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Phenotypic characterization of peptide transporter PEPT2 deficient mice [Elektronische Ressource] : assessing complex metabolic alterations by profiling techniques / Isabelle Frey

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Published 01 January 2007
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Lehrstuhl für Ernährungsphysiologie


Phenotypic characterization of peptide transporter PEPT2
deficient mice: assessing complex metabolic alterations by
profiling techniques



Isabelle Maria Anna Frey


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. Dirk Haller
Prüfer der Dissertation: 1. Univ.-Prof. Dr. Hannelore Daniel
2. Univ.- Prof. Dr. Michael Schemann
3. Dr. Francois Verrey
Universität Zürich / Schweiz
(schriftliche Beurteilung)


Die Dissertation wurde am 29.08.2007 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 10.12.2007 angenommen. Table of Contents
Table of Contents
Zusammenfassung 1
Summary 2
Introduction 3
1. Brief history of the discovery of peptide transport and peptide
transporters
2. The PTR superfamily 5
3. Molecular and genetic attributes of mammalian peptide transporters 6
4. Determinants of substrate recognition and transport by PEPT1 and
PEPT2 8
5. Localization, Function and Regulation of PEPT1 and PEPT2 10
6. The mouse as a model system for functional genomics 20
7. Application of profiling techniques for phenotyping of mutant mice 22
Aim of the Thesis 25
Materials 27
1. Laboratory animals
2. Chemicals and consumables
3. Instruments
Methods 28
1. Mouse husbandry, handling and sample collection 28
1.1 Mouse husbandry
1.2 Genotyping
1.3 Data sampling for weight, organ weight, litter size and lifespan 29
1.4 Collection of plasma, urine and tissue samples 29
1.5 Dietary intervention study 30
1.6 Blood pressure determination 31
2. Analysis of selected metabolites in body fluids and tissue 31
2.1 Protein, urea and creatinine concentration and osmolality of urine
samples 31
2.2 Free and peptide bound amino acids in urine, plasma and tissue 31 Table of Contents
2.3 Clinical chemical parameters in plasma and urine 32
2.4 Glucagon in plasma 33
2.5 Thiol compounds in urine, plasma and tissue
3. mRNA expression analysis of individual genes 34
3.1 Northern Blot analysis for mRNA expression of Pept1, Pht1 and Pht2 34
3.2 Gene expression analysis by qPCR
4. Characterization of kidney tissue by profiling techniques 35
4.1 Transcriptome analysis by cDNA microarrays 35
4.2 Proteome analysis by 2D-PAGE and MALDI-TOF-MS 36
4.3 Metabolite analysis by GC-TOF-MS 37
5. Statistics 38
Results 39
-/-1. Basal phenotypic characterization of Pept2 mice 39
1.1 Body weight and organ weight 39
1.2 Litter size and weight at weaning 40
1.3 Lifespan 42
1.4 Urine parameters and amino acids in urine 42
1.5 Compensatory regulation of related peptide transporters in the kidney 45
1.6 Clinical-chemical parameters and free amino acids in plasma 46
1.7 Blood pressure 49
1.8 Glucagon 50
2. Response to different dietary protein contents 51
2.1 Weight, food and water intake during the metabolic study
2.2 Clinical chemical plasma and urine parameters 52
2.3 Amino acids and dipeptides in urine 56
-/-2.4 Additional ninhydrine positive metabolites in Pept2 urine samples 59
2.5 Gene expression in kidney tissue 61
3. Characterization of kidney tissue by profiling techniques 64
3.1 Transcriptome analysis by cDNA microarrays and qPCR
3.2 Proteome analysis by 2D-PAGE and MALDI-TOF-MS 66
3.3 Metabolite analysis by GC-TOF-MS 70
3.4 Classification of processes and pathway analysis 72
4. Analysis of constituents of glutathione metabolism 78
4.1 Urinary excretion of Cys-Gly 78 Table of Contents
4.2 GSH in urine, plasma and kidney tissue 79
4.3 Analysis of changes at mRNA level of genes involved in GSH-
metabolism 80
Discussion 83
1. Basal phenotypic characterization 83
