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. 2009 Mar 4;29(9):2997-3008.
doi: 10.1523/JNEUROSCI.0354-09.2009.

Regulation of Glucose Transporter 3 Surface Expression by the AMP-activated Protein Kinase Mediates Tolerance to Glutamate Excitation in Neurons

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Free PMC article

Regulation of Glucose Transporter 3 Surface Expression by the AMP-activated Protein Kinase Mediates Tolerance to Glutamate Excitation in Neurons

Petronela Weisová et al. J Neurosci. .
Free PMC article

Abstract

Ischemic and excitotoxic events within the brain result in rapid and often unfavorable depletions in neuronal energy levels. Here, we investigated the signaling pathways activated in response to the energetic stress created by transient glutamate excitation in cerebellar granule neurons. We characterized a glucose dependent hyperpolarization of the mitochondrial membrane potential (Delta psi(m)) in the majority of neurons after transient glutamate excitation. Expression levels of the primary neuronal glucose transporters (GLUTs) isoforms 1, 3, 4, and 8 were found to be unaltered within a 24 h period after excitation. However, a significant increase only in GLUT3 surface expression was identified 30 min after excitation, with this high surface expression remaining significantly above control levels in many neurons for up to 4 h. Glutamate excitation induced a rapid alteration in the AMP:ATP ratio that was associated with the activation of the AMP-activated protein kinase (AMPK). Interestingly, pharmacological activation of AMPK with AICAR (5-aminoimidazole-4-carboxamide riboside) alone also increased GLUT3 surface expression, with a hyperpolarization of Delta psi(m) evident in many neurons. Notably, inhibition of the CaMKK (calmodulin-dependent protein kinase kinase) had little affect on GLUT translocation, whereas the inhibition or knockdown of AMPK (compound C, siRNA) activity prevented GLUT3 translocation to the cell surface after glutamate excitation. Furthermore, gene silencing of GLUT3 eradicated the increase in Delta psi(m) associated with transient glutamate excitation and potently sensitized neurons to excitotoxicity. In summary, our data suggest that the activation of AMPK and its regulation of cell surface GLUT3 expression is critical in mediating neuronal tolerance to excitotoxicity.

