164. Mitochondrial Bioenergetics

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164. Mitochondrial Bioenergetics

 

 

CATEGORY: Diet Nutrition Supplementation – 500 Courses

COURSE NUMBER: 164

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Syllabus

Title Page …………………………………………………………………………… i
Condition of Use …………………………………………………………………… ii
Certification of Thesis Work ……………………………………………………. iii

Abstract ………………………………………………………………………………………….. iv
Preface ……………………………………………………………………………………… vi
Acknowledgements …………………………………………………………… viii
Table of Contents ………………………………………………………………………… ix
List of Abbreviations ……………………………………………………………….. xx

List of Tables ………………………………………………………………………………………………………. xxiv
List of Figures ………………………………………………………………………………………………………. xxv
CHAPTER 1. GENERAL INTRODUCTION ……………………………………………………………….. 1

1.1. Overview ………………………………………………………………………………………………………………… 2
1.2. Energy homeostasis …………………………………………………………………………………………………. 3
1.2.1. Organismal energy homeostasis ……………………………………………………………………………… 3
1.2.2. Cellular energy homeostasis ………………………………………………………………………….. 5
1.3. Mitochondria structure and function …………………………………………………………………………… 6

1.3.1. Electron transport system ……………………………………………………………………………………….. 9
1.3.2. Oxidative phosphorylation ……………………………………………………………………………………. 13
1.3.2.1. Proton leak ………………………………………………………………………………………………………. 14
1.3.2.2. Electron leak and ROS ………………………………………………………………………………………. 14

1.3.3. Modulation of ETS respiratory activity and ROS production ……………………………………. 17
1.3.4. Modulation of mitochondrial function by environmental stress …………………………………. 20
1.4. Environmental stress ………………………………………………………………………………………………. 20

1.4.1. Temperature ……………………………………………………………………………………………………….. 21
1.4.1.1. Effects of temperature on fish physiology …………………………………………………………… 22
1.4.1.2. Effects of temperature on mitochondrial function …………………………………………………. 24
1.4.2. Hypoxia ………………………………………………………………………………………………… 25
1.4.2.1. Hypoxia in aquatic ecosystems …………………………………………………………………………… 25
1.4.2.2. Effects of hypoxia on physiology of aquatic organisms …………………………………………. 26
1.4.2.3. Hypoxia and mitochondrial function …………………………………………………………………… 28
1.4.2.4. Hypoxia-reoxygenation ……………………………………………………………………………………… 28
1.4.3. Copper ……………………………………………………………………………………………………………….. 29
1.4.3.1. Copper in the environment …………………………………………………………………………………. 29

1.4.3.2. Copper in biology …………………………………………………………………………………………….. 30
1.4.3.3. Copper homeostasis ………………………………………………………………………………………….. 32
1.4.3.4. Effects of Cu on mitochondrial function ……………………………………………………………… 33
1.5. Interactions of multiple stressors ……………………………………………………………………………… 34
1.5.1. Interactions of thermal stress, Cu and hypoxia ………………………………………………………… 34
1.5.2. Mitochondria as a target for multiple stressors ………………………………………………………… 36
1.6. Hypothesis and specific objectives …………………………………………………………………………… 36
CHAPTER 2. INTERACTIONS OF COPPER AND THERMAL STRESS ON
MITOCHONDRIAL BIOENERGETICS IN RAINBOW TROUT ………………………………. 38

2.1. Abstract ………………………………………………………………………………………………………………… 39
2.2. Introduction …………………………………………………………………………………………………………… 41
2.3 Materials and methods …………………………………………………………………………………………….. 44

2.3.1. Ethical considerations ………………………………………………………………………………………….. 44
2.3.2. Fish and isolation of hepatic mitochondria ……………………………………………………………… 44
2.3.3. Measurements of mitochondrial respiration ……………………………………………………………. 45
2.3.4. Complex I (NADH:ubiquinone oxidoreductase) activity ………………………………………….. 46
2.3.5. Mitochondrial swelling ………………………………………………………………………………………… 47
2.3.6. Mitochondrial membrane potential ………………………………………………………………………… 48
2.3.7. Q10 calculations …………………………………………………………………………………………………… 48
2.3.8. Statistical analysis………………………………………………………………………………………………… 49
2.4. Results ………………………………………………………………………………………………………………….. 49

2.4.1. Mitochondrial respiration ……………………………………………………………………………………… 49
2.4.2. Complex I activity ……………………………………………………………………………………………….. 55
2.4.3. Mitochondrial swelling ………………………………………………………………………………………… 55
2.4.4. Membrane potential …………………………………………………………………………………………….. 60
2.5. Discussion …………………………………………………………………………………………………………….. 65

2.5.1. Effects of Cu and temperature on mitochondrial bioenergetics ………………………………….. 65
2.5.2. Effect of Cu and temperature on mitochondrial complex I activity ……………………………. 69
2.5.3. Effect of Cu and temperature on mitochondrial swelling ………………………………………….. 70
2.5.4. Effect of Cu and temperature on membrane potential ………………………………………………. 72
2.6. Conclusions …………………………………………………………………………………………………………… 73

