63.Growth Hormone Effects on Mitochondria

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63.Growth Hormone Effects on Mitochondria

 

 

 

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS …………………………………………………………………………………………………. 2

DECLARATION ……………………………………………………………………………………………………………… 3

TABLE OF CONTENTS ………………………………………………………………………………………………………………….. 4

LIST OF ABBREVIATIONS …………………………………………………………………………………… 8

LIST OF FIGURES ………………………………………………………………………………………………………………………. 16

LIST OF TABLES ………………………………………………………………………………………………………………………… 18

LIST OF EQUATIONS ………………………………………………………………………………………………………….. 19

PROJECT ABSTRACT ………………………………………………………………………………………………………………….. 20

  1. INTRODUCTION ……………………………………………………………………………………………………………………. 22

1.1. PROJECT INTRODUCTION …………………………………………………………………………………………….. 23

1.2. PROJECT RATIONALE ………………………………………………………………………………………………………….. 26

  1. REVIEW OF THE LITERATURE …………………………………………………………………………………………………. 29

2.1. THE ROLES AND REGULATION OF MITOCHONDRIAL FUNCTION ……………………………………….. 30

2.1.1. Regulation of Oxidative Phosphorylation ………………………………………………………………30

2.1.2. Generation of Reactive Oxygen Species…………………………………………………………………32

2.1.3. Mitochondrial Regulation of Cell Death Pathways …………………………………………………33

2.1.4. The Regulation of Mitochondrial Respiration through Cell Signalling Pathways………..36

2.1.5. Regulation of Mitochondrial Gene Expression ……………………………………………………….41

2.1.5.1. Transcriptional Regulation ……………………………………………………………………..41

2.1.5.2. Post-Transcriptioal Regulation………………………………………………………………..44

2.1.6. Factors that Affect Mitochondrial Function: Aging, Exercise and Insulin Resistance ….46

2.2. GROWTH HORMONE AND IGF-1 ……………………………………………………………………………….. 48

2.2.1. Physiological Effects of Growth Hormone and IGF-1……………………………………………..52

2.2.2. The Signal Transduction Pathways of Growth Hormone and IGF-1………………………….54

2.2.3. The Effects of rhGH Administraion on Athletic Performance……………………………………56

2.3. GROWTH HORMONE AND IGF-1: IMPACT ON MITOCHONDRIAL FUNCTION ……………………….. 57

  1. OUTLINE OF PROJECT STUDIES: AIMS AND HYPOTHESES ………………………………………………………….. 64

3.1. OUTLINE OF STUDY ONE ……………………………………………………………………………………………………. 65

3.1.1. Outline of Variables Analysed in Study One …………………………………………………………..66

3.1.1.1. Mitochindrial Membrane Potential (Δψm)…………………………………………………66

3.1.1.2. Mitochondrial Superoxide Production ………………………………………………………66

3.1.1.3. Mitochondrial Permeability Transition Pore Activity………………………………….67

3.1.1.4. Cellular Viability……………………………………………………………………………………67

3.1.2. Study One Aims ………………………………………………………………………………………………….68

3.1.3. Study One Hypotheses…………………………………………………………………………………………69

3.2. OUTLINE OF STUDY TWO ………………………………………………………………………………………………….. 70

3.2.1. Outline of Variables Analysed in Study Two…………………………………………………………..72

3.2.1.1. Highly Reactive Oxygen Species Production ……………………………………………..725

3.2.1.2. Electron Transport Chain Activity ……………………………………………………………73

3.2.1. Study Two Aims ………………………………………………………………………………………………….74

3.2.2. Study Two Hypotheses…………………………………………………………………………………………74

3.3. OUTLINE OF STUDY THREE …………………………………………………………………………………………………. 75

3.3.1. Outline of Variables Analysed in Study Three ………………………………………………………..76

3.3.1.1. Bcl-2 and Bak Expression ……………………………………………………………………….76

3.3.1.2. Cytosolic and Mitochondrial miRNA Expression………………………………………..76

3.3.2. Study Three Aims………………………………………………………………………………………………..77

3.3.3. Study Three Hypotheses ………………………………………………………………………………………77

