Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2009
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | III, 129 S. Il., graph. Darst. |
Internformat
MARC
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| 245 | 1 | 0 | |a Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis |c vorgelegt von Sabine Jordan |
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Datensatz im Suchindex
| _version_ | 1804143377682268160 |
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| adam_text | Titel: Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis
Autor: Jordan, Sabine
Jahr: 2009
Table of Contents
TABLE OF CONTENTS
/ Introduction........................................................................................../
1.1 Diabetes mellitus....................................................................................................1
1.2 Molecular mechanisms of insulin signaling..........................................................2
1.3 Molecular mechanisms of insulin resistance.........................................................5
1.3.1 Mutation of insulin signaling genes 5
1.3.2 Transcriptional regulation of insulin signaling genes 6
1.3.3 Posttranslational modification of insulin signaling proteins 7
1.3.4 Ubiquitin-mediated degradation of insulin signaling proteins 8
1.4 Posttranscriptional gene silencing.........................................................................9
1.4.1 Biogenesis of miRNAs 9
1.4.2 Function of miRNAs 13
1.4.3 miRNAs as regulators of metabolism 15
1.5 Objectives.............................................................................................................17
2 Material and Methods.........................................................................18
2.1 Chemicals and Enzymes......................................................................................18
2.2 Molecular Biology................................................................................................18
2.2.1 Isolation of RNA 18
2.2.2 Purification of RNA 19
2.2.2.1 Gel extraction 19
2.2.2.2 Phenol-chloroform extraction 19
2.2.3 Cloning of small RNA molecules 19
2.2.4 Northern blot analysis 20
2.2.5 RT-PCR and quantitative real-time PCR 22
2.2.6 Isolation of genomic DNA 23
2.2.7 Polymerase Chain Reaction (PCR) 23
2.2.8 Agarose gel electrophoresis and DNA gel extraction 24
2.2.9 Cloning procedures 25
2.2.9.1 Generation of miR-143 expression vectors 25
2.2.9.2 Generation of an adenoviral vector 26
2.2.9.3 Generation of the miR-143 RMCE targeting vector 27
2.2.9.4 Generation of Iuciferase-reporter constructs 27
2.2.10 Competent cells and isolation of plasmid DNA 27
2.2.11 DNA sequencing 28
2.2.12 Southern blot analysis 28
2.3 Cell Biology_______________________.............................................................29
2.3.1 C2C12 myoblast cell culture 29
2.3.2 HEK 293 cell culture 29
2.3.3 HepG2 cell culture 30
2.3.4 ES cell culture 30
2.3.5 Dual luciferase reporter assay 31
2.3.6 Isolation of primary hepatocytes 31
2.4 Biochemistry ?-----------------------------?-------------------------------------------------32
2.4.1 Preparation of tissue protein extracts 32
2.4.2 Western blot analysis 32
I
Table of Contents
2.4.3 Enzyme-linked Immunosorbent Assay (ELISA) 33
2.4.4 Histological analysis and immunohistochemistry 33
2.4.5 In vivo SILAC 34
2.4.6 Lipid Analysis 34
2.5 Mouse Analysis....................................................................................................36
2.5.1 Animal care 36
2.5.2 Mouse lines 36
2.5.2.1 db/db mice 36
2.5.2.2 High fat diet (HFD) mice 36
2.5.2.3 miR-143DOX mice 37
2.5.2.4 shRNAUcZ mice 37
2.5.2.5 miR-143/145 KO mice 37
2.5.3 Doxycycline administration 37
2.5.4 Body weight and food intake 37
2.5.5 Collection of blood samples and determination of blood glucose levels 37
2.5.6 Glucose and insulin tolerance test 38
2.5.7 Homeostatic Model Assessment (HOMA) 38
2.5.8 Glucose-stimulated insulin secretion 38
2.5.9 Insulin signaling 38
2.5.10 Analysis of body composition 39
2.6 Computer Analysis...............................................................................................39
2.6.1 Computational analysis of miRNA library 39
2.6.2 Densitometrical analysis 39
2.6.3 Statistical methods 39
3 Results.................................................................................................40
3.1 Analysis of the miRNA expression profile in mouse liver..................................40
3.1.1 Generation of a liver-specific miRNA library 40
3.1.2 Verification of 32 cloned miRNAs by Northern blot analysis 43
3.1.3 Identification of an unknown miRNA 46
3.2 Analysis of hepatic miR-143 expression in insulin-resistant mice......................47
3.2.1 Increased hepatic expression of miR-143 in db/db mice 47
3.2.2 Increased hepatic expression of miR-143 in diet-induced obese mice 48
3.3 Conditional overexpression of miR-143 in vivo...................................................49
3.3.1 Generation of miR-143 expression vectors 50
3.3.1.1 Expression of miR-143 under control of a polll promoter in the context of random
intron sequences 50
3.3.1.2 Expression of miR-143 under control of a polll or polIII promoter in the context of
genomic sequences 52
3.3.1.3 Generation of an adenoviral vector for ectopic miR-143 expression 54
3.3.2 Generation of a transgenic mouse model for conditional miR-143 overexpression
57
3.3.2.1 Tissue-dependent variability of inducible miR-143 overexpression 59
3.3.2.2 Time course analysis of inducible miR-143 overexpression 60