Figures

Figure 1.
Figure 1.
Transient glutamate excitation induces an early hyperpolarization of Δψm that is not a function of increased mitochondrial biogenesis. CGNs were stimulated with glutamate (glutamate/glycine 100 μm/10 μm) for 10 min. A, Nuclei were stained with Hoechst at the indicated time points (0, 4, 16, and 24 h) with uniformly stained nuclei counted as healthy (viable neurons) and condensed nuclei counted as apoptotic (n = 4 experiments in triplicate, *p < 0.01, difference between sham and 4, 8, and 24 h) B, CGNs preloaded with TMRM and Fluo-4 AM were stimulated with glutamate. Traces are representative of the responses in neurons at postnatal day 15 from 10 separate experiments. C, Average TMRM fluorescence in neurons after glutamate excitation are represented as mean ± SEM. Fluorescent images for representative chosen at selected time points (0 and 120 min) after glutamate excitation. *p < 0.001, difference between sham (n = 63) and glutamate-treated neurons (n = 119). D, After glutamate excitation, the expression levels of COX IV were examined by Western blotting. α-Tubulin served as a loading control. Similar responses were observed in samples from three separate cultures. E, CGNs were stimulated with glutamate, loaded with 100 nm MitoTracker Green FM, and mitochondrial mass analyzed using FACs. Data are presented as mean ± SEM. No significant increase was identified. F, CGNs were stimulated with glutamate, and the mRNA expression of the transcription factor tfam and cofactor pgc-1α was analyzed by real-time qPCR. Expression levels were normalized to control cells and data are represented as mean ± SEM from three independent experiments, 2, 4, and 24 h). *, #p < 0.05, difference between sham and glutamate treated neurons at 4 h (n = 3 experiments in triplicate).
Figure 2.
Figure 2.
Reduced glucose availability attenuates the increase in Δψm after transient glutamate excitation and increases neuronal sensitivity to excitation. CGNs were stimulated with glutamate/glycine (100 μm/10 μm 10 min) in the presence of normal (15 mm) glucose or low (1 mm) extracellular glucose. A, Representative traces for whole-cell TMRM fluorescence in neurons during transient glutamate excitation in 1 mm (n = 26) or 15 mm (n = 31) extracellular glucose concentration. B, Quantification of the TMRM fluorescence in sham-treated neurons (n = 39) and glutamate-treated neurons with 15 mm (n = 26) or 1 mm glucose (n = 31). Data are presented as mean ± SEM. *p < 0.001, difference between sham-treated and glutamate-treated neurons in 15 mm glucose. C, Neurons were stimulated with glutamate and left to recover in media with different glucose concentrations (1 and 15 mm) for 24 h. Nuclei were stained with Hoechst, with uniformly stained nuclei counted as healthy/viable neurons and condensed nuclei scored as apoptotic. Data were presented as mean ± SEM. *p < 0.01, difference between sham- and glutamate-stimulated neurons in 15 mm glucose. #p < 0.01, difference between glutamate-stimulated neurons recovered in 15 mm glucose and those recovered in 1 mm glucose (n = 3 in triplicate).
Figure 3.
Figure 3.
Transient glutamate excitation did not induce a significant change in the expression levels of GLUT1, 3, 4, and 8 over 24 h. A, Representative gel of RT-PCR for semiquantitative assessment of different glucose transporter isoforms localized in the cerebellum. ND5 was used as a control for RNA loading and RT-PCR efficiency. B, CGNs were stimulated with glutamate/glycine (100 μm/10 μm) for 10 min and allowed to recover for indicated time periods. After treatment, mRNA expression of the predominant neuronal glucose transporters (glut1, 3, 4, and 8) were determined by real-time qPCR analysis. Expression levels were normalized to sham-treated neurons and data are represented as SEM from three separate experiments. Data were presented as mean ± SEM. ns, No significance. C, Western blot analysis of GLUT1, 3, 4, and 8 at indicated times of recovery after transient glutamate excitation. α-Tubulin served as a loading control. Similar results were observed in two additional experiments.
Figure 4.
Figure 4.
Transient glutamate excitation induces a rapid translocation of GLUT3 to the plasma membrane. A, Immunofluorescence of the cell surface expression of GLUT3 in sham- and glutamate- (glutamate and glycine 100 μm/10 μm, 10 min) treated neurons after 0.5, 1, 4, and 24 h. Nuclei were stained with DAPI (blue) and an Alexa Fluor 488-labeled secondary antibody (green) was used to visualize GLUT3 expression. B, Single-cell evaluation of cell surface GLUT3 expression in sham (1 h) and glutamate-treated neurons over a 24 h period. Data are presented as mean ± SEM. *p < 0.01, difference between sham- (n = 38) and glutamate-excited neurons after 0.5 (n = 43), 1 (n = 34), 4 h (n = 41), and 24 h (n = 39). C, Quantification of relative fluorescence intensity [arbitrary (Arb) units] by flow cytometry for GLUT3 surface expression. Cells were gated according to size. A right shift (gray–green) in distribution indicates an increase in GLUT3 cell surface fluorescence after stimulation with glutamate. Isotype control in blue. D, Population analysis (flow cytometry analysis) of GLUT3 surface expression for sham- and glutamate-stimulated neurons at the indicated time points. Data are presented as mean ± SEM. *p < 0.001, difference between sham-treated neurons and neurons 0.5 and 1 h after glutamate excitation. Experiments were performed in triplicate from three separate cultures. E, Flow cytometry analysis of the plasma membrane expression of other predominant neuronal glucose transporters (GLUT1, 4, and 8) for sham-treated and glutamate-stimulated neurons after 1 h. Results were obtained from three separate cultures and data are presented as mean ± SEM. For flow cytometry experiments a minimum of 104 events were collected per sample.
Figure 5.
Figure 5.
