Impact of 17BetaEstradiol on Beta-cell survival of female Munich Ins2C95S mutant mice [Elektronische Ressource] / Marion Susanne Schuster. Betreuer: Rüdiger Wanke

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Impact of 17βEstradiol on β-cell C95Ssurvival of female Munich Ins2 mutant mice Marion Susanne Schuster Aus dem Zentrum für Klinische Tiermedizin der Ludwig-Maximilians-Universität München Angefertigt unter der Leitung von Univ.- Prof. Dr. R. Wanke Impact of 17βEstradiol on β-cell C95Ssurvival of female Munich Ins2 mutant mice Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München von Marion Susanne Schuster aus Kirchheim/Teck München, 2011 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. Braun Berichterstatter: Univ.-Prof. Dr. Wanke Korreferent/en: Univ.-Prof. Dr. Göbel Univ.-Prof. Dr. Aigner Prof. Dr. Kaltner Univ.-Prof. Dr. Sutter Tag der Promotion: 12. Februar 2011 Meiner lieben Mutter und meiner Nichte Stefanie 1 INTRODUCTION 1 2 LITERATURE REVIEW 3 2.1 Diabetes mellitus 3 2.1.1 Definition and description 3 2.1.2 Classification and diagnosis criteria in human beings 4 2.1.2.1 Classification 4 2.1.2.2 Diagnosis criteria 5 2.1.3 Global and economical burden 7 2.1.3.1 Global burden of diabetes mellitus 7 2.1.3.2 Economical burden 8 2.

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Publié le 01 janvier 2011
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Impact of 17βEstradiol on β-cell
C95S
survival of female Munich Ins2
mutant mice














Marion Susanne Schuster




Aus dem Zentrum für Klinische Tiermedizin
der Ludwig-Maximilians-Universität München


Angefertigt unter der Leitung von
Univ.- Prof. Dr. R. Wanke






Impact of 17βEstradiol on β-cell
C95S
survival of female Munich Ins2
mutant mice







Inaugural-Dissertation
zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München

von
Marion Susanne Schuster
aus Kirchheim/Teck

München, 2011



Gedruckt mit Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München
















Dekan: Univ.-Prof. Dr. Braun

Berichterstatter: Univ.-Prof. Dr. Wanke

Korreferent/en: Univ.-Prof. Dr. Göbel
Univ.-Prof. Dr. Aigner
Prof. Dr. Kaltner
Univ.-Prof. Dr. Sutter






