Insulin-stimulated translocation of GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent - PubMed (original) (raw)

Insulin-stimulated translocation of GLUT4 to the plasma membrane in rat hippocampus is PI3-kinase dependent

C A Grillo et al. Brain Res. 2009.

Abstract

In the central nervous system (CNS) insulin mediates a variety of effects including feeding, metabolism and cognition. The cognitive enhancing effects of insulin are proposed to be mediated through activation of insulin receptors in the hippocampus, an important integration center for learning and memory in the mammalian brain. Since less is known regarding insulin signaling events in the hippocampus, the aim of the current study was to determine whether insulin stimulates similar signaling cascades and GLUT4 translocation in the rat hippocampus as has been described in peripheral tissues. Intracerebroventricular administration of insulin increases hippocampal insulin levels and also stimulates the phosphorylation of Akt in a time-dependent manner. Insulin also stimulates the translocation of GLUT4 to hippocampal plasma membranes in a time course that mirrors the increases in glucose uptake observed during the performance of hippocampal-dependent tasks. Insulin stimulated phosphorylation of Akt and translocation of GLUT4 were blocked by pretreatment with the PI3-kinase inhibitor LY294002. Confocal immunofluorescence determined that insulin stimulated phosphorylation of Akt was localized to neurons and colocalized with the insulin receptor and GLUT4 in the rat hippocampus, thereby identifying the functional anatomical substrates of insulin signaling in the hippocampus. These results demonstrate that insulin-stimulated translocation of GLUT4 to the plasma membrane in the rat hippocampus occurs via similar mechanisms as described in peripheral tissues and suggests that insulin-mediated translocation of GLUT4 may provide a mechanism through which hippocampal neurons rapidly increase glucose utilization during increases in neuronal activity associated with hippocampal-dependent learning.

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Figures

Figure 1

Figure 1

Intracerebroventricular insulin administration increases phosphorylation of Akt (pAkt) in the rat hippocampus in a time-dependent manner. Panel A. Representative immunoblot sof time-dependent increases in pAkt levels in total membrane extracts of rat hippocampus (top panel); icv insulin did not modulate total Akt levels in rat hippocampus (bottom panel). The zero time point rats were injected with saline. Panel B. Autoradiographic image analysis revealed that insulin significantly increased pAkt levels 30 minutes following icv injection, increases that were also observed at the 45 and 60 minute time points. Molecular weight standards are shown on the right. Data in Panel B are expressed as the pAkt/total Akt ratio and are based upon at least 6 rats/group. * = p ≤ 0.005 compared to saline control rats (zero time point); $ = p ≤ 0.02 compared to 45 minute time point.

Figure 2

Figure 2

Intracerebroventricular insulin administration increases plasma membrane association of GLUT4 in the rat hippocampus. Panel A. Representative immunoblots of GLUT4 immunoreactive bands in hippocampal plasma membrane fractions (PM; top panel) and total membrane fractions (T; bottom panel) 15, 30, 45 and 60 minutes following icv insulin treatment; zero time point rats were injected with saline. Panel B. Autoradiographic image analysis revealed that insulin increased plasma membrane association of GLUT4 in the rat hippocampus at the 15 and 30 minute post-insulin time points, increases that returned to baseline at the 45 minute time point. Molecular weight standards are shown on the left. Data in Panel B are expressed as the plasma membrane GLUT4/total membrane GLUT4 ratio and are based upon at least 6 rats/group. * = p ≤ 0.001 compared to saline control rats; $ = p ≤ 0.001 compared to 45 minute insulin group; # = p ≤ 0.001 compared to 60 minute insulin group.

Figure 3

Figure 3

Insulin receptor expression is unaffected by intracerebroventricular insulin administration. Panel A. Representative immunoblot of insulin receptor expression in hippocampal plasma membrane fractions isolated from icv-treated rats. Panel B. Autoradiographic analysis of hippocampal plasma membrane fractions of icv-treated rats revealed that insulin receptor expression is not modulated by insulin administration. Panel B. Pre-absorption of primary antisera with the peptide immunogen eliminated the 90 kDa band detected in immunoblot analysis. Con lanes indicate control conditions (i.e. primary antisera in the absence of blocking peptide); + indicates primary antisera pre-absorbed with blocking peptide. Data are expressed as percentage of saline-treated rats. Molecular weight standards are shown on the left.

Figure 4

Figure 4

Insulin mediated increases in pAkt in the rat hippocampus are inhibited by the PI3-kinase inhibitor LY294002. Panel A. Representative immunoblot of pAkt levels in total hippocampal membrane extracts isolated from saline-saline rats (Sal-Sal, open bars), saline-insulin rats (Sal-Ins, black bars) and LY294002-insulin rats (LY-Ins, grey bars). Panel B. Autoradiographic image analysis revealed that icv insulin significantly increased pAkt levels in rat hippocampus, increases that were attenuated by pre-treatment with the PI3-kinase inhibitor. Molecular weight standards are shown on the right. Data in Panel B are expressed as the pAkt/total Akt ratio and are based upon at least 6 rats/group. * = p ≤ 0.01 compared to Sal-Sal control rats; $ = p ≤ 0.01 compared to LY-Ins group.