Increased AMPK activity regulates the translocation of GLUT3 to the cell surface in response to both glutamate excitation and activation with AICAR. CGNs were exposed to glutamate/glycine (100 μm/10 μm) for 10 min or continuously with 2.5 mm AICAR. A, Western blot analysis of pAMPK (Thr172) and total AMPK at the times indicated after glutamate excitation. Actin was used as an additional loading control. The observed responses are similar to that obtained in four separate experiments. B, CGNs plated in 24-well plates were exposed to glutamate/glycine for 10 min and their ATP content measured (μmol ATP/mg protein) at the times indicated. (n = 3 experiments in triplicate; *p < 0.01, difference from sham-treated neurons). C, AMP and ATP levels were determined by HPLC for sham and glutamate (10 min glutamate/glycine 100 μm/10 μm and recovered for 5 min) treated neurons. Peak area is expressed as a % of initial sham values. *p < 0.01, difference between ATP levels in sham- and glutamate-treated neurons. #p < 0.001, difference between AMP levels in sham- and glutamate-treated neurons (lysates analyzed from 3 separate experiments). D, Western blot analysis of pAMPK (Thr172) and total AMPK expression in CGNs after incubation with AICAR (2.5 mm). Actin was used as an additional loading control. The observed responses are similar to that obtained in three separate experiments. E, Immunofluorescence of the cell surface expression of GLUT3 in vehicle treated and neurons 1 h after incubation with AICAR (2.5 mm). F, Flow cytometry analysis of GLUT3 surface expression for vehicle-treated neurons and neurons 1 h after glutamate excitation and neurons treated with AICAR for 1 h. (n = 3 in triplicate; data are presented as mean ± SEM). *p < 0.01, difference between vehicle treated and neurons stimulated with AICAR and glutamate. G, Neurons plated on Willco dishes were loaded with TMRM and continuously exposed to AICAR (2.5 mm). Quantification of TMRM fluorescent for neurons was performed in vehicle treated (n = 26) and neurons treated with AICAR (n = 23) at time 0, 30 min, 60 min, and 120 min. Representative images were taken at selected time points (0, 60, and 120 min) during AICAR exposure. Data are presented as mean ± SEM. *p < 0.01, difference from vehicle and AICAR treated neurons.
Figure 6.
Figure 6.
Inhibition of AMPK activity but not CaMKK activity significantly attenuates the translocation of GLUT3 to the cell surface after glutamate excitation. A, Western blot analysis of AMPK activity in sham treated and glutamate excited neurons plus and minus compound C. Total AMPK and actin served as a loading controls. Western blot is representative of three separate blots from three separate experiments. B, Flow cytometry analysis of GLUT3 surface expression in sham, vehicle treated, glutamate treated and neurons treated with glutamate preincubated with compound C (10 μm). Data are presented as mean ± SEM. *p < 0.001, difference between glutamate treated and neurons treated with glutamate in the presence of compound C. (n = 2 in triplicate). C, Western blot analysis of CGNs transfected with siRNA directed against AMPK and a nontargeting siRNA pool which acted as a control (control siRNA) (n = 3). D, Flow cytometry analysis of GLUT3 surface expression in sham treated control siRNA transfected neurons, glutamate treated control siRNA transfected neurons, sham treated AMPK siRNA transfected neurons and glutamate treated AMPK siRNA transfected neurons. *p < 0.001 difference between control siRNA and AMPK siRNA glutamate treated neurons (n = 3 in triplicate). E, Flow cytometry analysis of GLUT3 surface expression in sham and glutamate treated neurons plus or minus incubation with KN93 (10 μm for 16 h). Data are presented as mean ± SEM. No significant difference was identified between vehicle and KN93 treated neurons after glutamate excitation (n = 3 in triplicate).
Figure 7.
Figure 7.
Knockdown of GLUT3 with siRNA increases neuronal sensitivity to glutamate excitation and reduces the recovery and hyperpolarization of Δψm at a single cell level. PC12 cells and CGNs were transfected with siRNA directed against GLUT3 and a nontargeting siRNA pool which acted as a control (control siRNA). A, B, GLUT3 downregulation is shown for PC12 transfected with control siRNA and GLUT3 directed siRNA at a (A) gene (mRNA n = 3) and (B) protein level (Western blot, n = 3). Effect of GLUT3 gene silencing on the gene expression level of GLUT1 isoform is also presented. C, D, Immunofluorescence and quantification of GLUT3 surface expression in neurons transfected with Double-Promoter pFIV-H1/U6 siRNA expression vector containing green fluorescent protein (EGFP) and siRNA against GLUT3 or control siRNA 1 h after glutamate excitation. Data are presented as mean ± SEM. *p < 0.01, difference between nontransfected neurons (n = 34) and neurons transfected with the GLUT siRNA after glutamate excitation (n = 38). #p < 0.01, difference between control siRNA transfected neurons (n = 26) and neurons transfected with the GLUT siRNA after glutamate excitation. Sham (n = 22), control siRNA (n = 17). E, Representative TMRM fluorescence traces for neurons transfected with pFIV-H1/U6-copGFP siRNA vector containing siRNA against GLUT3 and those with control siRNA after glutamate excitation. F, Quantification of the TMRM fluorescence after 80 min for sham treated neurons (n = 36) and glutamate stimulated; nontransfected neurons (n = 42), control siRNA transfected neurons (n = 31) and GLUT3 siRNA transfected neurons (n = 48). Data are presented as mean ± SEM. *p < 0.01, difference between sham, and nontransfected neurons and control siRNA-transfected neurons stimulated with glutamate. #p < 0.001, difference between control siRNA transfected neurons and GLUT3 siRNA-transfected neurons exposed to glutamate. G, Quantification of neuronal viability for sham-treated GLUT3 siRNA-transfected neurons (n = 64), glutamate-stimulated control siRNA-transfected neurons (n = 71), and glutamate-stimulated GLUT3 siRNA-transfected neurons (n = 80). Data are presented as mean ± SEM. *p < 0.01, difference between sham and glutamate treated control siRNA transfected neurons. #p < 0.001, difference between glutamate-treated control siRNA-transfected neurons and glutamate-treated GLUT3 siRNA transfected neurons.
Figure 8.
Figure 8.
Modulations in AMPK activity, glucose transport, and Δψm in response to the energetic stress created by transient glutamate excitation. NMDA receptor activation induces a rapid influx of Na+ and Ca2+ ions. The removal of these ions by membrane ATPases rapidly decrease cellular ATP levels altering the AMP:ATP ratio significantly. This alteration in the AMP:ATP ratio and to a minor degree CAMKK activation lead to a rapid activation of AMPK. The activation of AMPK signaling pathway facilitates the translocation of GLUT3 to the plasma membrane, increasing glucose uptake and its metabolism resulting in an increase in Δψm and ATP production. The ability of neurons to recover an energetic equilibrium (ATP levels) appears to determine whether they will tolerate the excitotoxic event.

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