  1. METHODS OF ANALYSIS ………………………………………………………………………………………………………… 78

4.1. ISOLATION TECHNIQUES ……………………………………………………………………………………………………. 79

4.1.1. Cell Isolation: Density Gradient Centrifugation……………………………………………………..79

4.1.2. Mitochondrial Isolation: Magnetic Cell Sorting……………………………………………………..79

4.1.3. Total RNA Purification: Solid Phase Extraction……………………………………………………..80

4.2. FLOW CYTOMETRY ……………………………………………………………………………………………………………. 82

4.2.1. Measurement Parameters ……………………………………………………………………………………83

4.2.1.1. Light Scatter ………………………………………………………………………………………….83

4.2.1.2. Fluorescence …………………………………………………………………………………………83

4.2.2. Data Analysis …………………………………………………………………………………………………….85

4.2.3. Analysis of Variables…………………………………………………………………………………………..87

4.2.3.1. Mitochondrial Membrane Potential (Δψm)………………………………………………..87

4.2.3.2. Mitochondrial Superoxide Production and Cellular Viability ………………………89

4.2.3.3. Mitochondrial Permeability Transition Pore Activity………………………………….91

4.2.3.4. Highly Reactive Oxygen Species Production ……………………………………………..93

4.3. REAL-TIME RT-PCR ………………………………………………………………………………………………………….. 93

4.3.1. Assay Parameters……………………………………………………………………………………………….94

4.3.1.1. Reverse Transcription …………………………………………………………………………….94

4.3.1.1. Polymerase Chain Reaction …………………………………………………………………….95

4.3.1.2. Amplicon Detection ………………………………………………………………………………..96

4.3.2. Data Analysis …………………………………………………………………………………………………….97

4.3.2.1. Cycle Threshold……………………………………………………………………………………..97

4.3.2.2. Relative Quantification……………………………………………………………………………98

4.3.2.3. Data Normalization………………………………………………………………………………..99

  1. STUDY ONE:

THE EFFECT OF RECOMBINANT HUMAN GROWTH HORMONE AND INSULIN-LIKE GROWTH FACTOR-1

ON THE MITOCHONDRIAL FUNCTION AND VIABILITY OF PERIPHERAL BLOOD MONONUCLEAR CELLS

IN-VITRO ……………………………………………………………………………………………………………………………….. 100

5.1. ABSTRACT …………………………………………………………………………………………………………………….. 101

5.2. INTRODUCTION ………………………………………………………………………………………………………………. 102

5.3. METHOD …………………………………………………………………………………………………………………….. 104

5.3.1. Subjects …………………………………………………………………………………………………………..104

5.3.2. Reagents Used………………………………………………………………………………………………….105

5.3.3. Blood Sample Collection and PBMC Isolation ……………………………………………………..105

5.3.4. rhGH and rIGF-1 Treatment………………………………………………………………………………105

5.3.5. Analysis of Mitochondrial Membrane Potential ……………………………………………………106

5.3.6. Analysis of Mitochondrial Permeability Transition Pore Activity ……………………………1066

5.3.7. Analysis of Mitochondrial Superoxide Generation and Cellular Viability ………………..107

5.3.8. Analysis by Flow Cytometry……………………………………………………………………………….107

5.3.9. Statistical Analysis ……………………………………………………………………………………………108

5.4. RESULTS ………………………………………………………………………………………………………………………. 108

5.4.1. Mitochondrial Membrane Potential…………………………………………………………………….108

5.4.2. Mitochondrial Permeability Transition Pore Activity…………………………………………….110

5.4.3. Mitochondrial Superoxide Generation…………………………………………………………………113

5.4.4. Cellular Viability………………………………………………………………………………………………113

5.5. DISCUSSION ………………………………………………………………………………………………………………….. 118

  1. STUDY TWO:

THE EFFECT OF IN-VITRO ADMINISTRATION OF RECOMBINANT HUMAN GROWTH HORMONE AND INSULIN-LIKE GROWTH FACTOR-1 ON MITOCHONDRIAL VITALITY AND HIGHLY REACTIVE OXYGEN SPECIES PRODUCTION UNDER DIFFERING RESPIRATORY CONDITIONS IN PERIPHERAL BLOOD MONONUCLEAR CELLS …………………………………………………………………………………………………………… 125

