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

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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 .............................
Publié le : jeudi 1 janvier 2009
Lecture(s) : 21
Tags :
Source : NBN-RESOLVING.DE/URN:NBN:DE:HBZ:38-30539
Nombre de pages : 125
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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 energy homeostasis...............................................................15
Figure 5: POMC and AgRP neuron-restricted inactivation of the insulin receptor gene. .......39
ΔPOMC ΔAgRPFigure 6: General scheme of mice with POMC (IR ) and AgRP (IR ) neuron-
restricted inactivation of the insulin receptor gene. ..............................................40
Figure 7: Verification of Cre-mediated recombination in PomcCre and AgRPCre mice. .......41
Figure 8: Western blot analysis of insulin receptor expression..............42
POMC AgRPΔ ΔFigure 9: Insulin signaling in peripheral organs of control, IR and IR mice. .........42
POMCΔFigure 10: Regulation of the hypothalamic-pituitary-adrenal axis in IR mice. ..............43
ΔPOMC ΔAgRPFigure 11: Fertility of IR and IR mice. .................................................................44
∆POMC ∆AgRPFigure 12: PIP3 formation in hypothalamic neurons of control, IR and IR reporter
mice.....................................................................................................................45
Figure 13: Z/EG expression construct...................47
ΔPOMCFigure 14: Effects of insulin on electrical activity of control and IR -Z/EG neurons. .....48
AgRPΔFigure 15: Effects of insulin and tolbutamide on electrical activity of control and IR -
Z/EG neurons.......................................................................................................49
ΔPOMC ΔAgRPFigure 16: Body weight of IR and IR mice...........................50
ΔPOMC ΔAgRPFigure 17: Serum leptin concentrations in IR and IR mice....51
POMC AgRPΔ ΔFigure 18: Daily food intake of IR and IR mice....................................................51
ΔPOMC ΔAgRPFigure 19: Body weight, leptin levels and food intake of IR and IR female mice. .52
ΔPOMC ΔAgRP Figure 20: Epigonadal fat pad weights of female and male IR and IR mice...........53
POMC AgRPΔ ΔFigure 21: Hypothalamic neuropeptide expression in IR and IR male mice. .........54
ΔPOMC ΔAgRPFigure 22: Body length of IR and IR male mice....................................................55
ΔPOMCFigure 23: Fasting blood glucose levels and serum insulin concentrations in IR and
AgRPΔIR male mice................................................................................................56
POMC AgRPΔ ΔFigure 24: Glucose tolerance in IR and IR male mice...........................................57
Figure 25: General scheme of a euglycemic-hyperinsulinemic clamp...58
POMC AgRP Δ ΔFigure 26: Regulation of glucose homeostasis in IR and IR mice. .........................59
IV Figure Index
Figure 27: Insulin-stimulated tissue glucose uptake in euglycemic-hyperinsulinemic clamps.
............................................................................................................................60
Figure 28: Hepatic interleukin-6 expression under steady state and basal conditions in
euglycemic-hyperinsulinemic clamps...................................61
Figure 29: Hepatic G6Pase-expression under steady state and basal conditions in euglycemic-
hyperinsulinemic clamps......................................................................................62
Figure 30: Hepatic cytokine and chemokine expression at the end of euglycemic-
hyperinsulinemic clamps......................63
Figure 31: Restricted inactivation of the insulin receptor gene in tyrosine hydroxylase-
expressing cells....................................................................................................64
Figure 32: General scheme of mice with restricted inactivation of the insulin receptor gene in
ΔThtyrosine hydroxylase-expressing cells (IR ).......................................................64
Figure 33: Verification of Cre-mediated recombination in ThIRESCre mice. ........................65
∆ThFigure 34: PIP3 formation in neurons of control and IR reporter mice. .............................66
ΔThFigure 35: Fertility of IR mice..........................................................................................67
Th ΔFigure 36: Body weight, fat content and leptin levels of IR mice. .....................................69
∆ThFigure 37: Glucose tolerance and insulin sensitivity in IR mice........70
∆ThFigure 38: Serum insulin concentrations in IR mice. ........................................................71
∆ThFigure 39: Food intake of IR mice....................................................71
∆ThFigure 40: Basal metabolic rate and locomotor activity in IR mice...................................72
Figure 41: Expression levels of genes critically involved in dopamine signaling in the VTA,
∆ThNAc, and CPu of IR mice. ...............................................................................73
Figure 42: Long-term euglycemic-hyperinsulinemic clamps in C57BL/6 mice. ....................75
Figure 43: Synaptic input on dopaminergic neurons in C57BL/6 mice after long-term
euglycemic-hyperinsulinemic clamp studies.........................................................76
Figure 44: Model of hepatic-regulation via insulin action on AgRP neurons.83






V Table Index
Table Index

Table 1: Chemicals...............................................................................................................21
Table 2: Enzymes.................23
Table 3: Oligonucleotides used for genotyping.....................................................................25
Table 4: Probes used for Realtime PCR................26
Table 5: Primary antibodies used for Western blot analysis. .................................................32









VI Abbreviations
Abbreviations

°C degrees Celsius
3’ three prime end of DNA sequences
5’ five prime end of DNA sequences
A adenosine
a.m. ante meridiem
AAAD (Ddc) aromatic L-amino acid decarboxylase
ACTH adrenocorticotrophin
AgRP agouti-related peptide
Akt proteinkinase B
ARC arcuate nucleus
Avertin tribromoethyl alcohol and tert-amyl alcohol
Ba(OH) barium hydroxide 2
BAT brown adipose tissue
BMI body mass index
bp base pairs
C cytosine
c DNA concentration
CaCl calcium chloride 2
cAMP cyclic adenosine monophosphate
Ccl chemoligand (C-C motif)
cDNA complementary DNA
Ci Curie
CNS central nervous system
COMT catechol-O-methyl transferase
CPu caudate putamen
Cre site specific recombinase from phage P1 (causes recombination)
Cxcl chemoligand (C-X-C motif)
d deci
Da Dalton
DAPI 4',6-diamidino-2-phenylindole
DAT dopamine transporter
VII

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