Figure 5

Figure 5

LY294002 inhibits insulin stimulated GLUT4 translocation in the rat hippocampus. Panel A. Representative immunoblots of hippocampal GLUT4 in plasma membrane fractions (PM; top panel) and total membrane fractions (T; bottom panel) in saline-saline rats (Sal-Sal, open bars), saline-insulin rats (Sal-Ins, black bars) and LY294002-insulin rats (LY-Ins, grey bars). Panel B. Autoradiographic analysis revealed that insulin-stimulated translocation of GLUT4 to the plasma membrane was inhibited by the PI3-kinase inhibitor LY294002. Molecular weight standards are shown on the right. Data in Panel B are expressed as the plasma membrane GLUT4/total membrane GLUT4 ratio and are based upon at least 6 rats/group. * = p ≤ 0.05 compared to Sal-Sal control rats; $ = p ≤ 0.05 compared to LY-Ins group.

Figure 6

Figure 6

Radioimmunoassay (RIA) of insulin content in hippocampus and cortex following icv saline or insulin treatment. Tissue extracts were prepared for insulin RIA from saline-saline rats (Sal-Sal, open bars), saline-insulin rats (Sal-Ins, black bars) and LY294002-insulin rats (LY-Ins, grey bars). Hippocampal insulin levels were significantly increased in Sal-Ins rats and LY-Ins rats compared to control (Sal-Sal) rats. Conversely, insulin levels in rat cortex were unaffected by icv administration. Data based upon at least 6 rats per group. * = p ≤ 0.01 compared to saline-saline controls.

Figure 7

Figure 7

In vitro insulin stimulation of hippocampal slices increases plasma membrane association of GLUT4. Hippocampal slices were prepared and treated with artificial cerebrospinal fluid (Veh-Veh), artificial cerebrospinal fluid plus insulin (Veh-Ins) or artificial cerebrospinal fluid plus LY294002 prior to insulin treatment (LY-Ins). Slices were then used to isolate plasma membrane fractions. Insulin treatment increased hippocampal plasma membrane association of GLUT4, an effect that was blocked by the PI3-kinase inhibitor LY294002.

Figure 8

Figure 8

Confocal immunofluorescence for pAkt, NeuN and GFAP in the hippocampus of insulin-treated rats demonstrates that insulin stimulated phosphorylation of Akt is localized to neurons in the rat hippocampus. Panels A and D illustrate insulin stimulated pAkt (red fluorescence) in the CA3 region of the rat hippocampus. Panel B illustrates immunofluorescence for the neuron-specific marker NeuN (green fluorescence). Panel E depicts GFAP immunofluorescence in the CA3 region of the rat hippocampus (green fluorescence). Merged images demonstrates that pAkt is colocalized with NeuN (Panel C), while GFAP immunofluorescence exhibits little colocalization with pAkt (Panel F).

Figure 9

Figure 9

LY294002 inhibits insulin stimulated colocalization of pAkt with the insulin receptor in the rat hippocampus. The insulin receptor (IR; green immunofluorescence) exhibits the expected immunofluorescence profile in the CA3 region of the rat hippocampus in saline-treated rats (Panel A), insulin-treated rats (Panel D) and rats pre-treated with the PI3-kinase inhibitor LY294002 prior to insulin treatment (Panel G). Insulin treatment increases pAkt immunofluorescence in the CA3 region of the hippocampus (Panel E; red fluorescence), increases not observed in rats pre-treated with LY294002 (Panel H). The phosphorylated form of Akt was not detected in saline-treated rats (Panel B). The merged images illustrate that pAkt exhibits colocalization with the IR in the hippocampus of insulin-treated rats (Panel F).

Figure 10

Figure 10

Insulin stimulated phosphorylation of Akt is colocalized with GLUT4 in the rat hippocampus. GLUT4 (green immunofluorescence) exhibits the expected immunofluorescence distribution in the CA3 region of the rat hippocampus in saline-treated rats (Panel A), insulin-treated rats (Panel D) and rats pre-treated with the PI3-kinase inhibitor LY294002 prior to insulin treatment (Panel G). Insulin treatment increases pAkt immunofluorescence in the CA3 region of the hippocampus (Panel E; red fluorescence), increases not observed in rats pre-treated with LY294002 (Panel H). The phosphorylated form of Akt is not detected in saline-treated rats (Panel B). The merged images illustrate that pAkt exhibits colocalization with GLUT4 in the hippocampus of insulin-treated rats (Panel F).

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