The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death

Abstract

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal β-cell lines and human islet β-cells but not in α-cells. In human islets, glutamate-induced β-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of β-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced β-cell death, whereas GLT1 up-regulation promoted β-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that β-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

FIGURE 1.

Chronic incubation with glutamate induces β-cell apoptosis via ionotropic receptor activation and increased oxidative stress. A, dose-dependent effect of glutamate on β-cell viability. αTC1 and βTC3 cells were incubated for 5 days with the indicated glutamate concentrations, and viability was measured by the MTT assay. The relative growth rate (RGR) is presented as percentage of 0 mm glutamate (n = 7 with eight replicates). *, p < 0.05; **, p < 0.01 versus 0 mm glutamate. B, time-dependent effect of glutamate (Glu) on β-cell viability. βTC3 cells were incubated with 5 mm glutamate for the indicated times, and viability was measured by the MTT assay. Data are presented as percentage of each relative control (CTR) (without glutamate) (n = 4 with eight replicates). **, p < 0.01; ***, p < 0.001 versus relative control. C, glutamate increases β-cell apoptosis. βTC3 cell apoptosis was quantified by TUNEL assay after incubation with 0.5 mm glutamate for 5 days. Cell nuclei were labeled with propidium iodide, and data are presented as the number of TUNEL-positive cell per field (n = 3 in duplicate). Bar, 50 μm. *, p < 0.05. D, CNQX protects β-cells from glutamate toxicity. βTC3 cells were incubated for 5 days with or without 5 mm glutamate in the presence of ionotropic receptor antagonist CNQX (0.025 mm) or d-2-amino-5-phosphonovaleric acid (APV) (0.1 mm), and the viability was measured by MTT assay (n = 3 with eight replicates). *, p < 0.05; **, p < 0.01 versus control 0 mm glutamate; #, p < 0.05; ##, p < 0.01 versus control 5 mm glutamate. E, glutamate causes oxidative stress in β-cells. SH content in βTC3 lysates exposed to 5 mm glutamate for 5 days was quantified. *, p < 0.05. Inset, RT-PCR analysis on cDNA from βTC3 cells showing the transcript for the glutamate/cysteine exchanger subunit xCT. −, RT-PCR without enzymes. The error bars indicate standard errors.

FIGURE 2.

Expression of high affinity glutamate transporters in αTC1 and βTC3 cells. A, RT-PCR analysis on cDNA from αTC1 (α), βTC3 (β), and INS1 cells and brain using primers designed to amplify the transcripts for glutamate transporter subtypes of the SLC1 family or tubulin (TUB) as a housekeeping gene. −, negative controls without enzymes. Markers on the left indicate bp. B, immunoblot (IB) analysis of GLT1 expression in brain (P2 fraction) or βTC3 total cell extracts following 9% SDS-PAGE protein separation. Blots were stained with a specific anti-GLT1 antibody or a rabbit serum as a negative control (CTR). Markers on the left indicate kDa. **, GLT1 oligomer; *, GLT1 monomer. C, immunolocalization of GLT1 in βTC3 cells using an anti-GLT1 antibody. Bar, 5 μm. D, characterization of glutamate transport activity in αTC1 and βTC3 cells. The transport of d-[³H]aspartic acid was measured in the presence of NaCl (Na⁺-dependent) or choline chloride (ChCl) (Na⁺-independent). The Na⁺-dependent uptake activity in βTC3 cells was completely inhibited by 0.1 mm DHK, a selective GLT1 blocker, and 0.025 mm HIP-A, a non-selective glutamate transporter blocker. Data are expressed as cpm/well/15 min. **, p < 0.01 (n = 3 in triplicate). The error bars indicate standard errors.

FIGURE 3.

GLT1 transporter controls extracellular glutamate concentration and β-cell viability. A, incubation of βTC3 cells with the transporter inhibitor DHK for 5 days increases the extracellular glutamate concentration to 0.4 mm (n = 4 in triplicate). *, p < 0.05. B, GLT1 inhibition induces β-cell apoptosis. βTC3 cells were incubated with 0.1 mm DHK for 5 days, and cell apoptosis was determined by the TUNEL assay. Cell nuclei were labeled with propidium iodide (n = 3 in duplicate). Bar, 50 μm. *, p < 0.05. C, GLT1 knockdown increases β-cell apoptosis. βTC3 cells were transfected with control shRNA (SHC) or GLT1 shRNA (SH1 and SH3) to down-regulate GLT1 and incubated with or without 0.5 mm glutamate (Glu). Three days later, apoptosis was determined by the TUNEL assay. Cell nuclei were labeled with propidium iodide (n = 3 in duplicate). Bar, 50 μm. *, p < 0.05 versus control shRNA at 0 mm glutamate; **, p < 0.01 versus control shRNA at 0.5 mm glutamate. D, incubation of βTC3 cells with 10 or 100 μm CEF for 5 days increases the GLT1 expression. After incubation with the indicated CEF and glutamate concentrations for 5 days, 100 μg of each total cell lysate was immunoblotted (IB) with anti-GLT1 and anti-actin antibodies. A representative blot is shown. GLT1 expression was quantified by densitometry and normalized to actin content (n = 3). *, p < 0.05. E, ceftriaxone protects βTC3 cells from glutamate toxicity. Cells were incubated for 5 days with the indicated ceftriaxone and glutamate concentrations, and β-cell viability was determined by MTT assay (n = 3 with eight replicates). *, p < 0.05; ***, p < 0.001 versus 0 μm CEF; ##, p < 0.01 versus 0 μm CEF at 5 mm glutamate. CTR, control. The error bars indicate standard errors.

