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.


Available from: Jana Strakova, Aug 27, 2014
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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.
    Advances in Biological Regulation 10/2014; 57. DOI:10.1016/j.jbior.2014.09.007
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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.
    Biochemical and Biophysical Research Communications 05/2014; 449(3). DOI:10.1016/j.bbrc.2014.05.024 · 2.30 Impact Factor
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    • "By stimulating this cell line with an anti-CD3 mAb, a nearly fourfold PI5P increase was detected using a lipid mass assay (Guittard et al., 2009). This fold increase is in accordance with other reported results in response to insulin stimulation in other cell types (Sbrissa et al., 2004; Sarkes and Rameh, 2010). As TCRinduced PI5P elevations appear to be rapid (peaks at 2 min) and transient (Guittard et al., 2009), we suggest that there is a rapid recruitment of a specific lipid kinase/phosphatase to the plasma membrane upon TCR engagement, as observed previously for the class IA PI3K (Fabre et al., 2005). "
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    ABSTRACT: Phosphoinositides are critical regulators in cell biology. Phosphatidylinositol 4,5-bisphosphate, also known as PI(4,5)P2 or PIP2, was the first variety of phosphoinositide to enter in the T cell signaling scene. Phosphatidylinositol bis-phosphates are the substrates for different types of enzymes such as phospholipases C (β and γ isoforms) and phosphoinositide 3-kinases (PI3K class IA and IB) that are largely involved in signal transduction. However until recently, only a few studies highlighted phosphatidylinositol monophosphates as signaling molecules. This was mostly due to the difficulty of detection of some of these phosphoinositides, such as phosphatidylinositol 5-phosphate, also known as PI5P. Some compelling evidence argues for a role of PI5P in cell signaling and/or cell trafficking. Recently, we reported the detection of a PI5P increase upon TCR triggering. Here, we describe the current knowledge of the role of PI5P in T cell signaling. The future challenges that will be important to achieve in order to fully characterize the role of PI5P in T cell biology, will be discussed.
    Frontiers in Immunology 04/2013; 4:80. DOI:10.3389/fimmu.2013.00080
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