2. Response to different dietary protein contents 85
3. Characterization of kidney tissue by profiling techniques 89
3.1 Methodological aspects of the applied profiling techniques
3.2 Integrated analysis of kidney tissue at mRNA, protein and metabolite
level in combination with urine analysis reveals alterations in renal
amino acid and glutathione metabolism 100
4. Future perspectives 105
Appendix 109
1. Complete lists of differentially expressed genes 109
2. Oligonucleotide primers for qPCR 117
3. List of abbreviations 119
References 123
Erklärung 141
Curriculum Vitae 143
Danksagung 147
List of Tables
List of Tables
Table 1: PTR family members in animals, bacteria, plants and fungi 5
Table 2: Molecular characteristics of the human POT genes and murine Pept2 7
Table 3: Relative organ weights 40
Table 4: Number and mean age of deceased animals during 2 years of
follow-up 42
Table 5: Urine parameters in 24 h urine samples 43
Table 6: Free and dipeptide bound amino acids in urine of male and female
mice 44
Table 7: Clinical chemical parameters in male and female mice 47
Table 8: Free amino acids in plasma of male and female mice 48
Table 9: Body weight and food and water intake in response to different
protein content of the diet 52
Table 10: Clinical chemical parameters on diets with different protein content 53
Table 11: Urine parameters in response to diets with different protein content 55
Table 12: Free and dipeptide bound amino acids in urine 57
Table 13: Expression of genes involved in peptide and amino acid metabolism 62
Table 14: Changes in protein level in kidney tissue of PEPT2 deficient animals 67
Table 15: Metabolites in kidney tissue that contribute most to differentiation
between genotypes 71
Table 16: Differentially expressed genes involved in amino acid metabolism 73
Table 17: olved in fatty acid metabolism 74
Table 18: GSH in kidney tissue, urine and plasma 79
Table 19: Expression of genes involved in GSH metabolism 81
-/-Table 20: Genes with elevated mRNA levels in Pept2 animals as identified by
VarMixt 109
-/-Table 21: Genes with decreased mRNA levels in Pept2 animals as identified
by VarMixt 113
-/-Table 22: Differentially expressed genes in Pept2 animals as identified by PAM 116
Table 23: Primers for housekeeping genes 117
Table 24: Primers for genes involved in peptide and amino acid transport and
metabolism 117
Table 25: Primers for validation of microarray results 118
Table 26: Primers for genes involved in GSH metabolism List of Figures
List of Figures
Fig. 1: Main physiological function of mammalian PEPT1 and PEPT2 4
Fig. 2: Structural elements defining substrate affinity (from (49)) 9
Fig. 3: PEPT1 and PEPT2 in the context of the epithelial cell 13
Fig. 4: Renal accumulation of labelled model dipeptides 15
Fig. 5: Relationship between different "omics" disciplines (from (55) 22
Fig. 6: Monitoring of body weight over 25 weeks 39
Fig. 7: Litter size at birth and surviving pups at weaning 41
Fig. 8: Dipeptide bound glycine in male and female mice 45
Fig. 9: Expression of PHT1 in kidney 46
Fig. 10: Mean arterial blood pressure in male and female mice 49
Fig. 11: Plasma glucagon levels in male and female mice 50
Fig. 12: Dipeptide bound glycine and cystine in urine 58
-/-Fig. 13: Detection of additional ninhydrine positive metabolites in Pept2
urine samples 59
Fig. 14: Peak area of compound Y in relation to creatinine 60
Fig. 15: Expression of Eaf2 and Pept2 in the parental strains 129Sv and
C57Bl/6 66
Fig. 16: 2D-PAGE analysis of proteins from kidney tissue samples (see next
page) 68
Fig. 17: Multivariate data analysis of metabolites 70
Fig. 18: Biological processes (GO) affected by loss of PEPT2 72
Fig. 19: Pathways affected by loss of PEPT2 (see next page) 76
Fig. 20: Urinary cysteinyl-glycine in male and female mice 78
Fig. 21: Schema of GSH metabolism in epithelial cells of the proximal tubule 104