6.1. ABSTRACT …………………………………………………………………………………………………………………….. 126

6.2. INTRODUCTION ………………………………………………………………………………………………………………. 127

6.3. METHOD ……………………………………………………………………………………………………………………… 130

6.3.1. Subjects …………………………………………………………………………………………………………..130

6.3.2. Reagents Used………………………………………………………………………………………………….130

6.3.3. Blood Sample Collection and PBMC Isolation ……………………………………………………..130

6.3.4. rhGH and rIGF-1 Treatment………………………………………………………………………………131

6.3.5. Treatment with Respiratory Substrates and Inhibitors……………………………………………131

6.3.6. Analysis of Mitochondrial Membrane Potential ……………………………………………………131

6.3.7. Analysis of Highly Reactive Oxygen Species Generation………………………………………..132

6.3.8. Analysis by Flow Cytometry……………………………………………………………………………….132

6.3.9. Statistical Analysis ……………………………………………………………………………………………132

6.4. RESULTS ………………………………………………………………………………………………………………………. 133

6.4.1. Mitochondrial Membrane Potential…………………………………………………………………….133

6.4.2. Highly Reactive Oxygen Species Production ………………………………………………………..138

6.5. DISCUSSION ………………………………………………………………………………………………………………….. 143

  1. STUDY THREE:

THE EFFECT OF ONE WEEK’S ADMINISTRATION OF RECOMBINANT HUMAN GROWTH HORMONE ON

THE REGULATION OF MITOCHONDRIAL APOPTOSIS IN PERIPHERAL BLOOD MONONUCLEAR CELLS

IN-VIVO. AN EXPRESSION STUDY OF MITOCHONDRIAL AND CYTOSOLIC DERIVED MIRNA, MRNA AND

PROTEIN. ……………………………………………………………………………………………………………………….. 150

7.1. ABSTRACT ………………………………………………………………………………………………………………….. 151

7.2. INTRODUCTION ………………………………………………………………………………………………………….. 152

7.3. METHOD ……………………………………………………………………………………………………………………. 156

7.3.1. Subjects …………………………………………………………………………………………………………..156

7.3.2. Reagents Used………………………………………………………………………………………………….157

7.3.3. Experimental Design…………………………………………………………………………………………158

7.3.4. Blood Sample Collection and PBMC Isolation ……………………………………………………..158

7.3.5. Mitochondrial Isolation and Cytosolic RNA Decontamination………………………………..158

7.3.6. Total RNA and Protein Extraction from Cytosolic and Mitochondrial Lysates………….159

7.3.7. cDNA Synthesis from Cytosolic and Mitochondrial RNA ……………………………………….159

7.3.8. Real-time Polymerase Chain Reaction (RT-PCR) Analysis …………………………………….160

7.3.9. Determination of Bcl-2 and Bak Protein Concentrations ……………………………………….161

7.3.10. Statistical Analysis ……………………………………………………………………………………………162

7.4. RESULTS …………………………………………………………………………………………………………………….. 163

7.4.1. Bak / Bcl-2 Protein Concentrations …………………………………………………………………….163

7.4.2. Bak / Bcl-2 mRNA Expression…………………………………………………………………………….163

7.4.3. Cytosolic miR-125b / miR-181a miRNA Expression ………………………………………………166

7.4.4. Mitochondrial miR-125b / miR-181a miRNA Expression ……………………………………….167

7.5. DISCUSSION ……………………………………………………………………………………………………………….. 170

  1. FINAL DISCUSSION AND CONCLUSION ………………………………………………………………………………….. 180

8.1. GH AND IGF-1 MEDIATED EFFECTS ON MITOCHONDRIAL FUNCTION ……………………….. 181

8.2. IMPLICATIONS FOR CELLULAR VIABILITY ……………………………………………………………………. 186

8.3. REGULATION OF MITOCHONDRIAL MEDIATED APOPTOSIS BY RHGH: THE ROLE OF MIRNA ……. 188

8.4. CONCLUSIONS ……………………………………………………………………………………………………………….. 192

  1. REFERENCES ………………………………………………………………………………………………………………………. 193

Growth Hormone Effects on Mitochondria


The purpose of this study was to determine whether recombinant human growth hormone (rhGH) would show any significant effects on the expression of apoptosis regulating proteins in peripheral blood mononuclear cells (PBMCs). Additionally, the potential for post-transcriptional regulation of gene expression by miRNA was assessed in two cellular compartments, the cytosol and the mitochondria. Ten male subjects were subcutaneously injected with either rhGH (1 mg) or saline (0.9%) for seven consecutive days in a double-blinded fashion. Blood sampling was undertaken prior to treatment administration and over a period of three weeks following treatment cessation. Bcl-2 and Bak gene and protein expression levels were measured in PBMCs, while attention was also directed to the expression of miR-181a and miR-125b, known translational inhibitors of Bcl-2 and Bak respectively. Results showed that rhGH significantly decreased Bak protein concentrations compared to placebo samples for up to 8 days post treatment. While cytosolic miRNA expression was not found to be significantly affected by rhGH, measurement of the expression of miR-125b in mitochondrial fractions showed a significant down-regulation eight days post-rhGH administration. These findings suggest that rhGH induces short-term anti-apoptotic effects which may be partially mediated through a novel pathway that alters the concentration of mitochondrially-associated miRNAs.