Tag der Promotion: 12. Februar 2011















Meiner lieben Mutter
und meiner Nichte Stefanie














1 INTRODUCTION 1
2 LITERATURE REVIEW 3
2.1 Diabetes mellitus 3
2.1.1 Definition and description 3
2.1.2 Classification and diagnosis criteria in human beings 4
2.1.2.1 Classification 4
2.1.2.2 Diagnosis criteria 5
2.1.3 Global and economical burden 7
2.1.3.1 Global burden of diabetes mellitus 7
2.1.3.2 Economical burden 8
2.2 Animal models in diabetic research 9
2.2.1 The role of animal models for human diseases 9
2.2.2 Animal models for diabetes mellitus 10
2.2.3 ENU mutagenesis 10
2.2.3.1 Munich ENU Mouse Mutagenesis Project 11
2.2.3.2 ENU-induced hyperglycaemia models 12
2.3 Insulin gene mutations 13
2.3.1 Mutations in humans 13
C95S
2.3.2 The Munich Ins2 mutant mouse 15
C95S
2.3.2.1 Heterozygous Munich Ins2 mutant mice 15
C95S
2.3.2.2 Homozygous Munich Ins2 mutant mice 17
2.3.3 Akita mouse 17
2.3.3.1 Heterozygous Akita mutant mice 18
2.3.3.2 Homozygous Akita mutant mice 20
2.3.3.3 ER-stress in the Akita mouse 20
2.3.4 Ins1 and Ins2 null mutant mice 23
2.3.4.1 Double homozygous Ins1 and Ins2 null mutant mice 24
2.3.4.2 Single homozygous Ins1 and Ins2 null mutant mice 24
2.3.4.3 Single heterozygous Ins1 and Ins2 null mutant mice 25
2.4 Estrogen 26
2.4.1 Estrogen production and action 26
2.4.2 Estrogen receptors 28
2.4.2.1 Classical genomic estrogen receptors (ERα and ERβ) 29
2.4.2.2 Rapid estrogen signalling 30
2.4.2.3 Non-classical estrogen receptors 31
2.4.3 Role of estrogen in glucose homeostasis 32
2.4.4 Effects of estrogen on pancreas 33
2.4.4.1 Impact of estrogen on pancreatic insulin content 34
2.4.4.2 Enhancement of insulin secretion by estrogen 36
2.4.5 Effects of estrogen on skeletal muscle, adipose tissue and liver 38
2.4.5.1 Insulin sensitivity and insulin resistance 38
2.4.5.2 Modulation of GLUT4 expression by estrogen 39
2.4.6 Positive effects of estrogen on oxidative stress and apoptosis 39
2.4.7 Role of estrogen in endoplasmic reticulum stress 41
3 RESEARCH DESIGN AND METHODS 43
3.1 Research design 43
3.2 Materials and methods 43
3.2.1 Animals 43
3.2.2 Genotyping 45
3.2.3 Ovariectomy and pellet implantation 49
3.2.4 Body weight 50
3.2.5 Blood glucose concentration 50
3.2.5.1 Randomly fed mice 50
3.2.5.2 Fasted mice 50
3.2.6 Oral glucose tolerance test (OGTT) 50
3.2.7 Intraperitoneal insulin tolerance test 52
3.2.8 Serum insulin concentration 52
3.2.9 Serum estradiol concentration 52
3.2.10 Serum lipid peroxidation 53
3.2.11 Western blot analyses of isolated islets 53
3.2.11.1 Islet isolation 54
3.2.11.2 Islet protein content 57
3.2.11.3 SDS-PAGE 58
3.2.11.4 Western blot 59
3.2.11.5 Silver staining and drying 60
3.2.11.6 Western blot analysis 62
3.2.12 Necropsy and pancreas preparation 64
3.2.13 Immunohistochemistry of the pancreas 65
3.2.13.1 Insulin 65
3.2.13.2 Glucagon, somatostatin and pancreatic polypeptide 66
3.2.14 Quantitative stereological analyses 66
3.2.14.1 Quantification of the total pancreas volume (V ) 66 pan
3.2.14.2 Determination of the relative pancreas weight 67
3.2.14.3 Quantitative stereological parameters 67
3.2.15 Transmission electron microscopy (TEM) 68

4 RESULTS 72
4.1 Clinical investigations 72
4.1.1 Body weight 72
4.1.2 Blood glucose concentration 73
4.1.2.1 Randomly fed mice 73
4.1.2.2 Fourteen-hours fasted mice 74
4.1.3 Oral glucose tolerance test (OGTT) 75
4.1.4 Intraperitoneal insulin tolerance test (ipITT) 79
4.1.5 Serum insulin concentration 80
4.1.6 Serum estradiol concentration 82
4.2 Beta cell function indices 83
4.2.1 HOMA B and HOMA IR 84
4.2.2 Quicki 85
4.2.3 FGIR 86
4.3 Oxidative stress 86
4.4 Isolated pancreatic islets 88
4.4.1 ER-stress 88
4.4.1.1 BiP/β-actin 88
4.4.1.2 PeIF2alpha/β-actin 89
4.4.1.3 CHOP/β-actin 89
4.4.2 Apoptosis 90
4.4.3 Cell proliferation 91
4.5 Qualitative histological evaluations of the endocrine pancreas 92
4.5.1 Insulin 92
4.5.2 Glucagon, somatostatin and pancreatic polypeptide (PP) 94
4.5.3 Isolated β-cells 95
4.6 Quantitative stereological analyses of the endocrine pancreas 95
4.6.1 Total pancreas volume 95
4.6.2 Relative pancreas weight 96
4.6.3 Volume density of islets in the pancreas 97
4.6.4 Total islet volume 97
4.6.5 Volume density of β-cells in the islets 98
4.6.6 Total β-cell volume 98
4.6.7 Volume density of non-β-cells in the islets 99
4.6.8 Total volume of non-β-cells in the islets 99
4.6.9 Β-cell to non-β-cell ratio 100

