Identification of genetic polymorphisms for antioxidant defense system genes and study of their

188 Pages·2013·3.9 MB·English

UNIVERSIDAD DE GRANADA
PROGRAMA OFICIAL DE DOCTORADO EN
NUTRICIÓN Y TECNOLOGÍA DE LOS ALIMENTOS

Identification of genetic polymorphisms for antioxidant
defense system genes and study of their association with
obesity and metabolic syndrome features in children
Identificación de polimorfismos génicos relacionados con el sistema de defensa
antioxidante implicados en la obesidad y el síndrome metabólico en niños

Tesis Doctoral con Mención Internacional presentada por:

AZAHARA IRIS RUPÉREZ CANO
Bajo la dirección de los doctores:
ÁNGEL GIL HERNÁNDEZ
CONCEPCIÓN M. AGUILERA GARCÍA
MERCEDES GIL CAMPOS

Instituto de Nutrición y Universidad de Granada
Tecnología de los Dpto. de Bioquímica y

Alimentos Biología Molecular II
José Mataix Verdú Facultad de Farmacia
Editor: Editorial de la Universidad de Granada
Autor: Azahara Iris Rupérez Cano
D.L.: GR 1046-2014
ISBN: 978-84-9028-952-5

La doctoranda Azahara Iris Rupérez Cano y los directores de la tesis Ángel
Gil Hernández, Concepción M. Aguilera García (Instituto de Nutrición y Tecnología
de los Alimentos José Mataix Verdú, Departamento de Bioquímica y Biología
Molecular II, Universidad de Granada) y Mercedes Gil Campos (Unidad de
Metabolismo e Investigación Pediátrica, Instituto Maimónides de Investigación
Biomédica, Servicio de Pediatría, Hospital Universitario Reina Sofía, Córdoba)
garantizamos, al firmar esta tesis doctoral, que el trabajo ha sido realizado por el
doctorando bajo la dirección de los directores de la tesis y hasta donde nuestro
conocimiento alcanza, en la realización del trabajo, se han respetado los derechos
de otros autores a ser citados, cuando se han utilizado sus resultados o
publicaciones.

Granada, 19 de noviembre de 2013

Directores de la tesis Doctoranda

Fdo.: ÁNGEL GIL HERNÁNDEZ Fdo.: AZAHARA I. RUPÉREZ CANO

Fdo.: CONCEPCIÓN M. AGUILERA GARCÍA

Fdo.: MERCEDES GIL CAMPOS

TABLE OF CONTENTS
Acknowledgements ........................................................................................................................... 7
Grants and funding ............................................................................................................................ 9
List of publications ........................................................................................................................... 11
SUMMARY ........................................................................................................................................... 13
Introduction .............................................................................................................................. 13
Rationale and aims of the study ........................................................................................ 14
Methodology.............................................................................................................................. 14
Results ......................................................................................................................................... 15
Discussion .................................................................................................................................. 17
Conclusion .................................................................................................................................. 20
Limitations of the study ........................................................................................................ 20
RESUMEN GENERAL ...................................................................................................................... 21
Introducción .............................................................................................................................. 21
Fundamento y objetivos ....................................................................................................... 22
Metodología ............................................................................................................................... 22
Resultados .................................................................................................................................. 23
Discusión..................................................................................................................................... 25
Conclusión .................................................................................................................................. 28
Limitaciones del estudio ...................................................................................................... 28
References/Bibliografía ....................................................................................................... 29
INTRODUCTION............................................................................................................................... 31
Childhood obesity ................................................................................................................... 32
Metabolic syndrome in children ....................................................................................... 33
Oxidative stress in obesity................................................................................................... 35
Genetics of obesity .................................................................................................................. 37
Genetic studies ............................................................................................................. 38