CHAPTER 3. COPPER ALTERS THE EFFECT OF TEMPERATURE ON
MITOCHONDRIAL BIOENERGETICS IN RAINBOW TROUT ………………………………. 74
3.1. Abstract ………………………………………………………………………………………………………………… 75
3.2. Introduction …………………………………………………………………………………………………………… 76
3.3. Materials and methods ……………………………………………………………………………………………. 78

3.3.1. Fish ……………………………………………………………………………………………………………………. 78
3.3.2. Mitochondrial respiration ……………………………………………………………………………………… 79
3.3.3. Effect of temperature and Cu on mitochondrial respiration ………………………………………. 80
3.3.4. Q10 and activation energy calculations …………………………………………………………………… 81
3.3.5. Complex II (succinate:ubiquinone oxidoreductase) activity ……………………………………… 82
3.3.6. Data analysis ………………………………………………………………………………………………………. 83
3.4. Results ………………………………………………………………………………………………………………….. 83

3.4.1. Effect of temperature and Cu on mitochondrial respiration ………………………………………. 83
3.4.2. Mitochondrial complex II activity …………………………………………………………………………. 89
3.5. Discussion …………………………………………………………………………………………………………….. 89

CHAPTER 4. ALTERATIONS IN MITOCHONDRIAL ELECTRON TRANSPORT

SYSTEM ACTIVITY IN RESPONSE TO WARM ACCLIMATION, HYPOXIA-
REOXYGENATION AND COPPER IN RAINBOW TROUT ……………………………………. 97

4.1. Abstract ………………………………………………………………………………………………………………… 98
4.2. Introduction …………………………………………………………………………………………………………. 100
4.3. Materials and methods ………………………………………………………………………………………….. 103
4.3.1. Ethics ………………………………………………………………………………………………………………. 103
4.3.2. Experimental animals and warm acclimation ………………………………………………………… 103
4.3.3. Isolation of liver mitochondria …………………………………………………………………………….. 104

4.3.4. Measurements of mitochondrial respiration under normoxic conditions …………………… 104
4.3.5. Mitochondrial respiration and Cu exposure after hypoxia-reoxygenation …………………. 107
4.4. Statistical analysis…………………………………………………………………………………………………. 108
4.5. Results ………………………………………………………………………………………………………………… 109
4.5.1. Body and liver weights and mitochondrial protein …………………………………………………. 109
4.5.2. Effects of acute temperature rise on ETS respiratory function …………………………………. 109
4.5.3. Effects of warm acclimation, HRO and Cu on CI respiratory function ……………………… 112
4.5.4. Effects of warm acclimation, HRO and Cu on CII respiratory function ……………………. 115
4.5.5. Effects of warm acclimation, HRO and Cu on CIII respiratory function ………….,………. 117
4.5.6. Effects of warm acclimation, HRO and Cu on CIV respiratory function …………………… 119
4.6. Discussion …………………………………………………………………………………………………………… 122
4.6.1. Individual effects of warm temperature, HRO and Cu on mitochondrial respiration ….. 123
4.6.2. Effects of warm acclimation, HRO and Cu on coupling and phosphorylation

efficiencies ………………………………………………………………………………………………………………… 128

4.6.3. Interactions of warm acclimation, Cu and HRO on ETS respiratory activity …………….. 129
4.7. Conclusions ………………………………………………………………………………………………… 130
CHAPTER 5. EFFECTS OF COPPER, HYPOXIA AND ACUTE TEMPERATURE
SHIFTS FOLLOWING WARM ACCLIMATION ON MITOCHONDRIAL
OXIDATION IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) ……………………….. 132
5.1. Abstract ………………………………………………………………………………………………………………. 133

5.2. Introduction …………………………………………………………………………………………………………. 134
5.3. Material and methods ……………………………………………………………………………………………. 136

5.3.1. Fish and acclimation to warm temperature ……………………………………………………………. 136
5.3.2. Copper and hypoxia exposure ……………………………………………………………………………… 137
5.3.3. Mitochondrial isolation and respirometry ……………………………………………………………… 139
5.4. Statistical analysis ………………………………………………………………………………………………… 140
5.5. Results ………………………………………………………………………………………………………………… 140

5.5.1 Fish condition and plasma metabolites profile …………………………………………………………. 140
5.5.2 Effects of warm acclimation, acute temperature shift, Cu, hypoxia and Cu plus hypoxia
on ETS respiratory function ………………………………………………………………………………………….. 142

5.5.2.1 CI respiratory function ………………………………………………………………………………………. 142
5.5.2.2 CII respiratory function …………………………………………………………………………………….. 146
5.5.2.3 CIII respiratory function ……………………………………………………………………………………. 148
5.5.2.4 CIV respiratory function ……………………………………………………………………………………. 150
5.5.3 Thermal sensitivities of CI-IV maximal and basal mitochondrial respiration rates ………… 152
5.6 Discussion ……………………………………………………………………………………………………………. 155
5.6.1 Warm acclimation reduced fish condition ……………………………………………………………… 155