4.6.10 Volume density of isolated β-cells in the pancreas 101
4.6.11 Total volume of isolated β-cells 101
4.7 Transmission electron microscopy 102
5 DISCUSSION 107
5.1 Glucose homeostasis 107
5.1.1 Blood glucose and insulin secretion 107
5.1.2 Insulin sensitivity 113
5.2 Body weight 114
5.3 Oxidative stress 116
5.4 ER-stress 117
5.4.1 Islet isolation 117
5.4.2 ER-stress markers 118
5.5 Qualitative and quantitative morphological investigations of the endocrine
pancreas 120
5.5.1 Qualitative histological analyses 121
5.5.2 Quantitative stereological analyses 121
5.6 Electron microscopic findings in β-cells 128
5.7 Conclusion 131
6 PERSPECTIVE 133
7 SUMMARY 134
8 ZUSAMMENFASSUNG 136
9 REFERENCES 139
10 ACKNOWLEDGEMENTS 162 1 Introduction 1

1 Introduction
Diabetes mellitus is one of the most common human diseases worldwide with a still
increasing prevalence. Today there are more than 300 million people suffering from
diabetes and its accompanying diseases. It is expected, that the number of diabetic
patients will reach close to 500 million within 20 years, if nothing is done to slow
down the epidemic (International Diabetes Federation 2009).
Animal models, particularly mouse models, play an essential role for studying the
pathogenesis of diabetes mellitus and its complications. Transgenic and knock-out
mouse models used to be a common and powerful tool to explore the pathogenesis
of diabetes mellitus. In recent years, more and more mutant mouse models,
especially with point mutations in a diabetes relevant gene, have additionally been
established. Due to the fact that such mutations are also found in human patients,
these mutant mouse models are of great value in prospective diabetes research.
C95SThe Munich Ins2 mutant mouse and the Akita mutant mouse are two mouse
models, which possess point mutations in the Ins2 gene. These mutations lead to the
loss of the interchain and the intrachain disulfide bond of insulin 2, respectively
(Herbach et al. 2007, Wang et al. 1999). Extensive studies could demonstrate that
misfolded (pro-)insulin accumulates in β-cells of Akita mice, what leads to
endoplasmic reticulum (ER)-stress and β-cell dysfunction (Izumi et al. 2003,
Nozaki et al. 2004, Zuber et al. 2004). Moreover it could be shown that misfolded and
accumulated proteins can also induce β-cell apoptosis
(Scheuner and Kaufman 2008, Xu et al. 2005).
The disease pattern of both Ins2 mutant models is similar. It is characterized by a
progressive diabetic phenotype with severe hyperglycaemia, disturbed insulin
secretion, insulin resistance and profound β-cell loss of male mutant mice, and a
much milder disease-form with preserved β-cell mass of female mutant mice. This
considerable difference is probably based on several protective effects attributed to
estrogen, such as antioxidative effects (Katalinic et al. 2005, Le May et al. 2006),
enhancement of insulin sensitivity (Gonzalez et al. 2001, Lee et al. 1999), insulin
secretion (Balhuizen et al. 2010, Ropero et al. 1999) and diminution of ER-stress
(Kozlov et al. 2010). 1 Introduction 2

The aim of the present study was to investigate the impact of 17βEstradiol on the
C95S
survival of β-cells in female Munich Ins2 mutant mice. To exclude effects of other
ovarian hormones, one group of ovariectomized female mutant mice was
supplemented with 17βEstradiol long-term pellets. Sham-operated placebo-treated
mutant and wild-type mice served as controls.
Besides various clinical tests to determine β-cell function and insulin sensitivity,
isolated pancreatic islets were analysed with respect to ER-stress, islet-cell apoptosis
and cell proliferation, and further oxidative stress was investigated in serum samples.
Additionally, the effects of ovariectomy and estradiol supplementation on the
endocrine pancreas were determined, using qualitative histological as well as
quantitative stereological methods.