A Role for Phospholipase D in GLUT4 Glucose Transporter Translocation

Abstract

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitopetagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.Physiological glucose homeostasis in humans is largely dependent on the actions of the hormone insulin, particularly its ability to enhance glucose transport into fat and muscle. Insulin exerts this effect primarily through a process whereby sequestered, intracellular GLUT4 glucose transporter proteins are rapidly redistributed to cell surface membranes where they can catalyze glucose uptake into cells (1-3). In both the basal and insulin-stimulated states, GLUT4 proteins appear to cycle between intracellular membrane and plasma membrane locations, but exocytosis is relatively slow in the absence of insulin (4). The action of insulin to stimulate this process appears dependent on the activity of p85/p110-type phosphatidylinositol (PI) 1 3-kinase because it is blocked by inhibitors of this enzyme (5, 6) or by expression of dominant inhibitory constructs of the p85 regulatory subunit (7). Insulin receptor tyrosine kinase activity activates PI 3-kinase through recruitment of Src homology 2 domains of the p85 subunit to phosphotyrosine sites on insulin receptor substrate proteins (8). The concept that the 3Ј-polyphosphoinositide products of insulin-stimulated PI 3-kinase are key mediators of insulin action on GLUT4 translocation is consistent with extensive data supporting a major role of these lipids in membrane trafficking (9).Much effort has been expended attempting to identify downstream targets of the PI 3-kinase products, phosphatidylinositol 3-phosphate (PtdIns(3)P), PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 , that might connect insulin receptor signaling to GLUT4 glucose transporter trafficking (1). Some evidence (10, 11) has implicated Akt/ protein kinase B, which is thought to be activated in the presence of 3Ј-polyphosphoinositide through the actions of PDK1 and another unknown protein kinase (12), in GLUT4 regulation. However, other data do not support this view but i...

Phosphatidylinositol (3,4,5)-trisphosphate specifically interacts with the phox homology domain of phospholipase D1 and stimulates its activity

“…In addition, SWAP-70, a PtdIns(3,4,5)P 3 -dependent guanine nucleotide exchange factor (GEF) for Rac, can regulate membrane ruffling induced by epidermal growth factor (EGF) or PDGF (Shinohara et al, 2002). Although it was reported that the inhibition of PI3K activity results in the downregulation of PLD activity (Emoto et al, 2000;Kozawa et al, 1997;Standaert et al, 1996), the mechanism involved is unknown. Our finding that the PLD1 PX domain has affinity for PtdIns(3,4,5)P 3 is an important clue.…”

Exocytosis of CTLA-4 Is Dependent on Phospholipase D and ADP Ribosylation Factor-1 and Stimulated during Activation of Regulatory T Cells

“…GLUT4 is a recycling receptor that interacts with both AP-1 and AP-2 and recycles to a trans-Golgi network 38-negative compartment (33). Furthermore, PLD activity has also been implicated in translocation of GLUT4 to the PM (34). Perhaps most interestingly, both CTLA-4 and GLUT4 appear to use a storage compartment from which exocytosis is achieved rapidly upon stimulation.…”

Involvement of Phospholipase D in Sphingosine 1-Phosphate-induced Activation of Phosphatidylinositol 3-Kinase and Akt in Chinese Hamster Ovary Cells Overexpressing EDG3

“…Similar to PI3K, PLD is regulated by RalA, a downstream target of Ras, in plateletderived growth factor-and epidermal growth factor-stimulated cells (49,50), and PLD2 is tyrosine-phosphorylated by forming a physical complex with the epidermal growth factor receptor (51). Recent studies have demonstrated that PLD1 and PI3K play a role in GLUT4 translocation between the plasma membranes and intracellular vesicles in insulin-stimulated cells (52). Furthermore, several studies have demonstrated through the use of PI3K inhibitors that PI3K is involved in agoniststimulated PLD activation (53)(54)(55)(56).…”