Diabetes and Metabolism Journal

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

고농도 포도당에 노출된 INS-1세포와 백서 췌도 세포에서 포도당 산화 및 Reactive Oxygen Species (ROS) 생성

Yoon, Ji-Sung;Won, Kyu-Chang;Lee, Hyoung-Woo
윤지성;원규장;이형우

  • Published : 2006.07.31
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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 hyperglycemiainduced 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.

연구배경: 췌장 소도 베타 세포가 장기간 고혈당에 노출되었을 때 인슐린 분비능, 인슐린 mRNA 및 인슐린 gene transcription factor 등이 감소하는 것을 포도당 독성이라고 하며, 췌장 소도 내의 낮은 항산화 효소 및 포도당 대사 과정에서 reactive oxygen species (ROS)의 증가가 그 원인으로 보고되고 있고, 고농도 포도당에 의해 생성된 ROS는 미토콘드리아의 electron transport chain complex 저하제에 의해 감소된다는 보고들도 있다. 이에 연자 등은 고혈당에 노출된 췌장 소도에서 세포 내 ROS 생성장소를 알아보기 위해 본 연구를 시행하였다. 방법: INS-1 세포주 및 백서 췌장소도 세포를 고농도의 포도당에서 배양하고, 또한 미토콘드리아 전자전달계 복합체 저하제 및 산화성 인산화 반응 억제제인 TTFA, CCCP, $\alpha$CCA 등을 처리한 뒤 flow cytometer를 이용하여 세포 내 peroxide치를 측정하고, 인슐린 분비능 등을 측정하였다. 그리고 INS-1 세포주에 glyceraldehyde를 농도별로 24시간 배양하고 N-acetylcystein을 처리하여 세포 내 peroxide 및 인슐린 분비능을 측정하였다. 결과: INS-1 세포를 고농도 포도당 배지 (30 mM 포도당)에 배양하였을 때 세포 내 peroxide치는 증가하였고, 인슐린 분비능은 감소하였다 (P < 0.05). INS-1 세포를 고농도 포도당 배지(30 mM 포도당)에 전자전달계 복합체 저하제인 TTFA, CCCP, $\alpha$CCA 등과 함께 배양하였을 때 세포 내 peroxide는 감소하지 않았고, 인슐린 분비능도 회복되지않았다 (P < 0.05). INS-1 세포를 포도당 대사과정에서 미토콘드리아 진입 전구물질인 glyceraldehyde와 함께 5.6 mM 포도당 농도에서 배양하였을 때 세포 내 peroxide는 증가하였고, 인슐린 분비능은 감소하였다 (P < 0.05). 결론: 췌장 소도 세포의 포도당 독성에 ROS 생성이 중요한 역할을 하고, ROS 생성 장소로 미토콘드리아 이외에도 있다고 보여지며, glyceradehyde는 이러한 ROS 생성 및 베타세포 기능 저하에 영향을 미치는 물질로 생각할 수 있다.

Keywords

References

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