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Palmitoyl-CoA Inhibition of Mitochondrial ADP Sensitivity is Attenuated by Exercise Training in Human Skeletal Muscle
by
Alison Claire Ludzki
A Thesis
Presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Master of Science
in
Human Health and Nutritional Sciences
Guelph, Ontario, Canada
© Alison Claire Ludzki, August, 2014
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ABSTRACT
PALMITOYL-COA INHIBITION OF MITOCHONDRIAL ADP SENSITIVITY IS ATTENUATED BY EXERCISE TRAINING IN HUMAN SKELETAL MUSCLE
Alison Claire Ludzki Advisor: University of Guelph, 2014 Dr. Graham Holloway
A hallmark of improved metabolic control is a reduced free ADP requirement for a given
workload (increased ADP sensitivity). In contrast to in vivo data, in situ assessments suggest that
mitochondrial ADP sensitivity is decreased following exercise training, implying external
regulation that is not recapitulated in situ. One previously unexplored regulator is palmitoyl-CoA
(P-CoA), a lipid metabolism intermediate that inhibits the mitochondrial ADP transport protein
adenine nucleotide transferase (ANT). This thesis: 1) established reduced mitochondrial ADP
sensitivity following exercise training in middle aged males using permeabilized muscle fibre
bundles (PmFB), 2) determined a methodology to evaluate ADP kinetics in PmFB in the
presence of P-CoA, and 3) found increased mitochondrial ADP sensitivity in the presence of P-
CoA following training. These data suggest that P-CoA is a key regulator of oxidative
phosphorylation and direct future exploration of mitochondrial function towards the control of
ADP transport via ANT and the effects of exercise on the P-CoA-ANT interaction.
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Acknowledgements
Thanks to my advisor, Dr. Graham Holloway, for always making time for life advice and for
supporting my extracurricular endeavours. Thank you for pushing me to achieve my best,
selflessly cultivating my professional skillset, and reminding me to prioritize happiness. Most
importantly, thank you for modelling an awareness of how your work fits into the greater field,
which I hope to translate to my career.
To Dr. Lawrence Spriet, thank you for inspiring dedication, forward thinking, creativity,
and possibility. Through taking an interest in my career, you have inspired me to dream big. Dr.
George Heigenhauser, your passion and enthusiasm are unmatched and infectious, and I always
come away from conversations with you more excited about my work. Dr. David Wright, I have
much respect for your modesty and generosity, and you have been an important role model to me
during my Master’s schooling. Dr. Martin Gibala and Jenna Gillen, thank you for piquing my
interest in science and continuing to support my development as a physiologist.
Sabina Paglialunga and Eric Herbst, I cannot thank you enough for your teaching these
past two years. Your knowledge, mindfulness, willingness to help, and senses of humour made
this degree possible. To Brennan, Laelie, Jamie, Sarthak, and the rest of the past/present
Holloway lab members, I could not have asked for a better range of expertise or positive
influences from whom to learn. And to all the smiling faces in the HHNS department, thanks.
Thank you to my Speed River teammates for reminding me what it means to work hard
and test oneself every day. Finally, thank you to my family for always supporting my work, and
especially to my parents, for continuing to exemplify the social conscience and life long learning
that motivate my career path.
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Table of Contents List of Figures ............................................................................................................................................. vi List of Tables .............................................................................................................................................. vii
................................................................................ 1 CHAPTER 1 – REVIEW OF THE LITERATURE
................................................................................................................. 1 1.1 INTRODUCTION
................................................... 2 1.2 REGULATION OF SKELETAL MUSCLE METABOLISM
..................................................................... 2 1.2.1 Major Energy Stores for Skeletal Muscle
................................................................................. 3 1.2.2 Enzymatic Control of Metabolism
...................................................................... 4 1.2.3 Regulation of Carbohydrate Metabolism
....................................................................................... 7 1.2.4 Regulation of Fat Metabolism
......................................................................................................... 11 1.2.5 Fuel Interactions
.................................. 12 1.2.6 Skeletal Muscle Metabolic Adaptations to Endurance Training
................................................................................... 13 1.3 MITOCHONDRIAL RESPIRATION
.......................................................................................... 13 1.3.1 Oxidative Phosphorylation
................................................................................. 23 1.4 PALMITOYL-COA INTERACTIONS
................................................................ 24 1.5 CONCLUSIONS AND FUTURE DIRECTIONS
............................................................................................................ 25 CHAPTER 2: AIMS OF THESIS
CHAPTER 3: PALMITOYL-COA INHIBITION OF MITOCHONDRIAL ADP SENSITIVITY IS ................... 26 ATTENUATED WITH EXERCISE TRAINING IN HUMAN SKELETAL MUSCLE
........................................................................................................................ 26 3.1 METHODS
...................................................................................................................... 26 3.1.1 Subjects
