Research ReportInsulin-stimulated translocation of GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent
Introduction
The insulin receptor (IR) is a heterotetrameric protein consisting of two extracellular α subunits that provide the insulin-binding domain and the transmembrane-spanning β subunits (Jones and Clemmons, 1995). Insulin binding stimulates the tyrosine kinase activity of the β subunit, leading to the activation of intracellular signaling events. In peripheral tissues such as skeletal muscle, cardiac muscle and adipose cells, IR activation stimulates glucose uptake (Saltiel and Pessin, 2002). Cloning and characterization of an insulin-sensitive glucose transporter, GLUT4, helped to elucidate the mechanisms through which IR activation and signaling elicited increases in glucose uptake (Birnbaum, 1989, Charron et al., 1989). In particular, activation of the IR promotes the tyrosine phosphorylation of the IR substrate proteins, leading to the activation of phosphatidylinositol 3-kinase (PI3-kinase) and the generation of phosphatidylinositol (3,4,5)-triphosphate (PIP3; Saltiel and Pessin, 2002). PIP3 activates phosphoinositide-dependent protein kinase (PDK), which phosphorylates and activates Akt (also known as protein kinase B). Additional studies determined that insulin stimulated translocation of GLUT4 and increases in glucose uptake are PI3-kinase-dependent in peripheral tissues (Pessin et al., 1999).
The IR is also expressed in discrete neuronal populations in the CNS, including the hippocampus (Doré et al., 1997, Kar et al., 1993, Marks et al., 1991), an important integration center for learning and memory in the mammalian brain (McEwen and Sapolsky, 1995). Insulin improves cognitive performance, and these cognitive enhancing properties of insulin may be mediated through IRs expressed in the hippocampus. The IR and the GLUT4 exhibit overlapping distributions in the rat brain, suggesting that GLUT4 trafficking may be mediated by similar mechanisms in the CNS as has been described previously for peripheral tissues. In support of this hypothesis, we have previously demonstrated that glucose-mediated increases in plasma insulin levels stimulates GLUT4 translocation from the cytosol to the plasma membrane in the rat hippocampus (McEwen and Reagan, 2004, Piroli et al., 2007a). However, such analyses cannot differentiate between the potential effects of glucose and/or insulin or whether phosphorylation of Akt is an important intermediate in these trafficking events in the hippocampus, as it is in peripheral tissues. In order to more accurately address these questions, we examined the ability of icv insulin to stimulate GLUT4 trafficking to the plasma membrane and whether these signaling events were PI3-kinase dependent. In addition, we also examined the colocalization of the IR and GLUT4 with phospho-Akt (pAkt) immunoreactivity in the rat hippocampus following intracerebroventricular (icv) administration of insulin.
Section snippets
Time course of insulin-mediated GLUT4 signaling events in the hippocampus
The time course of insulin mediated phosphorylation of Akt in the hippocampus was examined after icv insulin injection using Western blotting techniques. As shown in Fig. 1, insulin exerted a long lasting elevation in pAkt levels, which started with a nonsignificant increase observed 15 min following icv insulin administration, followed by significant increases between 30 and 60 min post-icv insulin. Maximal increases in pAkt levels were observed 45 min following insulin administration (Fig. 1
Discussion
The results of the current study demonstrate that intracerebroventricular administration of insulin stimulates the translocation of the insulin-sensitive glucose transporter GLUT4 to the plasma membrane in the rat hippocampus in a time and PI3-kinase-dependent manner. Additionally, unlike the sustained increases in the pAkt levels, insulin-stimulated translocation of GLUT4 to the plasma membrane is transient and returns to baseline levels approximately 45 min after icv insulin administration.
Animal protocols
Adult male Sprague–Dawley rats (CD strain, Charles River) weighing 200–250 g were housed in accordance with all guidelines and regulations of The University of South Carolina Animal Care and Use Committee. Animals were maintained in a temperature-controlled room, with a light/dark cycle of 12/12 h (lights on at 0700 h). Rats were handled daily for 5 days and then underwent stereotaxic surgery. After an overnight fast, rats were anesthetized, placed in the stereotaxic apparatus, and insulin
Acknowledgments
Supported by Juvenile Diabetes Research Foundation grant number 2-03-675 (LPR) and NIH grant number NS047728 (LPR).
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These authors contributed equally to this manuscript.