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• W2061738346 abstract "Insulin receptors are highly enriched at neuronal synapses, but whose function remains unclear. Here we present evidence that brief incubations of rat hippocampal slices with insulin resulted in an increased protein expression of dendritic scaffolding protein postsynaptic density-95 (PSD-95) in area CA1. This insulin-induced increase in the PSD-95 protein expression was inhibited by the tyrosine kinase inhibitor, AG1024, phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, translational inhibitors, anisomycin and rapamycin, but not by LY303511 (an inactive analogue of LY294002), and transcriptional inhibitor, actinomycin D, suggesting that insulin regulates the translation of PSD-95 by activating the receptor tyrosine kinase-PI3K-mammalian target of rapamycin (mTOR) signaling pathway. A similar insulin-induced increase in the PSD-95 protein expression was detected after stimulation of the synaptic fractions isolated from the hippocampal neurons. Furthermore, insulin treatment did not affect the PSD-95 mRNA levels. In agreement, insulin rapidly induced the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1), protein kinase B (Akt), and mTOR, effects that were prevented by the AG1024 and LY294002. We also show that insulin stimulated the phosphorylation of 4E-binding protein 1 (4E-BP1) and p70S6 kinase (p70S6K) in a mTOR-dependent manner. Finally, we demonstrate the constitutive expression of PSD-95 mRNA in the synaptic fractions isolated from hippocampal neurons. Taken together, these findings suggest that activation of the PI3K-Akt-mTOR signaling pathway is essential for the insulin-induced up-regulation of local PSD-95 protein synthesis in neuronal dendrites and indicate a new molecular mechanism that may contribute to the modulation of synaptic function by insulin in hippocampal area CA1. Insulin receptors are highly enriched at neuronal synapses, but whose function remains unclear. Here we present evidence that brief incubations of rat hippocampal slices with insulin resulted in an increased protein expression of dendritic scaffolding protein postsynaptic density-95 (PSD-95) in area CA1. This insulin-induced increase in the PSD-95 protein expression was inhibited by the tyrosine kinase inhibitor, AG1024, phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin, translational inhibitors, anisomycin and rapamycin, but not by LY303511 (an inactive analogue of LY294002), and transcriptional inhibitor, actinomycin D, suggesting that insulin regulates the translation of PSD-95 by activating the receptor tyrosine kinase-PI3K-mammalian target of rapamycin (mTOR) signaling pathway. A similar insulin-induced increase in the PSD-95 protein expression was detected after stimulation of the synaptic fractions isolated from the hippocampal neurons. Furthermore, insulin treatment did not affect the PSD-95 mRNA levels. In agreement, insulin rapidly induced the phosphorylation of 3-phosphoinositide-dependent protein kinase-1 (PDK1), protein kinase B (Akt), and mTOR, effects that were prevented by the AG1024 and LY294002. We also show that insulin stimulated the phosphorylation of 4E-binding protein 1 (4E-BP1) and p70S6 kinase (p70S6K) in a mTOR-dependent manner. Finally, we demonstrate the constitutive expression of PSD-95 mRNA in the synaptic fractions isolated from hippocampal neurons. Taken together, these findings suggest that activation of the PI3K-Akt-mTOR signaling pathway is essential for the insulin-induced up-regulation of local PSD-95 protein synthesis in neuronal dendrites and indicate a new molecular mechanism that may contribute to the modulation of synaptic function by insulin in hippocampal area CA1. Insulin and its receptor are widely dispersed throughout the brain with the highest density located in the olfactory bulb, cerebral cortex, hypothalamus, and hippocampus, where they are thought to subserve a number of functions including regulation of glucose metabolism, food intake and body weight, fertility and reproduction, learning, memory, and attention (1Hill J.M. Lesniak M.A. Pert C.B. Roth J. Neuroscience. 