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  • Characterization of -cell Specific Knockout of UCP2

    By

    Sobia Sultan

    A thesis submitted in conformity with the requirements

    for the degree of Masters of Science

    Graduate Department of Physiology

    University of Toronto

    Copyright by Sobia Sultan (2010)

  • ii

    Abstract Characterization of -cell Specific Knockout of UCP2

    Sobia Sultan

    MSc. Thesis 2010

    Department of Physiology

    University of Toronto

    The whole body UCP2 knockout (UCP2/) have enhanced insulin secretion and higher ATP

    content. However, these changes could be due to indirect effects of extra-pancreatic deletion and

    therefore, generating beta-cell specific knockout mice (UCP2BKO) is essential. A 90%

    knockdown of UCP2 protein was observed in beta-cells of UCP2BKO mice. No significant

    differences were observed in body weight accumulation, fasting blood glucose, plasma insulin or

    glucagon. UCP2BKO had impaired oral glucose tolerance with no differences in insulin

    secretion or sensitivity. Enhanced ROS accumulation was observed in the beta-cells of

    UCP2BKO and upregulation of antioxidant enzyme genes. Morphometric analysis showed an

    increased glucagon positive area in the pancreata of UCP2BKO mice. Results obtained from

    UCP2BKO were contrary to the phenotype observed in UCP2/ mice. Overall, the

    characterization of UCP2BKO demonstrates that UCP2 in the beta-cell is involved in modulating

    ROS production.

  • iii

    Acknowledgements:

    I would like to extend my deepest gratitude to my supervisor, Dr. Michael B. Wheeler for giving

    me the opportunity to work on this project and for his ongoing support throughout my graduate

    studies. I would also like to thank Dr. Emma M. Allister for being my mentor and guiding me

    through every large and small endeavor.

    Furthermore, I would like to thank all the past and present members of the Wheeler lab for their

    invaluable recommendations.

    I am grateful for my supervisory committee members; Drs. Adria Giacca, Dominic Ng and Dr.

    Herbert Y. Gaisano for their important discussions.

    I would also like to thank the Banting and Best Diabetes Centre for funding my research over the

    years of 2008-2009.

    Lastly, I would like to thank my grandmother, parents, and the rest of my family members who

    have continued to support me throughout the years. Your love and guidance has made me a

    better person.

  • iv

    Table of Contents: Pg. No.

    Abstract ii

    Acknowledgements iii

    Table of Contents iv

    List of abbreviations viii

    List of figures x

    Chapter 1: Introduction

    1.1 Diabetes Mellitus 1

    1.1.1 The Epidemic 1

    1.1.2 Type 2 Diabetes Mellitus: description of the pathophysiology 2

    1.2 Glucose Homeostasis 4

    1.3 Glucose-Stimulated Insulin Secretion 6

    1.3.1 Glucose metabolism 6

    1.3.2 Insulin secretion via ATP production 7

    1.3.3 Insulin secretion: Amplification Pathway 9

    1.4 Uncoupling Proteins 10

    1.4.1 UCP1 10

    1.4.2 UCP3 11

    1.4.3 UCP2 11

    1.4.4 Role of UCP2 in insulin secretion and glucose homeostasis 12

    1.4.5 UCP2: Modulation of ROS & Cytoprotection 14

    1.4.6 Alternate functions of UCP2 17

    1.5 Cre-Lox Recombination system 19

    1.5.1 Background 19

    1.5.2 RIP-Cre model 20

    1.6 General Hypothesis 22

  • v

    Table of Contents Cont: Pg. No.

    Chapter 2: Creation and In Vivo characterization of UCP2BKO 2.1 Hypothesis 23

    2.2 Method and Materials 23

    2.2.1 Reagents 23

    2.2.2 Animal Breeding 23

    2.2.3 Pancreatic islet isolation and culture 25

    2.2.4 Islet cell dispersion 25

    2.2.5 RNA extraction & Reverse Transcription of animal tissues 26

    2.2.6 DNA extraction & Multiplex PCR 26

    2.2.7 Real-time PCR 26

    2.2.8 Immunostaining and immunofluorescence confocal microscopy 26

    2.2.9 Weight & Blood glucose measurements 27

    2.2.10 Measurement of fasting plasma glucagon 27

    2.2.11 Measurement of glucose tolerance 28

    2.2.12 Measurement of insulin sensitivity 28

    2.2.13 Statistical Analysis 28

    2.3 Results 29

    2.3.1 Genotyping 29

    2.3.2 Cre mRNA expression 21

    2.3.3 Effective deletion of UCP2 mRNA & protein 30

    2.3.4 Body Mass & Fasting Blood Glucose 32

    2.3.5 Plasma Insulin and Plasma Glucagon 33

    2.3.6 Assessing Glucose Tolerance: ipGTT & OGTT 35

    2.3.7 ITT 38

    Chapter 3: Characterization of UCP2KO: In Vitro Analysis 3.1 Hypothesis 40

    3.2 Method and Materials 40

  • vi

    Table of Contents Cont: Pg. No.

