Role for a Novel Signaling Intermediate, Phosphatidylinositol 5-Phosphate, in Insulin-Regulated F-Actin Stress Fiber Breakdown and GLUT4 Translocation

Wayne State University, Detroit, Michigan, United States
Endocrinology (Impact Factor: 4.5). 12/2004; 145(11):4853-65. DOI: 10.1210/en.2004-0489
Source: PubMed


The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.

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Available from: Jana Strakova, Aug 27, 2014
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    • "Mammalian PIKfyve, synthesizing phosphatidylinositol (PtdIns) 1 5P from PtdIns, and PtdIns(3,5)P 2 from PtdIns3P, represents a major node in PI signaling and membrane trafficking downstream of PtdIns3P [1] . PIKfyve controls both constitutive and regulated endocytic trafficking as evidenced by the presence of its lipid products in quiescent cells and their elevation upon activation of endosome dynamics by cues such as insulin, EGF or viral infection2345678. A hallmark of PIKfyve cellular inactivation, achieved by means of PIKfyve dominant-negative kinase-deficient mutants [9,10], pharmacological inhibition [5,11,12] , siRNA-mediated silenc- ing [13] or Cre-mediated gene disruption [14,15], is progressive dilation of endosomal membranes, culminating in the appearance of multiple translucent cytoplasmic vacuoles readily seen by light microscopy in dividing cells grown in complete media. "
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    ABSTRACT: The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34(fl/fl) mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34(wt), expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyve(wt) rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further present evidence for a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Jan 2015 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
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    • "Two of the many cellular effects of insulin are to promote F-actin stress fibre disassembly and to induce the trafficking of GLUT4 transporters to the plasma membrane (Ikonomov et al., 2002). Overexpression of the PI5P-generating enzyme PIKfyve or microinjection of PI5P mimics these insulin effects whilst sequestration of PI5P with the probe 3xPHD ING2 inhibits insulin-induced changes in the cytoskeleton and GLUT4 translocation (Sbrissa et al., 2004). Furthermore, when the PIKfyve inhibitor YM201636 is used at a concentration of 160 nM, a concentration that selectively impairs the production of PI5P over PI(3,5)P 2 , insulin-induced F-actin stress fibre disassembly is dramatically reduced (Sbrissa et al., 2012). "
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    ABSTRACT: The family of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) is emerging from a comparative backwater in inositide signalling into the mainstream, as is their substrate, phosphatidylinositol 5-phosphate (PI5P). Here we review some of the key questions about the PI5P4Ks, their localisation, interaction, and regulation and also we summarise our current understanding of how PI5P is synthesised and what its cellular functions might be. Finally, some of the evidence for the involvement of PI5P4Ks in pathology is discussed.
    Full-text · Article · Oct 2014 · Advances in Biological Regulation
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    • "This implies that the relative level of the PIP5K2B substrate, phosphatidylinositol-5-phosphate (PtdIns(5)P), seems to impact on PtdIns(3,4,5)P3 levels and Akt/ PKB phosphorylation. In support of this hypothesis, levels of PtdIns(5)P have been reported to be induced by insulin stimulation and that injection of PtdIns(5)P into cells could mimic insulin stimulation at the level of Akt/PKB phosphorylation, Glut4 translocation as well as F-Actin stress fiber breakdown in different cell systems [3] [4]. Genetic ablation of PIP5K2B in mice resulted in increased insulin sensitivity, improved weight and lipid profiles. "
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    ABSTRACT: Phosphatidylinositol-5-phosphate 4-kinase, type II, beta (PIP5K2B) is linked to the pathogenesis of obesity, insulin resistance and diabetes. Here, we describe the identification of a novel pyrimidine-2,4,-diamine PIP5K2B inhibitor, designated SAR088. The compound was identified by high-throughput screening and subsequently characterized in vitro and in vivo. SAR088 showed reasonable potency, selectivity and physicochemical properties in enzymatic and cellular assays. In vivo, SAR088 lowered blood glucose levels of obese and hyperglycemic male Zucker diabetic fatty rats treated for three weeks. Thus, SAR088 represents the first orally available and in vivo active PIP5K2B inhibitor and provides an excellent starting point for the development of potent and selective PIP5K2B inhibitors for the treatment of insulin resistance and diabetes.
    Preview · Article · May 2014 · Biochemical and Biophysical Research Communications
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