1986; 17: 1127-1138Crossref PubMed Scopus (323) Google Scholar, 2Wozniak M. Rydzewski B. Baker S.P. Raizada M.K. Neurochem. Int. 1993; 22: 1-10Crossref PubMed Scopus (194) Google Scholar, 3Wickelgren I. Science. 1998; 280: 517-519Crossref PubMed Scopus (151) Google Scholar, 4Stockhorst U. de Fries D. Steingrueber H.J. Scherbaum W.A. Physiol. Behav. 2004; 83: 47-54Crossref PubMed Scopus (157) Google Scholar). Brain insulin receptors are present in particularly high concentrations in neurons, and in much lower levels in glia (5Schwartz M.W. Figlewicz D.P. Baskin D.G. Woods S.C. Porte Jr., D. Endocr. Rev. 1992; 13: 387-414PubMed Google Scholar). Although the mRNA of insulin receptors is largely localized in neuronal somata, abundant insulin receptors are found in both cell bodies and synapses (5Schwartz M.W. Figlewicz D.P. Baskin D.G. Woods S.C. Porte Jr., D. Endocr. Rev. 1992; 13: 387-414PubMed Google Scholar, 6Marks J.L. Maddison J. Eastman C.J. J. Neurochem. 1998; 50: 774-781Crossref Scopus (22) Google Scholar, 7Abbott M.A. Wells D.G. Fallon J.R. J. Neurosci. 1999; 19: 7300-7308Crossref PubMed Google Scholar). However, very little is known about the functional significance of synaptic insulin receptors in the neurons. Recently, several studies have drawn links between insulin signaling and intracellular trafficking and plasma membrane expression of ion channels and neurotransmitter receptors at the central nervous system synapses. For example, it has been shown that insulin rapidly recruits functional GABAA receptors to postsynaptic domains in hippocampal neurons, resulting in a long-lasting enhancement of GABAA receptor-mediated synaptic transmission (8Wan Q. Xiong Z.G. Man H.Y. Ackerley C.A. Braunton J. Lu W.Y. Becker L.E. MacDonald J.F. Wang Y.T. Nature. 1997; 388: 686-690Crossref PubMed Scopus (454) Google Scholar). In addition, we and other investigators have provided evidence that insulin can promote the internalization of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors from the synaptic membrane of neurons, which causes a long-term depression of excitatory synaptic transmission in the hippocampus and cerebellum (9Man H.Y. Lin J.W. Ju W.H. Ahmadian G. Liu L. Becker L.E. Sheng M. Wang Y.T. Neuron. 2000; 25: 649-662Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 10Wang Y.T. Linden D.J. Neuron. 2000; 25: 635-647Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 11Huang C.C. You J.L. Lee C.C. Hsu K.S. Mol. Cell. Neurosci. 2003; 24: 831-841Crossref PubMed Scopus (33) Google Scholar, 12Huang C.C. Lee C.C. Hsu K.S. J. Neurochem. 2004; 89: 217-231Crossref PubMed Scopus (93) Google Scholar). Moreover, insulin enhances N-methyl-d-aspartate (NMDA) 1The abbreviations used are: NMDA, N-methyl-d-aspartate; PSD, postsynaptic density; PSD-95, protein postsynaptic density-95; aCSF, artificial cerebrospinal fluid; 4E-BP1, 4E-binding protein 1; eIF4E, eukaryotic initiator 4E; eIF4G, eukaryotic initiator 4G; p70S6K, p70S6 kinase; IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; LY303511, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one; AG1024, 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile; RT, reverse transcriptase. 1The abbreviations used are: NMDA, N-methyl-d-aspartate; PSD, postsynaptic density; PSD-95, protein postsynaptic density-95; aCSF, artificial cerebrospinal fluid; 4E-BP1, 4E-binding protein 1; eIF4E, eukaryotic initiator 4E; eIF4G, eukaryotic initiator 4G; p70S6K, p70S6 kinase; IGF-1, insulin-like growth factor-1; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; LY303511, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one; AG1024, 3-bromo-5-t-butyl-4-hydroxy-benzylidenemalonitrile; RT, reverse transcriptase. receptor-mediated synaptic transmission at the hippocampal CA1 synapses (13Liu L. Brown 3rd, J.C. Webster W.W. Morrisett R.A. Monaghan D.T. Neurosci. Lett. 1995; 192: 5-8Crossref PubMed Scopus (103) Google Scholar) and potentiates the activity of recombinant NMDA receptors expressed in Xenopus oocytes (14Liao G.Y. Leonard J.P. J. Neurochem. 1999; 73: 1510-1519Crossref PubMed Scopus (42) Google Scholar). Although these findings highlight the role of insulin signaling in modulating synaptic functions, it is not yet clear how exactly insulin contributes to these diverse actions at the molecular levels. The postsynaptic density (PSD) is a specialization of cytoskeleton at the synaptic junction and serves as an important organizer of the postsynaptic signal transduction machinery (15Ziff E.B. Neuron. 