    3.2.1 Reagents 40

    3.2.2 Animals 40

    3.2.3 Analysis of Membrane Potential 41

    3.2.4 Analysis of ATP levels in cells 41

    3.2.5 Measurement of glucose stimulated insulin secretion 41

    3.2.6 Pancreatic islet morphology 42

    3.2.7 Measurement of ROS 42

    3.2.8 Evaluating the mRNA expression of Anti-oxidant enzymes in -cells 42

    3.2.9 Statistical Analysis 43

    3.3 Results 43

    3.3.1 Membrane Potential 43

    3.3.2 ATP Levels 45

    3.3.3 Glucose Stimulated Insulin Secretion 45

    3.3.4 Islet Area 46

    3.3.5 Beta Cell area 47

    3.3.6 Alpha Cell area 48

    3.3.7 ROS accumulation 49

    3.3.8 Anti-oxidant enzyme expression in cells 50

    Chapter 4: Discussion & Conclusion 4.1 Summary of findings 52

    4.1.1 In Vivo Findings 52

    4.1.2 In Vitro Findings 52

    4.2 General Discussion 53

    4.2.1 UCP2BKO in comparison to previous global knockout models 53

    4.2.2 UCP2BKO and Oxidative Stress 57

    4.2.3 UCP2BKO and -cell area 58

    4.2.4 UCP2BKO and impaired glucose tolerance 60

  • vii

    Table of Contents Cont: Pg. No.

    4.3 Future Directions 62

    4.3.1 UCP2 in the Hypothalamus 62

    4.3.3 UCP2BKO on a High-Fat Diet 63

    4.4 Conclusions 64

    Reference List 66

  • viii

    List of Abbreviations

    ADP adenosine diphosphate

    ANOVA analysis of variance

    ATP adenosine triphosphate

    BP - basepair

    cDNA complementary deoxyribonucleic acid

    CNS central nervous system

    DM Diabetes Mellitus

    DCF dicholorofluorescein

    DNA deoxyribose nucleic acid

    EDTA ethylenediaminetetraacetic acid

    EGTA ethylene glycol tetraacetic acid

    ELISA enzyme-linked immunosorbent assays

    ETC electron transport chain

    FAD flavin adenine dinucleotide

    FFA free fatty acids

    FBS fetal bovine serum

    FITC Fluorescein isothiocyanate

    G-6-P glucose-6-phosphate

    GDP guanosine diphosphate

    GLUT glucose transporter

    GSIS glucose-stimulated insulin secretion

    mRNA messenger ribosomal nucleic acid

    NAD nicotinamide adenine dinucleotide

    NLS nuclear localization signal

    NO nitric oxide

    PI propidium iodide

    PBS phosphate buffered soluton

    PDX-1 pancreatic-duodenal homeobox factor-1

    PPAR- peroxisome proliferator-activated receptor

    RFU relative fluorescence unit

  • ix

    RIA radioimmunoassay

    RIP Rat Insulin Promoter

    RNA - ribosomal nucleic acid

    ROS reactive oxygen species

    RPMI Roswell Park Memorial Institute

    SREBP-1c Sterol Regulatory Element Binding Protein-1c

    STZ streptozotocin

    TCA cycle tricarboxylic acid cycle

    UCP uncoupling protein

    UCP2 uncoupling protein-2

    UCP2BKO beta-cell specific knockout of UCP2

    VDCC voltage dependent calcium channel

    WHO World Health Organization

  • x

    ListofFigures&Tables

    Figures:

    1. Control of Glucose Homeostasis in the Body

    2. Insulin Secretion in the Beta-Cell

    3. Structure of a generic uncoupling protein

    4. UCP2 in the Mitochondria

    5. UCP2 as a negative regulator of insulin secretion

    6. Proposed role of UCP2 in cytoprotection

    7. Cre-mediated DNA recombination

    8. Schematic diagram of the RIP-Cre transgene

    9. Recombination via Cre recombinase

    10. Genotyping for LoxUCP2 and Cre

    11. Cre expression in cDNA