Background
The purpose of this study was to investigate the mitochondrial effects exerted by physiological and supra-physiological concentrations of recombinant human growth hormone (rhGH) and recombinant insulin-like growth factor-1 (rIGF-1) under conditions of substrate saturation in peripheral blood mononuclear cells (PBMCs).


Methods
PBMCs from healthy male subjects were treated with either rhGH, at concentrations of 0.5, 5 and 50 μg/L, or rIGF-1 at concentrations of 100, 300 and 500 μg/L for 4 h. Mitochondrial membrane potential (Δψm) and mitochondrial levels of highly reactive oxygen species (hROS) were subsequently analysed. This analysis was performed by flow cytometry in digitonin permeabilized cells, following treatment with saturating concentrations of various respiratory substrate combinations and the use of specific electron transport chain (ETC.) complex inhibitors, enabling control over both the sites of electron entry into the ETC. at complexes I and II and the entry of electrons from reduced carriers involved in β-oxidation at the level of ubiquinol.

Results
Neither rhGH nor rIGF-1 exerted any significant effect on Δψm or the rate of hROS production in either lymphocyte or monocyte sub-populations under any of the respiratory conditions analysed.

Conclusion
That neither hormone was capable of attenuating levels of oxidative stress mediated via either complex I linked respiration or lipid-derived respiration could have serious health implications for the use of rhGH in healthy individuals, which is frequently associated with significant increases in the bioavailability of free fatty acids (FFA). Such elevated supplies of lipid-derived substrates to the mitochondria could lead to oxidative damage which would negatively impact mitochondrial function.


The time course of the effects of GH on state 3 respiration and fatty acid composition of liver mitochondria was investigated up to 48 h after an injecton of bovine GH to hypophysectomized rats. The respiratory rate increased significantly by 12 h, whereas the docosahexaenoic acid level, the total unsaturation index, and the ratio of arachidonic acid to linoleic acid of total phospholipids increased as early as 4 h after the hormone injection. A linear association was found between the respiratory rate and the three measures of fatty acid composition. It is concluded that the hormonal effects on respiration may be partly mediated through changes in fatty acid composition, which have an additive effect on increased synthesis of proteins and respiratory chain components.


Treatment with respiratory substrates and inhibitors
Following the experimental procedures and treatments, plasma membranes of these cells were permeabilized for the analysis of Δψm and hROS generation under differing respiratory conditions, a method adapted from the work of Pham et al. [27]. Sample cell aliquots were treated with digitonin at a concentration of 5 μg/mL for 5 mins, washed in PBS and resuspended in a respiratory buffer (0.25 M sucrose, 2 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, 0.1 % BSA, 1 mM ADP and 20 mM MOPS – pH adjusted to 7.4). The subsequent administration choice of substrate combinations with these cells gives rise to understanding either complex I, complex II or fatty acid mediated respiration.


Administration of the combination of pyruvate (5 mM) and malate (5 mM) (Pyr/Mal) activates isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex and malate dehydrogenase, which reduce NAD+ leading to the initiation of complex I mediated respiration. Complex II is not involved under these respiratory conditions, as malate equilibrates with fumarate at concentrations above 2 mM, inhibiting the conversion of succinate to fumarate and preventing the formation of FADH2 [28].


Administration of succinate (10 mM), in the presence of rotenone (20 μM) (Succ/Rot), activates SDH, which reduces FAD leading to the initiation of complex II mediated respiration. Rotenone acts to inhibit the flow of electrons through complex I and to prevent accumulation of oxaloacetate, a potent inhibitor of SDH [28]. Rotenone is also necessary to prevent reverse electron transfer to complex I, which stimulates the production of ROS [28].


Administration of the medium chain fatty acid octanoate (10 mM) and malate (2 mM) (Oct/Mal) mediate the transfer of electrons to reducing equivalents via β-oxidation. The use of octanoate, the oxidation of which is not carnitine dependent, to assess respiratory activity in isolated mitochondria has been previously demonstrated [29]. Additionally, malate at these concentrations acts as a “sparker” as it is known to significantly increase the rate of β-oxidation [28, 30].

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