Korean Diabetes J.  2010 Feb;34(1):47-54. 10.4093/kdj.2010.34.1.47.

The Effect of Glucose Fluctuation on Apoptosis and Function of INS-1 Pancreatic Beta Cells

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

• KMID: 2298045
• DOI: http://doi.org/10.4093/kdj.2010.34.1.47

Abstract

BACKGROUND
Blood glucose level continuously fluctuates within a certain range in the human body. In diabetes patients, the extent of such fluctuation is large, despite the strict control of blood glucose. Blood glucose fluctuation has been shown to mediate more adverse effects on vascular endothelial cells and diabetes complications than chronic hyperglycemia, which has been explained as due to oxidative stress. As few previous studies have reported the effects of chronic and intermittent hyperglycemia on the apoptosis and function of pancreatic beta cells, this study reported herein was performed to investigate such effects on these cells. METHODS: For chronic hyperglycemia, INS-1 cells were cultured for 5 days with changes of RPMI 1640 medium containing 33 mM glucose every 12 hours. For intermittent hyperglycemia, the medium containing 11 mM glucose was exchanged with the medium containing 33 mM glucose every 12 hours. Apoptosis was assessed by TUNEL assay Hoechst staining and cleaved caspase 3. Insulin secretory capacity was assessed, and the expression of Mn-SOD and Bcl-2 was measured by Western blotting. RESULTS: In comparison to the control group, INS-1 cells exposed to chronic hyperglycemia and intermittent hyperglycemia showed an increase in apoptosis. The apoptosis of INS-1 cells exposed to intermittent hyperglycemia increased significantly more than the apoptosis of INS-1 cells exposed to chronic hyperglycemia. In comparison to the control group, the insulin secretory capacity in the two hyperglycemic states was decreased, and more with intermittent hyperglycemia than with chronic hyperglycemia. The expression of Mn-SOD and Bcl-2 increased more with chronic hyperglycemia than with intermittent hyperglycemia. CONCLUSION: Intermittent hyperglycemia induced a higher degree of apoptosis and decreased the insulin secretory capacity more in pancreatic beta cells than chronic hyperglycemia. This activity may be mediated by the anti-oxidative enzyme Mn-SOD and the anti-apoptotic signal Bcl-2.

Keyword

Apoptosis; Bcl2 protein; Glucose; Mn-SOD; Pancreatic beta Cells

MeSH Terms

Apoptosis
Blood Glucose
Blotting, Western
Caspase 3
Diabetes Complications
Endothelial Cells
Glucose
Human Body
Humans
Hyperglycemia
In Situ Nick-End Labeling
Insulin
Insulin-Secreting Cells
Oxidative Stress
Superoxide Dismutase
Blood Glucose
Caspase 3
Glucose
Insulin
Superoxide Dismutase

Figure

• 

  Fig. 1 Expression of caspase 3 and cleaved caspase 3 in INS-1 cell. (A) Caspase 3 and cleaved caspase 3 were measured by Western blot analysis. (B, C) Densitometric analyses of Western blot are reported as means ± SD of the three different experiments. C, control; IHG, intermittent high glucose; SHG, sustained high glucose. aP < 0.05 vs. control, bP < 0.05 vs. IHG.

• 

  Fig. 2 Hoechst 33347 staining. (A) After INS-1 cell were incubated under control, IHG and SHG, they were stained with Hoechst 33347 to detect apoptosis morphologically. The red arrows represent cells with DNA condensation. (B) The numbers of cells with broken DNA per 300 cells in three different fields in each condition of one experiment were counted and repeated in three different experiments. The mean percentages of positive staining under control, IHG and SHG were presented by histogram. C, control; IHG, intermittent high glucose; SHG, sustained high glucose. aP < 0.05 vs. control, bP < 0.05 vs. IHG.

• 

  Fig. 3 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). (A) TUNEL stainings under control, IHG and SHG were analyzed by fluorescent in situ hybridization (FISH). (B) The percentage of TUNEL positive apoptotic cells / area was presented in histogram. C, control; IHG, intermittent high glucose; SHG, sustained high glucose. aP < 0.05 vs. control, bP < 0.05 vs. IHG.

• 

  Fig. 4 Insulin secretion from INS-1 cells in response to glucose (5 and 25 mM) after incubated for 5 days in control, IHG and SHG. Data are means ± SD of three separate experiments. C, control; IHG, intermittent high glucose; SHG, sustained high glucose. aP < 0.05 vs. control of 25 mM glucose, bP < 0.05 vs. IHG of 25 mM glucose.

• 

  Fig. 5 The changes of the expression of MnSOD and Bcl-2 in response to control, IHG and SHG. (A) Expression of Mn-SOD and Bcl-2 were evaluated by Western blot analysis. A representative experiment of three is shown. (B, C) Densitometric analyses of western blot are reported as means ± SD of the three different experiments. C, control; IHG, intermittent high glucose; SHG, sustained high glucose. aP < 0.05 vs. control, bP < 0.05 vs. IHG.

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