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고농도 지방산에 의한 INS-1 세포사에 철 대사가 미치는 영향

The effect of iron metabolism on palmitate-induced INS-1 cell death

초록/요약

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.

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목차

Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 24
A. MATERIALS 24
1. Reagents 24
B. METHODS 25
1. Cell Culture 25
2. Preparation of palmitate 25
3. Isolation of islets 25
4. MTT-bases viability assay 26
5. Measurement of cell death 26
6. Western blot analysis 27
7. RNA extraction and microarray analysis 27
8. Reverse transcriptase-polymerase chain reaction and quantitative PCR 28
9. DNA and RNA Transfection 28
10. Measurement of intracellular labile iron pool (LIP) 29
11. Aconitase activity assay 29
12. Oxygen consumption rate (OCR) 30
13. PA uptake 31
14. Quantitation of insulin 31
15. Immunocytochemistry 32
16. Statistics 32
Ⅲ. RESULTS [Part I] 36
A. Palmitate treatment reduces intracellular iron level in INS-1 beta cell. 36
B. Iron depletion and supplementation in palmitate-induced INS-1 cell death. 41
C. Toxic mediator of iron deficiency. 50
D. Beta-cell function by iron regulation. 60
E. Effect of iron regulation on lipotoxicity in other cells. 61
Ⅲ. RESULTS [Part II] 64
A. Control of PA-induced INS-1 cell death through the TCA cycle intermediate. 64
B. Role of SFA and SFC in various of cell death models. 71
C. The effect of aconitase inhibition on PA-induced lipotoxicity 76
D. SFC altered iron-regulating proteins. 84
E. Changes in intracellular signaling associated with SFC treatment. 88
F. Changes in oxygen consumption rate associated with SFC treatment. 98
G. SFC modulates CD36 translocation and reduces PA uptake. 100
H. SFA and SFC protect against PA-induced islet cell death. 103
Ⅳ. DISCUSSION [Part I] 104
Ⅳ. DISCUSSION [Part II] 110
Ⅴ. CONCLUSION 117
REFERENCES 118
국문요약 135

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