Genetic microdissection of insulin action on neurocircuits in control of glucose and energy homeostasis [Elektronische Ressource] / vorgelegt von Anne Christine Könner

Christine Könner

Sujets

• Gesundheit

Informations

• Publié par: universitat_zu_koln
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 15 Mo

Extrait

Genetic Microdissection of Insulin Action on
Neurocircuits in Control of Glucose and Energy
Homeostasis


Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln



vorgelegt von
Anne Christine Könner
aus Köln

Köln 2009













Berichterstatter: Prof. Dr. Jens C. Brüning
Prof. Dr. Wilhelm Krone
Prof. Dr. Tamas L. Horvath

Tag der mündlichen Prüfung: 23.10.2009















Wenn ein Tier oder Mensch seine ganze
Aufmerksamkeit und seinen ganzen Willen auf
eine bestimmte Sache richtet, dann erreicht er
sie auch. Das ist alles.
Hermann Hesse, Demian
Table of contents

Figure Index................................................................................................... IV
Table Index.... VI
AbbreviationsVII
1 Introduction.................................................................................................1
1.1 Obesity and Type 2 Diabetes Mellitus........1
1.2 Energy Homeostasis...................................2
1.3 Insulin........................................................................................3
1.3.1 History, Structure and Biosynthesis.....................................3
1.3.2 Metabolic Effects ................................3
1.4 The Insulin Receptor..................................5
1.4.1 Molecular Mechanisms of Insulin Receptor Signaling .........................................5
1.5 Leptin ........................................................................................................................7
1.5.1 Mechanisms of Leptin Receptor Signaling...........................8
1.6 Central Regulation of Energy Homeostasis..............................10
1.6.1 The Hypothalamus ............................................................................................11
1.6.2 The Central Melanocortin System and Regulation by Peripheral Hormones.......12
1.7 The Dopaminergic System of the Brain....................................16
1.7.1 Peripheral Hormonal Signals in Food Reward ...................................................18
1.8 Objectives................................................................................20
2 Materials and Methods.............21
2.1 Chemicals and Biological Material...........................................21
2.2 Molecular Biology ...................................................................23
2.2.1 Isolation of Genomic DNA................23
2.2.2 Quantification of Nucleic Acids.........................................24
2.2.3 Polymerase Chain Reaction (PCR) ....................................24
2.2.4 RNA Extraction, RT PCR and Quantitative Realtime PCR ................................25
2.3 Cell Biology ............................................26
2.3.1 Histological Analysis and Immunohistochemistry .............................................26
2.3.1.1 Immunohistochemistry...............................................26
2.3.1.2 Analysis of PIP3 formation in situ..............................................................28
2.3.2 Histomorphology...............................................................28
2.3.3 Electrophysiology29
I Table of contents
2.3.4 Electron Microscopy .........................................................................................29
2.4 Biochemistry............................................31
2.4.1 Enzyme-linked Immunosorbent Assay (ELISA) ................................................31
2.4.2 Protein Extraction..............................................................31
2.4.3 Western Blot Analysis.......................32
2.5 Mouse Experiments.................................................................33
2.5.1 Animal Care ......................................33
2.5.2 Mice..................................................................................33
2.5.3 Collection of Blood Samples and Determination of Blood Glucose Levels ........34
2.5.4 Food Intake and Indirect Calorimetry ................................................................34
2.5.5 Glucose and Insulin Tolerance Test...34
2.5.6 Insulin Signaling ...............................................................35
2.5.7 Analysis of Body Composition ..........................................................................35
2.5.8 Fertility Assessment..........................................................35
2.5.9 Restraint Stress..................................35
2.5.10 Euglycemic-Hyperinsulinemic Clamp Studies in Awake Mice.........................36
2.5.10.1 Catheter Implantation ...............................................................................36
2.5.10.2 Radioactive Euglycemic-Hyperinsulinemic Clamp Experiment ................36
2.5.10.2.1 Assays ...............................................................................................37
2.5.10.2.2 Calculations.......................37
2.5.10.3 Long-term euglycemic-hyperinsulinemic clamp experiment.....................38
2.6 Computer Analysis ..................................................................................................38
2.6.1 Densitometrical Analysis...................................................................................38
2.6.2 Statistical Methods............................38
3 Results........................................39
3.1 Generation of POMC and AgRP neuron-specific insulin receptor knockout mice.....39
3.2 Insulin hyperpolarizes AgRP and POMC neurons via K channel activation.........46 ATP
∆AgRP ∆POMC3.3 Normal body weight and energy homeostasis in IR and IR mice .............50
∆AgRP3.4 IR mice fail to fully suppress hepatic glucose production .................................55
∆AgRP3.5 Reduced hepatic IL-6 expression in IR mice ....................................................60
3.6 Inactivation of the insulin receptor gene in dopaminergic cells64
ΔTh3.7 Increased adiposity in IR mice.............................................68
ΔTh3.8 Unaltered glucose homeostasis in IR mice ...........................................................70
ThΔ3.9 Unaltered energy expenditure but increased food intake in IR mice .....................71
II Table of contents
3.10 Increased expression of tyrosine hydroxylase and dopamine receptor 2 in the ventral
∆Thtegmental area of IR mice ............................................................................................73
3.11 Insulin action decreases excitatory inputs on dopaminergic neurons in the VTA ....74
4 Discussion ..................................................................................................77
4.1 Role of central insulin signaling in regulation of energy homeostasis and glucose
metabolism.......................................................77
4.2 Inactivation of the insulin receptor gene specifically in POMC and AgRP neurons..78
4.2.1 Role of insulin action on POMC and AgRP neurons in the regulation of energy
homeostasis...................................................................................................................79
4.2.2 Role of insulin action on POMC and AgRP neurons in the regulation of glucose
metabolism...................80
4.3 Inactivation of the insulin receptor gene specifically in dopaminergic cells..............84
4.3.1 Role of insulin action on dopaminergic cells in the regulation of energy and
glucose homeostasis......................................................................................................85
4.3.2 Role of insulin action on dopaminergic cells in the regulation of gene expression
and synaptic plasticity...86
4.4 Perspectives.............................................................................................................87
5 Summary....89
6 Zusammenfassung.....................................................................................90
7 References..................................91
8 Acknowledgements..................108
9 Erklärung ................................................................109
10 Curriculum vitae...................110





III Figure Index
Figure Index

Figure 1: Insulin receptor signal transduction. ........................................................................7
Figure 2: Leptin receptor signal transduction..........9
Figure 3: Schematic anatomical structure of the hypothalamus.............12
Figure 4: Central regulation of e