Diabetes Metab J.  2016 Oct;40(5):396-405. 10.4093/dmj.2016.40.5.396.

Repeated Glucose Deprivation/Reperfusion Induced PC-12 Cell Death through the Involvement of FOXO Transcription Factor

• Han N
• Kim YJ
• Park SM
• Kim SM
• Lee JS
• Jung HS
• Lee EJ
• Kim TK
• Kim TN
• Kwon MJ
• Lee SH
• Kim MK
• Rhee BD
• Park JH

• KMID: 2354613
• DOI: http://doi.org/10.4093/dmj.2016.40.5.396

Abstract

BACKGROUND
Cognitive impairment and brain damage in diabetes is suggested to be associated with hypoglycemia. The mechanisms of hypoglycemia-induced neural death and apoptosis are not clear and reperfusion injury may be involved. Recent studies show that glucose deprivation/reperfusion induced more neuronal cell death than glucose deprivation itself. The forkhead box O (FOXO) transcription factors are implicated in the regulation of cell apoptosis and survival, but their role in neuronal cells remains unclear. We examined the role of FOXO transcription factors and the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt and apoptosis-related signaling pathways in PC-12 cells exposed to repeated glucose deprivation/reperfusion.
METHODS
PC-12 cells were exposed to control (Dulbecco's Modified Eagle Medium [DMEM] containing 25 mM glucose) or glucose deprivation/reperfusion (DMEM with 0 mM glucose for 6 hours and then DMEM with 25 mM glucose for 18 hours) for 5 days. MTT assay and Western blot analysis were performed for cell viability, apoptosis, and the expression of survival signaling pathways. FOXO3/4',6-diamidino-2-phenylindole staining was done to ascertain the involvement of FOXO transcription factors in glucose deprivation/reperfusion conditions.
RESULTS
Compared to PC-12 cells not exposed to hypoglycemia, cells exposed to glucose deprivation/reperfusion showed a reduction of cell viability, decreased expression of phosphorylated Akt and Bcl-2, and an increase of cleaved caspase-3 expression. Of note, FOXO3 protein was localized in the nuclei of glucose deprivation/reperfusion cells but not in the control cells.
CONCLUSION
Repeated glucose deprivation/reperfusion caused the neuronal cell death. Activated FOXO3 via the PI3K/Akt pathway in repeated glucose deprivation/reperfusion was involved in genes related to apoptosis.

Keyword

Apoptosis; Forkhead box O; PC-12 cell; Phosphatidylinositol 3-kinase/Akt pathway; Reperfusion

MeSH Terms

Apoptosis
Blotting, Western
Brain
Caspase 3
Cell Death*
Cell Survival
Cognition Disorders
Eagles
Glucose*
Hypoglycemia
Neurons
Phosphatidylinositol 3-Kinase
Reperfusion
Reperfusion Injury
Transcription Factors*
Caspase 3
Glucose
Phosphatidylinositol 3-Kinase
Transcription Factors

Figure

• 

  Fig. 1 Cell growth and cell viability in glucose deprivation for 1 hour/reperfusion for 23 hours and glucose deprivation for 6 hours/reperfusion for 18 hours for 5 days. (A) Representative light microscopic imaging (×400) of PC-12 cells not exposed to hypoglycemia, 0 mM glucose Dulbecco's Modified Eagle Medium (DMEM) for 1 hour, and 0 mM glucose DMEM for 6 hours for 5 days. (B) Quantification of cell viability used MTT assay in PC-12 cells exposed to 0 mM glucose DMEM for 1 hour and for 6 hours compared with the controls, not exposed to hypoglycemia. Scale bars represent 100 µm. Data are mean±standard error of mean. Statistical significance was tested using a Student t-test. The data shown represents three independent experiments. aRepresents P<0.05 vs. control.

• 

  Fig. 2 Cell growth and cell viability in PC-12 cells exposed to glucose deprivation/reperfusion. (A) Representative light microscopic imaging (×400) of PC-12 cells not exposed to hypoglycemia and exposed to repeated glucose deprivation/reperfusion for 5 days. (B) Quantification of cell viability in PC-12 cells exposed to repeated glucose deprivation/reperfusion and the controls by using MTT assay. The intact nuclei were counted on a haemocytometer. Scale bars represent 100 µm. Data are mean±standard error of mean. Statistical significance was tested using a Student t-test. (C) Representative 4',6-diamidino-2-phenylindole counterstaining of PC-12 cells exposed to repeated glucose deprivation-reperfusion for 5 days and control. The data shown represent three independent experiments. aRepresents P<0.05 vs. control.

• 

  Fig. 3 The nuclear localization of forkhead box O 3 (FOXO3) in PC-12 cells. (A) Representative FOXO3 staining in PC-12 cells. FOXO3 is localized to the nuclei (red arrowheads) in PC-12 cells exposed to repeated glucose deprivation/reperfusion while FOXO3 is localized to the cytoplasm in the controls. (B) Quantification of nuclear localization of FOXO3 in PC-12 cells exposed to repeated glucose deprivation/reperfusion and the controls. Scale bars represent 200 µm. Data are mean±standard error of mean. Statistical significance was tested using an unpaired, two-tailed Student t-test. The data shown represent three independent experiments. DAPI, 4',6-diamidino-2-phenylindole. aP<0.05 compared with control.

• 

  Fig. 4 Protein expressions of Akt, phosphorylated Akt (phospho-Akt), caspase-3, cleaved caspase-3, and Bcl-2 in PC-12 cells exposed to repeated glucose deprivation/reperfusion condition. (A) The expressions of phospho-Akt, Akt, cleaved caspase-3, and Bcl-2 were assessed by Western blot. (B) Quantification of phospho-Akt/Akt, cleaved caspase-3/caspase-3 and Bcl-2 expressions. Data are mean±standard error of mean from three independent experiments. Statistical significance was tested using an unpaired, two-tailed Student t-test. aRepresents P<0.05.

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