췌장 베타세포의 인슐린 생성 및 분비기능에 관여하는 PARK2 유전자의 역활
Role of PARK2 Gene in Maintenance of Pancreatic Beta Cell Functions
- 주제(키워드) genetic association , single nucleotide polymorphism (SNP) , mitochondiral dysfunction , mitochondrial quality control , mitophagy , PARK2 , parkin , fasting blood glucose , insulin secretion , pancreatic beta-cell , type 2 diabetes
- 발행기관 아주대학교
- 지도교수 정선용
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000017620
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Type 2 diabetes (T2D) is characterized by decreased insulin secretion and action. Although many studies have reported the genetic loci/genes for susceptibility to T2D, the vast majority of genetic factors in T2D are still unknown. Mitophagy, a selective elimination system for impaired mitochondria, plays a key role in mitochondrial quality control. Defects in mitophagy that contribute to causing mitochondrial dysfunction have been recently suggested to be involved in the pathogenesis of T2D as well as in neurodegenerative diseases. By performing a candidate-gene association analysis of the single nucleotide polymorphisms (SNPs) in the 28 mitophagy-related genes with glucose- and insulin-related quantitative traits in the Korean study cohort (7,551 subjects), I found that two SNPs, rs10455889 and rs9365294, in the PARK2 gene encoding an E3 ubiquitin-protein ligase (parkin) were significantly associated with fasting plasma glucose levels and insulin secretion indices in response to glucose stimulation in men (3,747 subjects). Replication analysis in two other Korean cohorts and a European-descendent cohort revealed that genetic variants in the PARK2 gene were associated with glucose homeostasis-related quantitative traits in three cohorts. One variant showed a meaningful linkage disequilibrium correlation to the two discovered PARK2 SNPs. The PARK2 gene is known to be mutated in certain cases of familial Parkinson’s disease. Nevertheless, my results indicated a close relationship between the PARK2 gene and pancreatic β-cell dysfunction-related T2D. In order to prove this finding experimentally, I first investigated whether the PARK2 gene is expressed in the pancreatic tissues. Gene expression profiling of the Park2 gene in rats showed its predominant expression in the pancreatic islets as well as in the brain, skeletal muscle, and liver tissues. Next, I performed a functional analysis to investigate whether the alterations in PARK2 gene expression have an influence on insulin-producing pancreatic β-cell functions. Downregulation of the Park2 gene by RNA interference in rat INS-1 β-cells resulted in a significant decrease in the transcriptional and translational insulin gene (Ins1) expression levels and glucose-stimulated insulin secretion. The mRNA levels of the genes Pdx1, Mafa, Mafb, Nkx2-2, Nkx6-1, Pax4, and Irs1 that encode the pancreatic transcription factors were significantly decreased in the Park2-depleted INS-1 β-cells. In addition, the INS-1 β-cells lacking parkin exhibited decreased intracellular ATP level and mitochondrial membrane potential (m∆ψ) but increased reactive oxygen species production and mitochondrial fragmentation. This finding is particularly important because I have provided both population-based statistical and experimental evidence to support the claim that the mitophagy-regulating PARK2 gene plays an important role in the maintenance of pancreatic β-cell function as well as in the maintenance of neuronal cell function.
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ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES v
LIST OF TABLES vii
I. INTRODUCTION 1
II. MATERIALS AND METHODS 10
1. Chemicals & Reagents 10
2. Korean Association Resource (KARE) study cohort 10
3. Health2 Type 2 Diabetes (Health2 T2D) Study cohort 12
4. Korean Child & Adolescent cohort Study (KoCAS) cohort 12
5. Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) cohort
13
6. Genotyping and SNP selection 13
7. Cell culture 14
8. Isolation of rat tissues and pancreatic islet cells 14
9. Immunofluorescence in rat pancreas tissue 15
10. Gene silencing by small interfering RNAs 16
11. Quantification of intracellular and extracellular insulin contents 16
12. RNA isolation, cDNA synthesis and real-time RT-PCR 17
13. Western blotting 18
14. Measurement of intracellular ATP content 19
15. JC-1 staining and fluorescence activated cell sorter (FACS) analysis 19
16. Flow-cytometric measurement of reactive oxygen species 20
17. Confocal microscopy 20
18. Electron microscopy 21
19. Subcellular fractionation 21
20. Statistical analysis 22
III. RESULTS 24
1. Association of PARK2 SNPs with quantitative glycemic traits 24
2. Predominant expression of parkin in pancreatic islets in rats 57
3. Decrease in insulin production and secretion by knockdown of Park2 in rat INS-1 β-
cells 61
4. Alteration in mitochondrial ATP generation by knockdown of Park2 in rat INS-1 β-
cells 69
5. Alteration in mitochondrial function by knockdown of Park2 in rat INS-1 β-cells
71
6. Alteration of parkin expression may contribute to β-cell dysfunction in high-fat diet
mice 79
IV. DISCUSSION 81
V. CONCLUSION 96
REFERENCES 97
국문요약 118