3.4 Metabolic phenotyping of mice overexpressing miR-143________...------------61
3.4.1 Conditional overexpression of miR-143 does not alter energy homeostasis 61
3.4.2 Conditional overexpression of miR-143 impairs glucose metabolism 63
3.4.2.1 Impaired glucose metabolism in miR-143 x does not arise from p-cell
dysfunction 65
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3.4.2.2 Conditional overexpression of an unrelated small RNA does not affect glucose
metabolism 67
3.4.2.3 Conditional overexpression of miR-143 mimics the effects of diet-induced obesity
on glucose metabolism 68
3.4.3 Impaired insulin-stimulated AKT-activation in the liver of miR-143DOX mice 70
3.5 Identification of miR-143 targets.........................................................................72
3.5.1 In vitro assay suggests IRS-1 as an miR-143 target 72
3.5.2 Unaltered expression and activation of IRS-1 during miR-143 overexpression in
vivo 74
3.5.3 In vivo SILAC identifies oxysterol-binding protein-related protein 8 (ORP8) as a
miR-143 target 75
3.5.4 miR-143-mediated downregulation of ORP8 alters hepatic cholesterol and lipid
metabolism 77
3.5.4.1 Altered hepatic lipid composition in miR-MS00* mice suggests cellular 25-OH-
cholesterol accumulation 78
3.5.4.2 25-OH-cholesterol inhibits insulin-stimulated AKT activation 82
3.6 Metabolic analysis of miR-143-deficient mice.....................................................83
3.6.1 Increased expression of miR-143/145 targets in the liver of miR-143/145 knockout
mice 84
3.6.2 miR-143/145 knockout mice show improved glucose homeostasis 85
3.6.3 Enhanced insulin signaling in the liver of miR-143/145 knockout mice 86
4 Discussion...........................................................................................88
4.1 Hepatic miRNA expression profile reveals obesity-induced upregulation of
miR-143................................................................................................................88
4.2 Conditional overexpression of miR-143 in vivo...................................................91
4.3 Overexpression of miR-143 impairs glucose homeostasis and hepatic insulin
signaling...............................................................................................................93
4.4 miR-143 targets oxysterol-binding protein-related protein 8 (ORP8)...............95
4.5 Loss-of-function analysis confirms role of miR-143 in glucose metabolism in
vivo........................................................................................................................99
4.6 Perspectives........................................................................................................101
5 Summary...........................................................................................102
6 Zusammenfassung............................................................................103
7 References.........................................................................................104
8 Figure Index.....................................................................................119
9 Table Index.......................................................................................121
10 List of Abbreviations.........................................................................122
Acknowledgements........................................................................................127
Eidesstattliche Erklarung..............................................................................128
Curriculum Vitae...........................................................................................129
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| any_adam_object | 1 |
| author | Jordan, Sabine 1981- |
| author_GND | (DE-588)14133360X |
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| bvnumber | BV036726632 |
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| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 616 - Diseases |
| dewey-raw | 616.4 |
| dewey-search | 616.4 |
| dewey-sort | 3616.4 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
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| spelling | Jordan, Sabine 1981- Verfasser (DE-588)14133360X aut Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis vorgelegt von Sabine Jordan 2009 III, 129 S. Il., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Köln, Univ., Diss., 2009 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020644387&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Jordan, Sabine 1981- Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis |
| subject_GND | (DE-588)4113937-9 |
| title | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis |
| title_auth | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis |
| title_exact_search | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis |
| title_full | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis vorgelegt von Sabine Jordan |
| title_fullStr | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis vorgelegt von Sabine Jordan |
| title_full_unstemmed | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis vorgelegt von Sabine Jordan |
| title_short | Obesity induced increase in hepatic expression of microRNA miR-143 impairs glucose homeostasis |
| title_sort | obesity induced increase in hepatic expression of microrna mir 143 impairs glucose homeostasis |
| topic_facet | Hochschulschrift |
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