulated with EGF.

Acetylcysteine↗

PubMedDate not supplied

CD151 gene delivery activates PI3K/Akt pathway and promotes neovascularization after myocardial infarction in rats.

Our previous study showed that CD151 promotes neovascularization and improves blood perfusion in a rat hindlimb ischemia model. In this study, we investigated whether CD151 promotes neovascularization and improves ventricular function after myocardial infarction in rats and the mechanisms involved. Rats were subjected to sham surgery or coronary artery ligation. We used rAAV for direct delivery of the human CD151 gene into the rat myocardium. At 4 weeks after coronary artery ligation, human CD151 mRNA was detected using RT-PCR. Measurement of capillary density was evaluated using immunostaining for von Willebrand factor, and hemodynamic variables and physiological parameters were monitored. Western blot analysis for CD151, PI3K, phosphor-Akt, total Akt, phosphor-eNOS, and total eNOS was performed. In addition, we also observed the effect of CD151 on the expression of VEGF using Western blot analysis. CD151 gene delivery could increase the expression of CD151 at gene and protein levels. Overexpression of CD151 could increase the number of microvessels in the ischemic myocardium and significantly improved the hemodynamic variables after myocardial infarction. In addition, CD151 could activate the PI3K pathway, including activation of Akt and eNOS, but did not affect the expression of VEGF. This study suggested that CD151 could promote neovascularization and improve ventricular function after myocardial infarction in rats. The mechanism may be that CD151 can activate the PI3K pathway and promote neovascularization via the PI3K pathway, without affecting ischemia-induced VEGF expression.

Animals↗

PubMed2003

SHIP2: an emerging target for the treatment of type 2 diabetes mellitus.

With the rapid increase in the number of patients developing type 2 diabetes mellitus and the lack of optimal therapies, much focus has been placed on the insulin-signaling pathway in the discovery of novel drug targets. Phosphatidyl Inositol 3-Kinase (PI3K) is central to mediating insulin-s metabolic effects. PI3K catalyzes the generation of phosphatidyl inositol (3,4,5) triphosphate (PIP(3)). Inhibition of PI3K activity results in a blockade of insulin signaling including glucose uptake and glyocogen synthesis. Thus, PIP(3) is a critical mediator of insulin action. A family of phosphatidyl inositol phosphatases have been identified that counter-regulate PI3K activity by hydrolyzing PIP(3) to phosphatidyl inositol bisphosphate at either the 3' or 5' position of the inositol ring. Mice lacking one of these enzymes, Src-Homology Inositol Phosphatase-2 (SHIP2), demonstrate increased insulin sensitivity, suggesting that pharmacological inhibition of SHIP2 could alleviate insulin resistance. Recent studies demonstrate elevated SHIP2 expression is associated with insulin resistance in human patients. Comparing the studies on SHIP2 and other phosphatases suggests how inhibition of SHIP2 leads to increased insulin sensitivity without deleterious effects. This review focuses on the emergence of SHIP2 as a target in the insulin-signaling pathway for the treatment of type 2 diabetes.

Animals↗

PubMed2000

Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats.

To determine whether impaired Akt (protein kinase B or rac) activation contributes to insulin resistance in vivo, we examined the expression, phosphorylation, and kinase activities of Akt1 and Akt2 isoforms in insulin target tissues of insulin-resistant obese Zucker rats. In lean rats, insulin (10 U/kg i.v. x 2.5 min) stimulated Akt1 activity 6.2-, 8.8-, and 4.4-fold and Akt2 activity 5.4-, 9.3-, and 1.8-fold in muscle, liver, and adipose tissue, respectively. In obese rats, insulin-stimulated Akt1 activity decreased 30% in muscle and 21% in adipose tissue but increased 37% in liver compared with lean littermates. Insulin-stimulated Akt2 activity decreased 29% in muscle and 37% in liver but increased 24% in adipose tissue. Akt2 protein levels were reduced 56% in muscle and 35% in liver of obese rats, but Akt1 expression was unaltered. Phosphoinositide 3-kinase (PI3K) activity associated with insulin receptor substrate (IRS)-1 or phosphotyrosine was reduced 67-86% in tissues of obese rats because of lower IRS-1 protein levels and reduced insulin receptor and IRS-1 phosphorylation. In adipose tissue of obese rats, in spite of an 86% reduction in insulin-stimulated PI3K activity, activation of Akt2 was increased. Maximal insulin-stimulated (100 nmol/l) glucose transport was reduced 70% in isolated adipocytes, with a rightward shift in the insulin dose response for transport and for Akt1 stimulation but normal sensitivity for Akt2. These findings suggest that PI3K-dependent effects on glucose transport in adipocytes are not mediated primarily by Akt2. Akt1 and Akt2 activations by insulin have a similar time course and are maximal by 2.5 min in adipocytes of both lean and obese rats. We conclude that 1) activation of Akt1 and Akt2 in vivo is much less impaired than activation of PI3K in this insulin-resistant state, and 2) the mechanisms for divergent alterations in insulin action on Akt1 and Akt2 activities in tissues of insulin-resistant obese rats involve tissue- and isoform-specific changes in both expression and activation.

Adipose Tissue↗

PubMed2003

Insulin-stimulated protein kinase C lambda/zeta activity is reduced in skeletal muscle of humans with obesity and type 2 diabetes: reversal with weight reduction.

In humans with obesity or type 2 diabetes, insulin target tissues are resistant to many actions of insulin. The atypical protein kinase C (PKC) isoforms lambda and zeta are downstream of phosphatidylinositol-3 kinase (PI3K) and are required for maximal insulin stimulation of glucose uptake. Phosphoinositide-dependent protein kinase-1 (PDK-1), also downstream of PI3K, mediates activation of atypical PKC isoforms and Akt. To determine whether impaired PKClambda/zeta or PDK-1 activation plays a role in the pathogenesis of insulin resistance, we measured the activities of PKClambda/zeta and PDK-1 in vastus lateralis muscle of lean, obese, and obese/type 2 diabetic humans. Biopsies were taken after an overnight fast and after a 3-h hyperinsulinemic-euglycemic clamp. Obese subjects were also studied after weight loss on a very-low-calorie diet. Insulin-stimulated glucose disposal rate is reduced 26% in obese subjects and 62% in diabetic subjects (both comparisons P < 0.001). Insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and PI3K activity are impaired 40-50% in diabetic subjects compared with lean or obese subjects. Insulin stimulates PKClambda/zeta activity approximately 2.3-fold in lean subjects; the increment above basal is reduced 57% in obese and 65% in diabetic subjects. PKClambda/zeta protein amount is decreased 46% in diabetic subjects but is normal in obese nondiabetic subjects, indicating impaired insulin action on PKClambda/zeta. Importantly, weight loss in obese subjects normalizes PKClambda/zeta activation and increases IRS-1 phosphorylation and PI3K activity. Insulin also stimulates PDK-1 activity approximately twofold with no impairment in obese or diabetic subjects. In contrast to our previous data on Akt, reduced insulin-stimulated PKClambda/zeta activity could play a role in the pathogenesis of insulin resistance in muscle of obese and type 2 diabetic subjects.

3-Phosphoinositide-Dependent Protein Kinases↗