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AIMS OF THE STUDY..................................................................................................................... 41
Background and justification ............................................................................................. 42
Hypothesis ................................................................................................................................. 43
Aims .............................................................................................................................................. 43
General Aims ................................................................................................................ 43
Specific Aims................................................................................................................. 43
METHODOLOGY .............................................................................................................................. 45
Experimental design .............................................................................................................. 46
Anthropometry ........................................................................................................................ 46
Biochemical measurements................................................................................................ 47
Adipokines and biomarkers of inflammation and endothelial damage ........... 48
Antioxidant defense system evaluation ......................................................................... 50
Activity of the antioxidant enzymes in erythrocytes ................................... 50
Determination of paraoxonase 1 activities ...................................................... 51
SNP selection and genotyping ........................................................................................... 53
SNP selection ................................................................................................................ 53
Sample genotyping..................................................................................................... 55
Statistical analysis .................................................................................................................. 57
CHAPTER 1. LITERATURE REVIEW: Genetics of oxidative stress in obesity......... 59
Abstract ....................................................................................................................................... 60
Introduction .............................................................................................................................. 61
Methodology ............................................................................................................................. 62
Oxidative stress in obesity .................................................................................................. 62
Enzymatic antioxidant defence genes ............................................................................ 64
Glutathione peroxidases .......................................................................................... 64
Catalase ........................................................................................................................... 66
Paraoxonases ............................................................................................................... 66
Peroxiredoxins ............................................................................................................ 67
Superoxide dismutases ............................................................................................ 68
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The ROS producer: NADPH oxidase ................................................................................ 69
ROS response mechanisms: transcription factors ..................................................... 70
PPARγ .............................................................................................................................. 70
PGC1α .............................................................................................................................. 71
NRF2 ................................................................................................................................. 72
Conclusions ................................................................................................................................ 75
Limitations ................................................................................................................................. 75
Acknowledgments .................................................................................................................. 75
Funding........................................................................................................................................ 76
Disclosure statement ............................................................................................................. 76
References .................................................................................................................................. 80
CHAPTER 2. GENERAL RESULTS .............................................................................................. 89
General characteristics of the population under study ........................................... 90
Genotyping results .................................................................................................................. 94
Genotype frequency ................................................................................................... 94
Association with obesity .......................................................................................... 94
Association of gene-specific SNPs with obesity related
phenotypes and other biomarkers ...................................................................... 96
CHAPTER 3. Paraoxonase 1 activity and genetic variation in childhood
obesity ................................................................................................................................................ 109
Abstract .................................................................................................................................... 110
Introduction ........................................................................................................................... 110
Experimental methods ....................................................................................................... 111
Study population ...................................................................................................... 111
Anthropometric and biochemical measurements ...................................... 111
DNA isolation and genotyping ............................................................................ 111
Determination of paraoxonase 1 activities ................................................... 112
Statistical analyses .................................................................................................. 112

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Results ...................................................................................................................................... 112
Patient characteristics ........................................................................................... 112
Paraoxonase 1 activities in childhood obesity and their
correlations with metabolic, cardiovascular risk and oxidative
stress biomarkers .................................................................................................... 112
Association of paraoxonase 1 SNPs with the incidence of
childhood obesity and related traits ................................................................ 113
Effects of paraoxonase 1 SNPs on paraoxonase 1 activities .................. 114
Discussion ............................................................................................................................... 115
Conclusion............................................................................................................................... 117
Acknowledgements ............................................................................................................. 117
References............................................................................................................................... 117
Supplementary material ................................................................................................... 119
CHAPTER 4. Are catalase -844A/G polymorphism and activity associated
with childhood obesity? ............................................................................................................. 123
Abstract .................................................................................................................................... 124
Introduction ........................................................................................................................... 124
Innovation ............................................................................................................................... 124
Anthropometric and biochemical characteristics of the study population . 125
Correlation of erythrocyte CAT activity with obesity, insulin resistance
and oxidative stress biomarkers in plasma .............................................................. 125
Association of CAT SNPs with childhood obesity ................................................... 126
Association of the SNP -844A/G with obesity and insulin resistance
biomarkers ............................................................................................................................. 126
Conclusion............................................................................................................................... 126
Notes ......................................................................................................................................... 127
Study population ..................................................................................................... 127
Anthropometric and biochemical measurements ..................................... 128
DNA isolation and genotyping ........................................................................... 128
Statistical analysis ................................................................................................... 128
Acknowledgments ............................................................................................................... 128
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References ............................................................................................................................... 128
CONCLUSIONS ................................................................................................................................ 131
CONCLUSIONES ............................................................................................................................. 135
REFERENCES .................................................................................................................................. 139
Abbreviations .................................................................................................................................. 145
List of tables ..................................................................................................................................... 148
List of figures ................................................................................................................................... 151
APPENDIX ........................................................................................................................................ 153
Additional publication I ..................................................................................................... 154
Additional publication II ................................................................................................... 160
Table of genotyped SNPs ................................................................................................... 166
Legend .......................................................................................................................... 179
Curriculum Vitae .................................................................................................................. 180

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