1997; 19: 1163-1174Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). The PSD forms a disc that consists of cytoskeletal and regulatory proteins, some of which contact the cytoplasmic domains of ion channels or neurotransmitter receptors in the postsynaptic membrane (16Ehlers M.D. Mammen A.L. Lau L.F. Huganir R.L. Curr. Opin. Cell Biol. 1996; 8: 484-489Crossref PubMed Scopus (90) Google Scholar). One of fundamental structural proteins within the PSD is PSD-95, a 95-kDa scaffolding protein containing multiple PSD-95/Discs large/zona occluens-1 domains to anchor and associate glutamate receptors with other functional proteins in the PSD (17Hering H. Sheng M. Nat. Rev. Neurosci. 2001; 2: 880-888Crossref PubMed Scopus (691) Google Scholar, 18Kim E. Sheng M. Nat. Rev. Neurosci. 2004; 5: 771-781Crossref PubMed Scopus (1194) Google Scholar). Although the function of the PSD-95 protein at the synapses is not yet clear, evidence from the PSD-95 mutant or expression studies has demonstrated that PSD-95 may play a modulatory role in control of the synaptic transmission (19El-Husseini A.E. Schnell E. Chetkovich D.M. Nicoll R.A. Bredt D.S. Science. 2000; 290: 1364-1368Crossref PubMed Google Scholar), bidirectional synaptic plasticity (20Migaud M. Charlesworth P. Dempster M. Webster L.C. Watabe A.M. Makhinson M. He Y. Ramsay M.F. Morris R.G. Morrison J.H. O'Dell T.J. Grant S.G. Nature. 1998; 396: 433-439Crossref PubMed Scopus (948) Google Scholar, 21Ehrlich I. Malinow R. J. Neurosci. 2004; 24: 916-927Crossref PubMed Scopus (413) Google Scholar), maturation of excitatory synapses (19El-Husseini A.E. Schnell E. Chetkovich D.M. Nicoll R.A. Bredt D.S. Science. 2000; 290: 1364-1368Crossref PubMed Google Scholar, 22Stein V. House D.R. Bredt D.S. Nicoll R.A. J. Neurosci. 2003; 23: 5503-5506Crossref PubMed Google Scholar), and drug addition (23Yao W.D. Gainetdinov R.R. Arbuckle M.I. Sotnikova T.D. Cyr M. Beaulieu J.M. Torres G.E. Grant S.G. Caron M.G. Neuron. 2004; 41: 625-638Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). A pressing question that follows from these observations is whether and how PSD-95 protein can be elevated at synaptic sites under physiological conditions to modulate the synaptic function. A recent study has demonstrated that estrogen rapidly stimulates dendritic PSD-95 protein synthesis in NG108-15 neuroblastoma cells (24Akama K.T. McEwen B.S. J. Neurosci. 2003; 23: 2333-2339Crossref PubMed Google Scholar). Given that insulin receptor and its substrate are components of PSD fractions and are concentrated at the synapses in the hippocampal neurons, it is of particular interest to examine the relationship between insulin receptor activation and PSD-95 protein synthesis. Our results reveal the first evidence that brief insulin treatment substantially increases the synthesis of PSD-95 protein in local dendritic compartments via the activation of PI3K-Akt-mTOR signaling pathway. This event may provide a potentially important way to modulate synaptic transmission and plasticity in hippocampal area CA1. Hippocampal Slice Preparations—All experiments were performed according to the guidelines laid down by the Institutional Animal Care and Use Committee of National Cheng Kung University. Hippocampal slices (400 μm thick) were obtained from 28- to 35-day-old young male Sprague-Dawley rats by the procedures described previously (25Huang C.C. Liang Y.C. Hsu K.S. J. Neurosci. 1999; 19: 9728-9738Crossref PubMed Google Scholar). In brief, animals were killed by decapitation under halothane anesthesia, and the hippocampi were removed, placed in ice-cold artificial CSF (aCSF) solution and cut with a Leica VT1000S tissue slicer (Leica, Nussloch, Germany) in 400-μm thick transverse slices. After preparation, slices were placed in a holding chamber of aCSF oxygenated with 95% O2, 5% CO2 and kept at room temperature for at least 1 h before experiments. The composition of the aCSF solution was 117 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgCl2, 25 mm NaHCO3, 1.2 mm NaH2PO4, and 11 mm glucose 11 at pH 7.3-7.4, and equilibrated with 95% O2, 5% CO2. Slices then were exposed to different compounds of interest for the indicated times and snap frozen over dry ice. Preparation of Synaptoneurosomes—Synaptoneurosome fractions were prepared as described previously (26Wells D.G. Dong X. Quinlan E.M. Huang Y.S. Bear M.F. Richter J.D. Fallon J.R. J. Neurosci. 