........................................................................................................... 26 3.1.2 Blood Samples
......................................................................................................... 27 3.1.3 VO2peak Testing
......................................................................................................... 27 3.1.4 Muscle Samples
........................................................................................................ 28 3.1.5 Exercise Training
....................................................................................................................... 28 3.1.6 Animals
.............................................................. 28 3.1.7 Preparation of Permeabilized Fibre Bundles
.................................................... 29 3.1.8 Mitochondrial Respiration in Permeabilized Fibres
........................................................................................................... 30 3.1.9 Blood Analyses
....................................................................................................... 31 3.1.10 Western Blotting
................................................................................................................... 31 3.1.11 Statistics
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.......................................................................................................................... 32 3.2 RESULTS
3.2.1 Six Weeks of Combined HIT and Endurance Training Significantly Improves Muscle ............................. 32 Oxidative Capacity, Glucose Tolerance, and Cardiorespiratory Fitness
3.2.2 Improved Mitochondrial Capacity and Reduced Sensitivity Following Exercise ................................................................................................................................ 36 Training
................................................. 38 3.2.3 Increased ANT1 Protein Content Following Training
.............. 39 3.2.4 Palmitoyl-CoA Rapidly Inhibits Mitochondrial Respiration in Rodent PmFB
3.2.5 Exercise Training Improves Mitochondrial ADP Sensitivity and Capacity in the .................................................................................................. 41 Presence of Palmitoyl-CoA
.................................................................................................................... 43 CHAPTER 4: DISCUSSION
... 43 4.1 MITOCHONDRIAL ADP TRANSPORT AND REGULATION OF ENERGY BALANCE
.................... 44 4.2 PALMITOYL-COA AS A REGULATOR OF MITOCHONDRIAL FUNCTION
.............. 45 4.3 EXERCISE IMPROVES APPARENT ADP Km IN THE PRESENCE OF P-COA
4.4 MITOCHONDRIAL ADP AVAILABILITY AND THE REGULATION OF MEMBRANE ............................................................................................................................ 48 POTENTIAL
..................................................... 51 4.5 FATTY ACID OXIDATION IN METABOLIC DISEASE
.................................................................................................... 53 4.6 FUTURE DIRECTIONS
.................................................................................................................. 56 4.7 CONCLUSION
REFERENCES .......................................................................................................................................... 58 APPENDICES ........................................................................................................................................... 68
Appendix A: Exercise Training Progression ............................................................................ 68 Appendix B: Mitochondrial Respiration Experiments .............................................................. 69 Appendix C: Blood Handling Procedures ................................................................................ 75 Appendix D: Supplementary Figures ...................................................................................... 76
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List of Figures Figure 1 – Contribution of carbohydrate and fat at various exercise intensities. ........................... 3
Figure 2 – Mechanism of mitochondrial LCFA transport. ........................................................... 10
Figure 3 – Improved ADP sensitivity in the trained individual. ................................................... 13
Figure 4 – Regulation of mitochondrial respiration. ..................................................................... 14
Figure 5 – Key substrates and products of the TCA cycle. .......................................................... 16
Figure 6 – Electron transport chain. .............................................................................................. 18
Figure 7 – Mechanisms of mitochondrial ADP transport. ............................................................ 20
Figure 8 – Overview of experimental design. ............................................................................... 27
Figure 9 – Training improves the OGTT response 72 hours following the last exercise bout. .... 35
Figure 10 – Training improves mitochondrial capacity in permeabilized muscle fibres. ............ 36
Figure 11 – Mitochondrial ADP sensitivity is reduced following exercise training. ................... 37
Figure 12 – Increase in ANT1 and no change in ANT2 isoform content following 6 weeks of training. .................................................................................................................................. 38
Figure 13 – Respiration is acutely inhibited by P-CoA and recovered by DNP. .......................... 39
Figure 14 – ADP and L-carnitine prevent inhibition of mitochondrial respiration by P-CoA. .... 40
Figure 15 – Mitochondrial ADP sensitivity in the presence of P-CoA is improved with training. ............................................................................................................................................... 42
