Chonnam Med J.  2018 Jan;54(1):24-30. 10.4068/cmj.2018.54.1.24.

Antidiabetic Drug Metformin Protects Neuronal Cells against Quinolinic Acid-Induced Excitotoxicity by Decreasing Intracellular Calcium

Affiliations
  • 1Department of Physiology, Chonnam National University Medical School, Gwangju, Korea. hoonpark@jnu.ac.kr
  • 2Research Institute of Medical Sciences, Chonnam National University, Gwangju, Korea.

Abstract

The antidiabetic drug metformin has been found to have beneficial effects in various neurological disorders; however, the molecular mechanisms underlying these effects remain unclear. Here we report that metformin protects neuronal cells from quinolinic acid (QUIN)-induced excitotoxicity. For this, we pretreated N18D3 neuronal cells with metformin prior to QUIN for 24 h. We found that pretreating the cells with metformin significantly improved cell survival rate in a concentration-dependent manner and reduced apoptotic cell death, as revealed by a MTT assay and DAPI staining, respectively. Calcium imaging using fluo-4 showed that metformin (100 µM) inhibited the intracellular calcium increase that was induced by QUIN. In addition, mRNA expression of pro-apoptotic genes, p21 and Bax, was decreased and of anti-apoptotic genes, Bcl-2 and Bcl-xl, was increased with metformin treatment compared to QUIN-induced cells. The immunoreactivity of phosphorylated ERK1/2 was elevated in cells treated with metformin, indicating the ERK1/2 signaling pathway in the neuroprotective effects of metformin in QUIN-induced cell death. Collectively, our data demonstrates that metformin exerts its neuroprotective effects by inhibiting intracellular calcium increases, allowing it to regulate ERK1/2 signaling and modulate cell survival and death genes.

Keyword

Apoptosis; Metformin; Quinolinic Acid; Neuroprotection; Calcium

MeSH Terms

Apoptosis
Calcium*
Cell Death
Cell Survival
Genes, bcl-2
Metformin*
Nervous System Diseases
Neurons*
Neuroprotection
Neuroprotective Agents
Quinolinic Acid
RNA, Messenger
Calcium
Metformin
Neuroprotective Agents
Quinolinic Acid
RNA, Messenger

Figure

  • FIG. 1 Effects of metformin in QUIN-induced excitotoxicity. (A) We treated metformin (1, 10, 25, 50, or 100 µM) for 2 h prior to exposing them to QUIN (30 mM) for 24 h in N18D3 cells. Pretreatment with metformin improved the cell viability. (B) DAPI staining showed the condensation and fragmentation, which characterized features of apoptosis. Untreated cells (control) and those treated with metformin alone displayed a round or oval shape. The treatment of QUIN (30 mM) caused apoptotic-like cell, whereas, metformin pretreatment prevented such changes from occurring. Scale bar indicates 20 µm. (C) Apoptotic cell population was reduced by metformin pretreatment. *p<0.05 and **p<0.01 compared with QUIN (30 mM)-treated cells; #p<0.05 compared with control.

  • FIG. 2 Changes in intracellular calcium ([Ca2+]i) induced by QUIN or metformin. (A) The [Ca2+]i were elevated with QUIN (30 mM) alone and reduced by pretreatment with metformin (100 µM). Scale bar indicates 20 µm. (B) Time course curve of fluo-4 fluorescence intensity demonstrated that metformin (100 µM) significantly blocked the QUIN-induced elevations in [Ca2+]i. **p<0.01 compared with QUIN-treated cells.

  • FIG. 3 Modulation of anti-apoptotic and pro-apoptotic genes by QUIN and metformin. (A) The cells were incubated with QUIN (30 mM) and/or metformin (10 or 100 µM) for 6 h. Cellular mRNA expression for p21, Bax, Bcl-2, and Bcl-xl was analyzed using RT-PCR. The pro-apoptotic gene expression was reduced by metformin pretreatment, whereas the expression of anti-apoptotic genes was elevated. (B) The expression level of each gene was normalized to GAPDH. *p<0.05 and **p<0.01 compared with QUIN-treated cells; #p<0.05 compared with control.

  • FIG. 4 Expression of MAP kinase signaling pathway. (A) To evaluate the MAP kinase expression, western blot analysis was performed following treatment of metformin (10 or 100 µM) and/or QUIN (30 mM) for 6 h in N18D3 cells. The JNK, ERK1/2, and p38 MAP kinase proteins were investigated and the phosphorylated ERK1/2 was increased. (B–D) The expression level of each gene was normalized to GAPDH. *p<0.05 compared with QUIN-treated cells; #p<0.05 compared with control.


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