2001; 21: 9541-9548Crossref PubMed Google Scholar). Briefly, the microdissected CA1 regions were homogenized in ice-cold Ca2+, Mg2+-free buffer (50 mm HEPES, 100 mm NaCl, and 3 mm KAc, pH 7.4) with RNase inhibitor (15 units/ml) and centrifuged at 2000 × g for 1 min. Supernatants were passed through two 100-μm nylon mesh filters, followed by a 5-μm pore filter. The filtrate was then centrifuged at 1000 × g for 10 min and then was gently resuspended with same buffer at a protein concentration of 2 mg/ml. Western Blotting—For each experimental group, homogenates from at least three slices were pooled. The microdissected CA1 regions were lysed in ice-cold Tris-HCl buffer solution (pH 7.4) containing a mixture of protein phosphatase and proteinase inhibitors (50 mm Tris-HCl, 100 mm NaCl, 15 mm sodium pyrophosphate, 50 mm sodium fluoride, 1 mm sodium orthovanadate, 5 mm EGTA, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μm microcystin-LR, 1 μm okadaic acid, 0.5% Triton X-100, 2 mm benzamidine, 60 μg/ml aprotinin, and 60 μg/ml leupeptin) to avoid dephosphorylation and degradation of proteins, and ground with a pellet pestle (Kontes Glassware, Vineland, NJ). Samples were sonicated and spun down at 15,000 × g at 4 °C for 10 min. The supernatant was then assayed for total protein concentration using the Bio-Rad Bradford Protein Assay Kit. Each sample was separated in 10% SDS-PAGE gels. Following the transfer on nitrocellulose membranes, blots were blocked in Tris-HCl buffer solution containing 3% bovine serum albumin and 0.01% Tween 20 for 1 h and then blotted for 2 h at room temperature with antibodies that recognize PSD-95 (1:1000; Upstate Biotechnology, Lake Placid, NY), phosphorylated PDK1 (1: 1000; Cell Signaling Technologies, Beverly, MA), phosphorylated Akt (1:1000; Cell Signaling Technologies), phosphorylated mTOR (1:1000; Cell Signaling Technologies), phosphorylated p70S6K (1:1000; Cell Signaling Technologies), or phosphorylated 4E-BP1 (1:1000; Cell Signaling Technologies). It was then probed with horseradish peroxidase-conjugated secondary antibody for 1 h and developed using the ECL immunoblotting detection system. The immunoblots using phosphorylation site-specific antibodies were subsequently stripped and reprobed with the following antibodies: anti-PDK1 antibody (1:1000), anti-Akt antibody (1:1000), anti-mTOR (1:1000), anti-p70S6K, or anti-4E-BP1 antibody (1:1000) that were purchased from Cell Signaling Technologies. Immunoblots were analyzed by densitometry. Distribution of PSD-95 mRNA in Synaptoneurosome—To determine relative distribution of PSD-95 mRNA between crude homogenate and synaptoneurosome, we used RT-PCR analysis. Total RNA was isolated from the microdissected hippocampal CA1 region or synaptoneurosome using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. We used 1-2 μg of total RNA in reverse transcription reactions performed using a commercially available cDNA synthesis kit (Invitrogen). RT-PCR was performed as described previously. Crude homogenate samples were amplified for 30 cycles, and synaptoneurosome samples were amplified for 45 cycles. Each cycle consisted of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, and extension at 72 °C for 30 s. After amplification, equal volumes of crude hippocampal CA1 region and synaptoneurosome PCR products were subjected to electrophoresis on a 1% (w/v) agarose gel and visualized with ethidium bromide. The integrated density value obtained from the synaptoneurosome sample was divided by those obtained from the crude hippocampal CA1 region sample to obtain a ratio indicating the relative distribution in the synaptoneurosome for each particular mRNA species. The primers used in this analysis are: PSD-95, 5′-CGAGGATGCCGTGGCAGCC-3′ (forward) and 5′-CATGGCTGTGGGGTAGTCAGTGCC-3′ (reverse); histone H1, 5′-GGTGGCTTTCAAGAAGACCAA-3′ (forward) and 