5.6.2 Warm acclimation, Cu and hypoxia induced anaerobic metabolism ……………………………. 156
5.6.3 Warm acclimation reduced maximal CI-IV mitochondrial respiration rates and altered
effects of acute temperature change, hypoxia and Cu ………………………………………………………. 158
5.6.4 Warm acclimation reduced basal mitochondrial respiration and altered the effects
and interactions of acute temperature shift, hypoxia and Cu ………………………………………………. 160
5.6.5 RCR was more responsive to warm acclimation, acute temperature shift, hypoxia
and Cu than the P/O ratio ……………………………………………………………………………………………… 161

5.6.6 ETS thermal sensitivity displayed anterior-distal dichotomy with inverse responses
of state 3 versus state 4 to temperature, hypoxia and Cu ………………………………………………….. 162
5.7 Conclusions ………………………………………………………………………………………………………….. 163

CHAPTER 6. TRANSCRIPTIONAL AND FUNCTIONAL IMPACTS OF MULTIPLE
STRESSORS ON ENERGY METABOLISM AND STRESS RESPONSE IN RAINBOW
TROUT (ONCORHYNCHUS MYKISS) ……………………………………………………………………… 164
6.1 Abstract ……………………………………………………………………………………………………………….. 165

6.2 Introduction ………………………………………………………………………………………………………….. 166
6.3 Material and methods …………………………………………………………………………………………….. 169
6.3.1 Fish and experimental procedures ………………………………………………………………………… 169
6.3.2 Isolation of hepatic and gill mitochondria ……………………………………………………………… 170
6.3.3 Measurements of ETS enzyme activities in liver and gill mitochondria …………………….. 170
6.3.3.1 Complex I (NADH:ubiquinone oxidoreductase) activity ………………………………………. 170
6.3.3.2 Complex II (succinate:ubiquinone oxidoreductase) activity ………………………………….. 171
6.3.3.3 Complex III (ubiquinol:cytochrome c oxidoreductase) activity ……………………………… 171
6.3.3.4 Complex IV (cytochrome c oxidase; COX) activity …………………………………………….. 172
6.3.4 Gene expression analyses ……………………………………………………………………………………. 172
6.3.4.1 RNA extraction ……………………………………………………………………………………………….. 173
6.3.4.2 Gene Expression ……………………………………………………………………………………………… 174
6.4 Statistical analysis …………………………………………………………………………………………………. 177
6.5 Results …………………………………………………………………………………………………………………. 177

6.5.1 Liver ETS CI-CIV enzyme activities …………………………………………………………………….. 177
6.5.2 Gill ETS CI-CIV enzyme activities ………………………………………………………………………. 180
6.5.3 Relative expression of energy metabolism and stress/metals response genes ……………… 180
6.5.3.1 Liver mitochondria energy metabolism and stress/metals response genes ……………….. 180
6.5.3.2 Whole liver energy metabolism and stress/metals response genes ………………………….. 183
6.5.3.3 Gill energy metabolism and stress/metals response genes …………………………………….. 185
6.5.4 COX4-2/COX4-1 ratio ………………………………………………………………………………………… 187
6.6 Discussion …………………………………………………………………………………………………………… 187

6.6.1 Effect of warm acclimation, hypoxia and Cu on activities of ETS enzymes ………………. 189
6.6.2 Expression of energy metabolism and stress/metal responsive genes ………………………… 191
6.6.2.1 Effect of warm acclimation, hypoxia and Cu on COX4-1 and 2 gene expression …….. 192
6.6.2.2 Effect of warm acclimation, hypoxia and Cu on AMPKα1 gene expression ……………. 195
6.6.2.3 Effect of warm acclimation, hypoxia and Cu on MTA and B gene expression ………… 197
6.7 Conclusions ………………………………………………………………………………………………………….. 199
CHAPTER 7: GENERAL DISCUSSION AND FUTURE DIRECTIONS …………………… 200
7.1 General discussion ………………………………………………………………………………………………… 201

7.1.1 Acute temperature changes exacerbate the effects of Cu on mitochondrial function ……. 201
7.1.2 Acute temperature shifts and warm acclimation differentially alter mitochondrial
oxidation …………………………………………………………………………………………………………………… 202
7.1.2.1 Warm acclimation, Cu and HRO act additively to impair ETS respiratory activity ….. 203
7.1.3 Warm acclimation alter effects and interactions of acute temperature change, hypoxia
and Cu ………………………………………………………………………………………………………………………. 203
7.1.4 Biochemical and transcriptional responses to warm acclimation, hypoxia and Cu ………. 204
7.2 Future directions ……………………………………………………………………….. 205
8. REFERENCES ……………………………………………………………………….. 208

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