FIGURE 4.

GLT1 is expressed in islet of Langerhans and localizes to plasma membrane of β-cells. A, immunohistochemistry staining of human or Cercopithecus pancreas sections with the anti-GLT1 antibody. 40× image magnifications are shown. B, immunofluorescence staining of human pancreas sections with anti-GLT1 (red) and hormones (green) as markers of different endocrine cell types. Bar, 10 μm. In the inset, a particular region is shown at higher magnification (2×). The yellow/orange staining indicates co-localization between the transporter and hormones. C, scatter plot analysis of GLT1 (red; channel 1) and hormone (green; channel 2) staining generated from B with ImageJ software reveals partial co-localization of GLT1 with chromogranin and insulin. D, intensity correlation analysis of single channel images from B supports co-localization between GLT1 and insulin or chromogranin at the plasma membrane. The PDM value was calculated in each location of the image. The analysis was performed on the entire islet for chromogranin and insulin and on the region shown at higher magnification for glucagon and somatostatin. Results are presented in pseudocolored images. A PDM scale bar is inserted. A positive PDM value is indicative of dependent staining (co-localization), and a negative value is indicative of segregated staining. Bar, 10 μm.

FIGURE 5.

GLT1 is expressed in human isolated islets and modulates hormone secretion. A, determination of GLT1A expression and tubulin (TUB) in human isolated islets by means of RT-PCR. (C), negative control without enzymes. Left, DNA marker (M). C, control. B, immunoprecipitation (IP) of total human islet extracts with a rabbit serum (IgG) or the GLT1 antibody. 50 μg of brain and 100 μg of human islet extracts (Lys) were loaded in the same gel. Markers on the right indicate kDa. **, oligomer; *, monomer; ○, nonspecific band. C, immunolocalization of GLT1 (green) and insulin (red) on dispersed human β-cells seeded on glass coverslips. Bar, 10 μm. D, d-[³H]aspartic acid uptake measurements in batches of 40 human islets. 0.3 mm DHK was added to the uptake solution. Data are expressed as percentage of the Na⁺-dependent uptake (NaCl) (three different islet preparations in triplicate). **, p < 0.01. E, acute glutamate (Glu) and DHK application modulates the stimulated glucagon secretion. Human islets were preconditioned in 3.3 or 16.7 mm glucose for 1 h (Preconditioning) and then stimulated with 3.3 or 16.7 mm glucose for 1 h (Stimulus) in the presence of 0.5 mm glutamate, 0.1 mm DHK, or both glutamate and DHK as specified. The glucagon released over the 1-h stimulus was measured. Data were normalized to glucagon content in the islet and are expressed as -fold increase over 3.3–3.3 mm glucose (islets were kept in 3.3 mm glucose and then transferred to a solution containing 3.3 mm glucose) (three different islet preparations in triplicate). **, p < 0.01 versus 3.3–3.3 mm glucose; #, p < 0.05 versus 16.7–3.3 mm glucose, 0 mm glutamate, and 0 mm DHK. F, acute glutamate and DHK application does not affect the stimulated insulin release. Human islets were first preconditioned in 3.3 or 16.7 mm glucose (Preconditioning) and then stimulated with 3.3 or 16.7 mm glucose for 1 h (Stimulus) in the presence of 0.5 mm glutamate, 0.1 mm DHK, or both glutamate and DHK as specified. The insulin released over the 1-h stimulus was measured. Data were normalized to insulin content, and data are expressed as -fold increase over 3.3–3.3 mm glucose (three different islet preparations in triplicate). **, p < 0.01 versus 3.3–3.3 mm glucose. ChCl, choline chloride. The error bars indicate standard errors.

FIGURE 6.

