J Korean Diabetes Assoc.  2006 Jul;30(4):246-253. 10.4093/jkda.2006.30.4.246.

Glucose Oxidation and Production of Reactive Oxygen Species (ROS) in INS-1 Cells and Rat Islet Cells Exposed to High Glucose

• Yoon JS
• Won KC
• Lee HW

• KMID: 2177594
• DOI: http://doi.org/10.4093/jkda.2006.30.4.246

Abstract

BACKGROUND: Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes beta cell dysfunction that is a process known as glucose toxicity. It has been reported that hyperglycemia increases the production of reactive oxygen species (ROS) in human islets and that ROS accumulation causes beta cell dysfunction associated with low capacity of intrinsic antioxidant enzymes. Also it has been postulated that this increase in ROS is prevented by an inhibitor of electron transport chain complex. The purpose of this study were to determine whether prolonged exposure of pancreatic islets to supraphysiologic glucose concentrations disrupts the intracellular balance between ROS thereby causing defective insulin secretion and to evaluate the site of hyperglycemia-induced ROS production.
METHODS
INS-1 cells & rat islets were incubated in increasing concentrations of glucose and either an inhibitor of complex I & II (TTFA), an uncoupler of oxidative phosphorylation (CCCP), aCCA, etc and also incubated in increasing concentration of glyceraldehyde and N-acetylcystein. Then intracellular peroxide levels by flow cytometric analysis and glucose induced insulin secretion were detected.
RESULTS
We observed that incubation with 30 mM glucose increased intracellular peroxide levels but decreased glucose-stimulated insulin secretion (GSIS) (P < 0.05). Exposure to TTFA, CCCP, aCCA did not reduce this increased intracellular peroxide levels, and did not increase GSIS (P < 0.05). 24-h incubation with glyceraldehyde at 5.6 mM glucose increased intracellular peroxide levels and decreased insulin content.
CONCLUSION
These observations indicate that there might be other origins in which ROS species are produced besides electron transport chain in mitochondria and glyceraldehyde may be a key molecule to produce ROS, and induce beta cell dysfunction.

Keyword

Diabetes Mellitus; Glucose toxicity; Glyceraldehyde; Oxidative stress; Reactive oxygen species

MeSH Terms

Animals
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Diabetes Mellitus
Electron Transport
Glucose*
Glyceraldehyde
Humans
Hyperglycemia
Insulin
Islets of Langerhans*
Mitochondria
Oxidative Phosphorylation
Oxidative Stress
Rats*
Reactive Oxygen Species*
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Glucose
Glyceraldehyde
Insulin
Reactive Oxygen Species

Figure

• 

  Fig. 1 Intracellular peroxide level (A, B) and GSIS (C, D) in INS-1 cells and rat islet cells after 3 days subculture. Data are mean ± SE from 3 separate experiments. *p < 0.05.

• 

  Fig. 2 Effects of several agents that affect mitochondrial metabolism on formation of hyperglycemia-induced intracellular peroxide in INS-1 cells. Cells were incubated in 5.6 mM glucose, 30 mM glucose alone, and 30 mM glucose plus either Mannoheptulose (MH), Methylsuccinate, aCCA (a-cyano-4-hydroxycinnamic acid), Rotenone, TTFA (Thenoyltrifluoroacetone), CCCP (Carbonyl cyanide chlorophenyl hydrazone), Azaserine, and intracellular peroxide levels were quantified. Data are mean ± SE from 3 separate experiments. *p < 0.05 vs control. **p < 0.05 vs 30 mM glucose.

• 

  Fig. 3 Intracellular peroxide level in islets after 24 h culture with glyceraldehyde. Data are mean ± SE from 3 separate experiments. *p < 0.05 vs control. **p < 0.05 vs 5 mM.

• 

  Fig. 4 GSIS (A) & Insulin content (B) in islets after 24 h culture with glyceraldehyde. Data are mean ± SE from 3 separate experiments. *p < 0.05 vs 0 mM.

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