5′-TGAGGTCTGTTTGCTGTCCTT-3′ (reverse); and α-CAMKII, 5′-TTCAGGGGCAATAGCAAGCAACAC-3′ (forward) and 5′-ATGGGGAGGGAGAGCACGAAGATT-3′ (reverse). Real-time PCR—Real-time PCR was performed on the Roche Light-Cycler instrument using the DNA Master SYBR Green 1 (Roche Norge AS). Each SYBR Green reaction (20 μl in volume) contained 2 ng of cDNA template, 0.5 μm of the PCR primers, and additional MgCl2 (final concentration of 4 mm). Each reaction mixture was cycled 30-40 times under the following parameters: 95 °C for 5 s; 62 °C for 5 s; 72 °C for 26 s; and 83 °C for 1 s. The acquired fluorescence signal was quantified by the fit-point method using LightCycler Data Analysis software according to the manufacturer's instructions. Changes in concentration of the amplified target were detected as differences in threshold cycle between samples. Drug Application—All drugs were applied by dissolving them to the desired final concentrations in the aCSF. Appropriate stock solutions of drugs were made and diluted with aCSF just before application. AG1024, LY294002, LY303511, wortmannin, anisomycin, actinomycin D, and rapamycin were dissolved in dimethyl sulfoxide (Me2SO) stock solution and stored at -20 °C until the day of the experiment. The concentration of Me2SO in the aCSF was 0.05%, which alone had no effect on the basal insulin signaling cascade or PSD-95 level (data not shown). Insulin and IGF-1 were purchased from Sigma, LY294002, wortmannin, anisomycin, and actinomycin D were obtained from Tocris Cookson Ltd. (Bristol, UK), and AG1024, LY303511, and monoclonal antibody for insulin-like growth factor-1 (IGF-1) receptor were purchased from Calbiochem (La Jolla, CA). Data Analysis—All of the values reported are mean ± S.E. The significance of the difference between the mean was calculated by Student's t test. Probability values of p < 0.05 were considered to represent significant differences. Activation of Insulin Receptors Increases the PSD-95 Protein Expression in Hippocampal Area CA1—The initial set of experiments was designed to investigate whether the activation of insulin receptors regulates the PSD-95 protein expression. Incubation of hippocampal slices with insulin (0.1-3 μm) for 10 min resulted in dose-dependent increases in levels of PSD-95 protein as measured on Western blotting of homogenates from area CA1 (Fig. 1A). At a concentration of 0.5 μm, the level of PSD-95 protein was increased by 46.3 ± 5.4% of control 10 min after washout of insulin. Because 0.5 μm insulin could consistently increase PSD-95 protein expression, we chose this treatment protocol to identify the underlying mechanisms of this event in all subsequent experiments. In a time course analysis, PSD-95 protein levels remained elevated for at least 30 after washout of insulin (Fig. 1B). In addition, the effect of insulin on PSD-95 protein expression appears to be specific, because the levels of another PDZ-containing scaffold protein SAP97 were not altered after insulin treatment (Fig. 1C). IGF-1 receptors are structurally very similar to insulin receptors and insulin binds to both insulin and IGF-1 receptors, although the former is bound with about 40-fold greater affinity (27Rechler M.M. Nissley S.P. Annu. Rev. Physiol. 1985; 47: 425-442Crossref PubMed Google Scholar, 28Heidenreich K.A. Ann. N. Y. Acad. Sci. 1993; 692: 72-88Crossref PubMed Scopus (55) Google Scholar). Further experiments were performed to determine whether the increases in PSD-95 protein by insulin were mediated by the activation of IGF-1 receptors. To directly test this possibility, hippocampal slices were pretreated with a monoclonal antibody directed against the IGF-1 receptor (5 μg/ml), which has been shown to block IGF-1 binding and its biological activity (10Wang Y.T. Linden D.J. Neuron. 2000; 25: 635-647Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 12Huang C.C. Lee C.C. Hsu K.S. J. Neurochem. 