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고농도 지방산에 의한 INS-1 세포사에 철 대사가 미치는 영향
- Alternative Title
- The effect of iron metabolism on palmitate-induced INS-1 cell death
- Author(s)
- Jung Ik Rak
- Alternative Author(s)
- Ik Rak Jung
- Advisor
- 강엽
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2015-08
- Language
- eng
- Keyword
- Apoptosis; Endoplasmic reticulum (ER) stress; Lipotoxicity; Iron; INS-1 cell; Palmitate; Transferrin receptor; Soduim fluorocitrate
- Alternative Abstract
- High level of plasma free fatty acid (FFA) was thought to contribute to the loss of pancreatic beta-cells in type 2 diabetes. In particular, saturated FFAs such as palmitate or stearate were able to induce apoptosis in cultured beta cells (lipotoxicity). Endoplasmic reticulum (ER) stress was reported to be a critical mediator for the FFA-induced lipotoxicity. Recently, disorders in mitochondrial respiratory metabolism were reported to be involved in the lipotoxicity. Since iron was a critical component for respiratory metabolism, I studied to determine whether abnormal iron metabolism is involved in palmitate (PA)-induced beta cell death. Immunoblotting analysis showed that treatment of INS-1 cells with palmitate reduced level of transferrin receptor (TfR), but increased level of heavy chain ferritin (FTH). In accordance with TfR down-regulation and FTH up-regulation, palmitate reduced intracellular labile iron pool. Whereas iron depletion through treatment with iron-chelators such as deferoxamine (DFO) or deferasirox (DS) augmented PA-induced cell death, iron supplementation through treatment with FeCl3, FeSO4, or holo-transferrin significantly protected PA-induced death. Furthermore, overexpression of TfR1 reduced PA-induced death whereas knockdown of TfR1 augmented the death. In particular, treatment with DFO increased level of ER stress markers such as phospho-PERK, phospho-eIF2α, CCAAT/enhancer binding protein homologous protein (CHOP) and phospho-c-Jun N-terminal kinase (p-JNK), and furthermore, treatment with chemical chaperone significantly protected DFO-induced cell death. Iron supplementation also demonstrated protective effect on PA-induced primary islet cell death. Collectively data suggest that iron depletion plays a role in PA-induced beta cell death through induction of ER stress and that attempts to block iron depletion may be a maneuver to prevent beta cell loss in type 2 diabetes. Chronic exposure to palmitate leads to mitochondrial dysfunction. Furthermore, reduced levels of tricarboxylic acid (TCA) cycle intermediates have been observed during PA-induced lipotoxicity. It has been demonstrated previously that artificial inhibition of TCA cycling, through treatment with inhibitors of pyruvate carboxylase and carnitin palmitoyltrasferase 1 augment PA-induced INS-1 cell death, whereas activation of TCA cycling through treatment with glutamate dehydrogenase (GDH) activators reduced PA-induced cell death. Therefore, the present study aimed to identify new drugs able to control PA-induced INS-1 cell death by regulating TCA cycle intermediates. Sodium fluorocitrate (SFC), a known aconitase inhibitor, completely protected against PA-induced reductions in INS-1 cell viability and increased INS-1 cell death. Furthermore, SFC ameliorated reductions in insulin signaling and increased ER stress marker expression during PA treatment. However, SFC did not protect against the INS-1 cell death induced by a thapsigargin, streptozotocin, H2O2 and cytokine mixture. To understand how SFC completely protects against PA-induced INS-1 cell death, I investigated the effect of genetic knockdown of aconitase, by small interference RNA (siRNA), on PA-induced cell death. Aconitase siRNA efficiently reduced aconitase mRNA but PA-induced INS-1 cell death was further increased by aconitase knockdown. Reduced activity of cytosolic aconitase acts in a similar way to an iron regulatory protein. I demonstrated previously that an increase in the labile iron pool reduces PA-induced INS-1 cell death; however, in the present study ferritin (a known iron storage protein) levels were decreased, and transferrin receptor 1 (a known iron uptake protein) levels increased, by SFC treatment, such that the intracellular iron pool was diminished. On the other hand, PA uptake and oxygen consumption rate were both decreased by SFC treatment. It is assumed that SFC may interrupt the translocation of CD36 into the cell membrane. Collectively, these data suggest that SFC prevents PA-induced lipotoxicity by reducing PA uptake, rather than by regulating TCA cycle intermediates or iron metabolism.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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