Glutamate incubation and GLT1 inhibition induce β-cell apoptosis in human isolated islets. A, chronic incubation with glutamate (Glu) or DHK increases cell apoptosis in human islets. Batches of 40 islets were incubated for 3 days with the indicated treatments in basal glucose (Glc) (3.3 mm), and cell apoptosis was determined by ELISA. Data were normalized for protein content and are expressed as -fold increase over control (CTR) (three different islet preparations in triplicate) *, p < 0.05 versus control. B, apoptosis was confined to β-cells as shown by double immunofluorescence staining with TUNEL assay (green) and insulin (red). Bar, 10 μm. C, ultrastructural characterization of human isolated islets exposed to elevated glutamate concentration. Panel a, low power view (×2,800) of endocrine cells present in an islet cultured in the absence of glutamate supplementation. Nuclei and cytoplasm are well preserved without significant alterations. In particular, β- (panel b) and α-cells (panel c) are both well granulated and do not show degenerative features in both nuclei and cytoplasm. Conversely, in islets exposed to 5 mm glutamate (panel d; low power view; ×2,200), numerous β-cells show apoptotic nuclei (some of them indicated with arrows) characterized by peripheral condensation and margination of nuclear chromatin and by aggregation of nuclear chromatin in dense masses. In the cytoplasm, predominant lysosomal development and autophagic vacuoles are also observed. Remarkably, degenerative features are only found in β-cells (panel e) and are lacking in α-cells (panel f) (panels b, c, e, and f, ×10,000). D, quantification of apoptosis/degeneration in α- and β-cells by electron microscopy analysis. Data are expressed as a percentage of total α- or β-cells (n = 3 different islet preparations). *, p < 0.05. The error bars indicate standard errors.

FIGURE 7.

Chronic incubation with glutamate or DHK affects insulin and proinsulin release. A, batches of 25 human islets were incubated with 0.5 mm glutamate (Glu), 5 mm glutamate, and/or 0.1 mm DHK as indicated. Three days later, the insulin secretion was measured over a 1-h period at basal 3.3 mm (Basal release) or stimulatory 16.7 mm glucose (Stimulates release) concentrations in static incubations. Insulin content in the medium and the lysate was determined by ELISA. Data are normalized for the insulin content and are expressed as -fold increase over basal release (control (CTR), 3.3 mm glucose) (n = 3 different islet preparations in triplicate). *, p < 0.05; **, p < 0.01 versus control in basal glucose concentrations. B, human islets were incubated as described above, and insulin and proinsulin secretion was measured over a 1-h period in static incubations at basal 3.3 mm or stimulatory 16.7 mm glucose concentrations. Insulin and proinsulin contents were determined by ELISA, and the proinsulin to insulin ratio was calculated. Data are expressed as -fold increase over control (three different islet preparations in triplicate).*, p < 0.05. The error bars indicate standard errors.

FIGURE 8.

Glutamate induces oxidative stress in human islets of Langerhans. A, CNQX does not prevent glutamate toxicity in human islets. Islets were incubated for 3 days with 0.5 mm glutamate in the presence of 0.025 mm CNQX, and apoptosis was determined by ELISA (three different islet preparations in duplicate) **, p < 0.01. B, quantification of human β-cell apoptosis after 3-day incubation with 0.5 mm glutamate (Glu) and/or 0.025 mm CNQX by electron microscopy analysis. C, RT-PCR analysis of human islets showing the transcript for the xCT subunit of the glutamate/cysteine exchanger. −, RT-PCR without enzymes. M, markers in bp. D, incubation of human islets with 5 mm glutamate or 16.7 mm glucose (Glc) for 3 days increases the amount of 4-HNE-modified proteins. 100 μg of each total islet extract was immunoblotted (IB) with anti-4-HNE and anti-actin antibodies. A representative blot is shown. 4-HNE signal was quantified by densitometry and normalized to actin content. Molecular weight markers (in kDa) are shown on the left. Arrowheads indicate 4-HNE-modified proteins induced by chronic glutamate incubation (three different islet preparations). *, p < 0.05; **, p < 0.01. E, dispersed islets incubated for 3 days with or without 0.5 mm glutamate were double stained with insulin and 4-HNE. 4-HNE-positive cells are expressed as the percentage of insulin-positive cells (two different islet preparations in triplicate). *, p < 0.05. CTR, control. The error bars indicate standard errors.

FIGURE 9.

Proposed events leading to progressive β-cell death by glutamate toxicity in diabetes mellitus. The extracellular glutamate concentration in the islet of Langerhans is controlled by the activity of GLT1. In diabetes mellitus, a combination of insults may trigger β-cell death and consequently modify the extracellular glutamate concentration by increasing its release (via α-cells) and decreasing its clearance (via β-cells). Increased glutamate levels may cause β-cell death, thus triggering a vicious cycle that further increases the glutamate levels in the islet of Langerhans and β-cell death.