2004; 89: 217-231Crossref PubMed Scopus (93) Google Scholar, 29Kull Jr., F.C. Jacobs S. Su Y.F. Svoboda M.E. Van Wyk J.J. Cuatrecasas P. J. Biol. Chem. 1993; 258: 6561-6566Abstract Full Text PDF Google Scholar). This treatment had no effect on the insulin-induced increases in PSD-95 protein expression (Fig. 1D). The lack of effect of anti-IGF-1 receptor antibody on the effect of insulin is not because of the inefficacy of our treatment protocol to block IGF-1 receptors, because the same treatment protocol completely blocked the increases in PSD-95 protein expression induced by IGF-1 (50 ng/ml) (Fig. 1D). To further determine whether this event specifically required insulin receptor activation, we used AG1024, a tyrosine kinase inhibitor that shows some specificity toward the insulin receptors (30Parrizas M. Gazit A. Levitzki A. Wertheimer E. LeRoith D. Endocrinology. 1997; 138: 1427-1433Crossref PubMed Scopus (0) Google Scholar), to find out whether or not the action of insulin on PSD-95 protein expression was mediated through the insulin receptors. As shown in Fig. 2A, AG1024 (10 μm) successfully prevented the insulin-induced increases in PSD-95 protein expression. These results indicate that activation of insulin receptor signaling serves to regulate the expression of PSD-95 protein in the hippocampal area CA1. Insulin-induced Increases in New PSD-95 Protein Expression Are PI3K-Akt-mTOR Dependent—The activation of insulin receptors by insulin elicits a large cascade of signal transduction events. To define the specific signaling pathway that contributes to the insulin-induced increases in the PSD-95 protein expression, the requirement for PI3K activation was first examined. Pretreatment of the hippocampal slices with the specific PI3K inhibitor, LY294002 (20 μm) (31Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar), completely blocked insulin-induced increases in the PSD-95 protein expression (Fig. 2A). Similar results were also observed with the use of another structurally unrelated PI3K inhibitor, wortmannin (5 μm) (data not shown). In contrast, the inactive analogue of LY294002, LY303511 (20 μm), did not affect the action of insulin (Fig. 2A). None of LY294002, wortmannin, or LY303511 alone altered the basal levels of PSD-95 protein. These results support that the activation of PI3K is required for the insulin-induced increases in PSD-95 protein expression in hippocampal area CA1. To determine whether the enhanced PSD-95 protein expression by insulin was because of an increase in the new protein synthesis, we pretreated the hippocampal slices with the protein synthesis inhibitor, anisomycin (20 μm), 60 min before insulin stimulation. As shown in Fig. 2B, anisomycin pretreatment completely blocked the insulin-induced increases in PSD-95 protein expression. To evaluate the possible contribution of mRNA synthesis to this event, we examined the effect of the transcription inhibitor, actinomycin D (25 μm), on the action of insulin. In contrast to the translation inhibitor, actinomycin D pretreatment for 60 min had no effect on the increases in PSD-95 protein level by insulin (Fig. 2B). We also examined whether insulin affected the levels of PSD-95 mRNA determined by quantitative real-time RT-PCR, but we observed no effect (Fig. 3), a result consistent with the suggestion that the increases in PSD-95 protein induced by insulin were derived from an activation of mRNA translation, and furthermore, that this mRNA was already present at the time of the stimulation. The mTOR signaling pathway has been shown to play a crucial role in regulating several components of the translational machinery in mammalian cells (32Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar), and insulin can stimulate this pathway to modulate the activity of several translation regulatory factors (33Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3377) Google Scholar). We next asked if the increases in PSD-95 protein expression by insulin occur through a mTOR-coupled mechanism, so the mTOR inhibitor, rapamycin, was employed. As shown in Fig. 2B, pretreatment of the hippocampal slices with rapamycin (200 nm) for 60 min completely blocked insulin-induced increases in PSD-95 protein expression. Rapamycin alone failed to alter the basal levels of PSD-95 protein. We conclude therefore that insulin stimulates the new PSD-95 protein synthesis in a rapamycin-sensitive and PI3K-dependent manner. To further ascertain the requirement for the activation of the PI3K-Akt-mTOR signaling pathway in insulin-induced increases in PSD-95 protein