Iron overload by transferrin receptor protein 1 regulation plays an important role in palmitate-induced insulin resistance in human skeletal muscle cells
Iron overload by transferrin receptor protein 1 regulation plays an important role in palmitate-induced insulin resistance in human skeletal muscle cells
- 주제(키워드) Iron overload , Insulin resistance , Transferrin receptor , calcium , Human skeletal muscle myoblasts
- 발행기관 아주대학교
- 지도교수 이관우
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028658
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Free fatty acid is considered one of the major pathogenic factors inducing insulin resistance. The association between iron disturbances and insulin resistance has recently begun to receive a lot of attention. Although skeletal muscle is a major tissue for iron utilization and storage, the role of iron in palmitate (PA)-induced insulin resistance is unknown. We investigated the molecular mechanism underlying iron dysregulation in PA-induced insulin resistance. Intracellular iron was measured using calcein AM and transferrin-GFP. Iron and calcium chelator or siRNA were used to investigate the effects of iron metabolism on insulin sensitivity. Intracellular calcium was detected using Fluo-3 AM. PA induced insulin resistance and simultaneously increased intracellular iron. The iron chelator, deferoxamine dramatically inhibited PA-induced insulin resistance, and iron donors impaired insulin sensitivity by activating JNK. PA upregulated tfR1 through IRP2. Knockdown of tfR1 and IRP2 prevented PA-induced iron uptake and insulin resistance. PA also translocated the tfR1-GFP protein complex by stimulating calcium influx, but the BAPTA-AM dramatically reduced iron overload by inhibiting transferrin translocation and subsequent insulin resistance. PA induces insulin resistance through iron overload, reducing intracellular iron protected cells against insulin resistance. Therefore, blocking iron overload may be a useful strategy for preventing insulin resistance and diabetes.
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ⅠINTRODUCTION 2
ⅠI METERIALS AND METHODS 27
A.METERIALS 28
B.METHODS 29
Preparation of PA 29
Cell culture 29
Myotube staining 29
Immunoblot analysis 30
RNA isolation, quantitative real-time PCR, and semi-quantitative reverse transcriptase-PCR 30
Transfection of siRNA 31
Uptake of 2-NBDG by HSMMs 31
Measurement of intracellular labile iron pool 31
Animal model and insulin tolerance tests 32
Measurement of iron concentration in skeletal muscles 32
Study subjects and skeletal muscle biopsy 33
Measurement of intracellular calcium levels 33
Analysis of GFP-labeled iron and Tf complex uptake 34
Statistical analyse 34
Table 1. Nucleotide sequences of sets for real-time PCR 35
Table 2. Nucleotide sequences of sets and reaction condition for semi-quantitative PCR 36
Table 3. Nucleotide wequence of siRNA 37
Ⅲ.RESULTS 38
A. Human skeletal muscle myoblasts differentiation 39
B. PA treatment induces insulin resistance in HSMMs 41
C. PA increases intracellular iron levels 43
D. Excess iron induces insulin resistance 45
E. Knockdown of tfR1 protects cells from excess iron-induced insulin resistance 50
F. TfR1 is overexpressed in diabetic muscle 52
G. IRP2 is essential for tfR1 induction by PA stimulation 54
H. Calcium flux regulates tfR1-mediated iron overload to induce insulin resistance 58
I. Excess intracellular iron stimulates JNK activation, which is inhibited by DFO 67
Ⅳ.DISCUSSION 70
Ⅴ.CONCLUSION 76
Ⅵ.REFERENCES 78
국문요약 92
