Diabetes Metab J.  2011 Oct;35(5):480-488. 10.4093/dmj.2011.35.5.480.

The Effects of Glyburide on Apoptosis and Endoplasmic Reticulum Stress in INS-1 Cells in a Glucolipotoxic Condition

• Kwon MJ
• Chung HS
• Yoon CS
• Ko JH
• Jun HJ
• Kim TK
• Lee SH
• Ko KS
• Rhee BD
• Kim MK
• Park JH

• KMID: 1857503
• DOI: http://doi.org/10.4093/dmj.2011.35.5.480

Abstract

BACKGROUND
beta-cell death due to endoplasmic reticulum (ER) stress has been regarded as an important pathogenic component of type 2 diabetes. The possibility has been suggested that sulfonylurea, currently being used as one of the main oral hypoglycemic agents of type 2 diabetes, increases ER stress, which could lead to sulfonylurea failure. The authors of the present study examined ER stress of beta-cells in a glucolipotoxic condition using glyburide (GB) in an environment mimicking type 2 diabetes.
METHODS
Apoptosis was induced by adding various concentrations of GB (0.001 to 200 microM) to a glucolipotoxic condition using 33 mM glucose, and the effects of varied concentrations of palmitate were evaluated via annexin V staining. The markers of ER stress and pro-apoptotic markers were assessed by Western blotting and semi-quantitative reverse transcription-polymerase chain reaction. Additionally, the anti-apoptotic markers were evaluated.
RESULTS
Addition of any concentration of GB in 150 microM palmitate and 33 mM glucose did not increase apoptosis. The expression of phosphorylated eukaryotic initiation factor (eIF-2alpha) was increased and cleaved caspase 3 was decreased by adding GB to a glucolipotoxic condition. However, other ER stress-associated markers such as Bip-1, X-box binding protein-1, ATF-4 and C/EBP-homologous protein transcription factor and anti-apoptotic markers phosphor-p85 phosphatidylinositol 3-kinase and phosphorylation of Akt did not change significantly.
CONCLUSION
GB did not show further deleterious effects on the degree of apoptosis or ER stress of INS-1 cells in a glucolipotoxic condition. Increased phosphorylation of eIF-2alpha may attenuate ER stress for adaptation to increased ER protein load.

Keyword

Apoptosis; Endoplasmic reticulum stress; Glyburide; Insulin-secreting cells

MeSH Terms

Annexin A5
Apoptosis
Blotting, Western
Caspase 3
Endoplasmic Reticulum
Endoplasmic Reticulum Stress
Eukaryotic Initiation Factor-2
Glucose
Glyburide
Hypoglycemic Agents
Insulin-Secreting Cells
Peptide Initiation Factors
Phosphatidylinositol 3-Kinase
Phosphorylation
Transcription Factors
Annexin A5
Caspase 3
Eukaryotic Initiation Factor-2
Glucose
Glyburide
Hypoglycemic Agents
Peptide Initiation Factors
Phosphatidylinositol 3-Kinase
Transcription Factors

Figure

• 

  Fig. 1 Apoptosis in INS-1 cells after incubation for 24 hours in culture media with 33 mM glucose and various concentrations of palmitate according to annexin V staining. Values are presented as mean±standard deviation of five repetitions.

• 

  Fig. 2 The effect on apoptosis of INS-1 cells at various concentrations of glyburide in a glucolipotoxic condition. Apoptosis was evaluated using annexin V staining. The experiments were performed in triplicate. Values are presented as mean±standard deviation and aP<0.05.

• 

  Fig. 3 Changes in endoplasmic reticulum stress markers with the addition of glyburide to a glucolipotoxic condition. Bip-1 (A), ATF-4 (B), and XBP-1 (C) were evaluated using reverse transcription-polymerase chain reaction. Phosphorylated eukaryotic initiation factor (eIF)-2α and eIF-2α were analyzed by Western blotting, and the phosphorylation rate was assessed (D). The experiments were performed in triplicate. Values are presented as mean±standard deviation and aP<0.05 for results under a glucolipotoxic condition.

• 

  Fig. 4 Changes in endoplasmic reticulum stress markers representing the pro-apoptotic pathway by adding glyburide to a glucolipotoxic condition. Functional caspase 3 (A) and cleaved caspase 3 (B) were evaluated according to Western blotting. CHOP (C) was assessed by reverse transcription-polymerase chain reaction. The experiments were performed in triplicate. Values are presented as mean±standard deviation and aP<0.05 for results under a glucolipotoxic condition.

• 

  Fig. 5 Changes in PI3K (A) and phosphorylated Akt (B), markers representing anti-apoptotic defense, due to the addition of glyburide to a glucolipotoxic condition. The experiments were performed in triplicate. Values are presented as mean±standard deviation.

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