expression, we used phospho-specific antibodies to measure the relative levels of phosphorylated, active forms of PDK1, Akt, and mTOR after hippocampal slices were treated with insulin. For PDK1, we used an antibody specific for phosphorylated serine 241, which is on the activation loop of PDK1 and is essential for kinase activity (34Casamayor A. Morrice N.A. Alessi D.R. Biochem. J. 1999; 342: 287-292Crossref PubMed Scopus (285) Google Scholar). For Akt, we used an antibody specific for phosphorylated serine 473, which is necessary for maximal activation of Akt (35Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2476) Google Scholar). For mTOR, we used an antibody specific for phosphorylated serine 2448, which has been shown to be important in the control of mTOR activity (36Brunn G.J. Fadden P. Haystead T.A. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 32547-32550Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 37Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (404) Google Scholar). As expected, stimulation of the hippocampal slices with 0.5 μm insulin for 10 min caused a marked increase in the phosphorylation of PDK1, Akt, and mTOR as measured on Western blots of homogenates from the CA1 area (Fig. 4). In addition, the insulin-induced increase in the phosphorylation of PDK1, Akt, and mTOR was transient, returning to control levels within 20-30 min after washout of insulin. It is well documented that PDK1, Akt, and mTOR are downstream effectors of PI3K. Therefore, we determined whether LY294002 could block the insulin-induced increases in the phosphorylation of PDK1, Akt, and mTOR. We found that LY294002 (20 μm) but not LY303511 (20 μm) completely blocked the increases in PDK1, Akt, and mTOR phosphorylation induced by insulin (Fig. 5). Neither LY294002 nor LY303511 alone altered the basal levels of PDK1, Akt, and mTOR phosphorylation. Similarly, tyrosine kinase inhibitor AG1024 (10 μm) pretreatment also completely blocked the insulin-induced increases in the phosphorylation of PDK1, Akt, and mTOR (Fig. 5). Together, these results indicate that PI3K acted upstream of PDK1, Akt, and mTOR to initiate the insulin receptor signaling in hippocampal area CA1. Insulin Stimulates the Phosphorylation of 4E-BP1 and p70S6K—Because activation of mTOR can contribute to translational initiation by phosphorylation of 4E-binding protein 1 (4E-BP1) that binds to and represses the function of the cap-binding translation factor eIF4E, we asked if this regulatory protein may have a role in insulin-induced increases in PSD-95 protein synthesis. We used an antibody specific for phosphorylated threonine 70, a site whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eIF4E complex (38Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1162) Google Scholar). Incubation of the hippocampal slices with 0.5 μm insulin for 10 min increased phosphorylation of 4E-BP1 (Fig. 6). Furthermore, the insulin-induced increases in the phosphorylation of 4E-BP1 were completely blocked by AG1024, LY294002, and rapamycin pretreatment, respectively. LY303511 failed to alter the increases in 4E-BP1 phosphorylation induced by insulin (data not shown). mTOR activation may also regulate translation by direct or indirect phosphorylation of other translation-related protein factors such as p70S6K (38Gingras A.C. Raught B. Gygi S.P. Niedzwiecka A. Miron M. Burley S.K. Polakiewicz R.D. Wyslouch-Cieszynska A. Aebersold R. Sonenberg N. Genes Dev. 2001; 15: 2852-2864Crossref PubMed Scopus (1162) Google Scholar). We then asked if the increases in PSD-95 protein expression by insulin are p70S6K-dependent, so the phosphorylation state of p70S6K after insulin treatme" @default.
• W2061738346 created "2016-06-24" @default.
• W2061738346 creator A5018506745 @default.
• W2061738346 creator A5031754749 @default.
• W2061738346 creator A5052365004 @default.
• W2061738346 creator A5076906135 @default.
• W2061738346 date "2005-05-01" @default.
• W2061738346 modified "2023-10-08" @default.
• W2061738346 title "Insulin Stimulates Postsynaptic Density-95 Protein Translation via the Phosphoinositide 3-Kinase-Akt-Mammalian Target of Rapamycin Signaling Pathway" @default.
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