Figure 16 – Exercise training reduces mitochondrial ADP sensitivity to P-CoA – a model. ....... 46
Figure 17 – Propensity for succinate-supported mitochondrial ROS emission before and after exercise training. .................................................................................................................... 49
Figure 18 – Reductions in markers of oxidative stress following 6 weeks exercise training ....... 50
Figure 19 – P-CoA inhibits ADP attenuation of ROS emission ................................................... 50
Figure 20 – Effects of P-CoA on mitochondrial respiration. ........................................................ 52
Figure 21 – Lower apparent ADP Km corresponds to higher insulin sensitivity (HOMA). ........ 55
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List of Tables Table 1 – Characterization of Training Protocol. ......................................................................... 35
Table 2 – Subject characteristics and training responses. ............................................................. 36
viii
List of Abbreviations
Acetyl-CoA Carboxylase Acyl-CoA Synthetase Adenosine Diphosphate Free Adenosine Diphosphate Adenosine Monophosphate AMPK-Activated Protein Kinase Adenine Nucleotide Transferase Adipose Triglyceride Lipase Adenosine Triphosphate Area Under the Curve Blebbistatin Body Mass Index Calcium Citric Acid Cycle Carnitine/Acyl-Carnitine Translocase Carnitine Palmitoyl Transferase Citrate Synthase Electron Transport Chain Electron Transferring Flavoprotein Fatty Acid Fatty Acid Binding Protein Flavin Adenine Nucleotide Fatty Acid Translocase/Cluster of Differentiation 36 Fatty Acid Transport Protein Free Fatty Acid Glucose 1-Phosphate Glycerol 3-Phosphate Glucose 6-Phosphate Glucose Transporter Hydrogen Water High-density Lipoprotein Homeostatic Model Assessment High-sensitivity C-reactive Protein Hormone Sensitive Lipase Inosine Monophosphate Intramuscular Triglyceride Potasssium Michaelis-Menten Constant L-Carnitine Long Chain Fatty Acid Low-density Lipoprotein Lysine Malonyl-CoA
ACC ACS ADP ADPf AMP AMPK ANT ATGL ATP AUC BLEB BMI Ca2+ CAC CACT CPT CS ETC ETF FA FABP FAD FAT/CD36 FATP FFA G1P G3P G6P GLUT H+ H2O HDL HOMA HS-CRP HSL IMP IMTG K+ Km LCarn LCFA LDL Lys M-CoA
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Mitochondrial Respiration Medium Monoglyceride Lipase Nicotinamide Adenine Dinucleotide Ammonium Non-esterified Fatty Acids Oxygen Oral Glucose Tolerance Test Oxidative Phosphorylation Inorganic Phosphate Palmitoyl-CoA Pyruvate Dehydrogenase Pyruvate Dehydrogenase Kinase Pyruvate Dehydrogenase Phosphatase Glycogen Phosphorylase Permeabilized Muscle Fibre Bundle Respiratory Control Ratio Reactive Oxygen Species Triglycerides Voltage Dependent Anion Channel Maximal Workload Zucker Diabetic Fatty
MiR05 MGL NAD NH4
+
NEFA O2 OGTT OXPHOS Pi P-CoA PDH PDK PDP PHOS PmFB RCR ROS TG VDAC Wmax ZDF
1
CHAPTER 1 – REVIEW OF THE LITERATURE
1.1 INTRODUCTION An impressive feature of human physiology is the ability to regulate energy production to
maintain adenosine triphosphate (ATP) homeostasis throughout varying demands. The regulation
of fuel selection and metabolism is well characterized in human skeletal muscle and the
discovery of mitochondrial biogenesis in response to exercise training has identified muscle
oxidative capacity as a limiting factor in aerobic metabolism and whole body health (Holloszy &
Coyle, 1984). However, the observation that metabolic control can be improved independent of
mitochondrial capacity suggests that there are other important mechanisms in energy
homeostasis (Green et al., 1992; Phillips et al., 1996a). Ex vivo studies of mitochondrial function
have identified adenosine diphosphate (ADP) availability as a major regulator of mitochondrial
respiration (Lardy & Wellman, 1952; Chance & Williams, 1955). Surprisingly, training appears
to decrease the sensitivity of mitochondria to submaximal ADP concentrations (Walsh et al.,
2001a; Guerrero et al., 2005). This paradox suggests that the regulation of mitochondrial ADP
sensitivity warrants further investigation. Palmitoyl-CoA (P-CoA) is a lipid metabolism
intermediate that has been shown to interact with the ADP transport protein adenine nucleotide
transferase (ANT). Interestingly, P-CoA concentrations are elevated by acute exercise and
diabetes, and recent work has shown that mitochondrial ADP sensitivity is regulated by acute
exercise (Perry et al., 2012) and a diabetic state (Smith et al., 2013). These data suggest that
P-CoA may represent a regulator of mitochondrial ADP sensitivity and overall metabolic control
that remains largely unexplored. This review will provide background on the regulation of
substrate provision to mitochondria, will outline the existing literature on mitochondrial ADP
sensitivity, and will introduce the potential for P-CoA to influence this aspect of aerobic health.
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1.2 REGULATION OF SKELETAL MUSCLE METABOLISM
1.2.1 Major Energy Stores for Skeletal Muscle Skeletal muscle is impressively designed to meet an enormous range of energy requirements by
varying ATP production rates over 100-fold from rest to maximal exercise (Spriet & Howlett,
1999). With the exception of prolonged exercise and fasting, ATP production is primarily fuelled
by carbohydrate and fat (Cermak & van Loon, 2013). These fuels are each stored in two
locations: liver and skeletal muscle glycogen for carbohydrate, and adipose tissue and
intramuscular triglyceride (IMTG) for fat. Fat stores comprise around 50 times the amount of
energy available in carbohydrate stores, and fat contains a greater amount of energy per unit
weight compared with carbohydrate (Jeppesen & Kiens, 2012). However, carbohydrate is a
preferred fuel source in situations of high demand because of its rapid rate of catabolism and
superior ATP yield per unit of oxygen utilized (Cermak & van Loon, 2013). In general, fat has a
greater relative contribution to energy provision during conditions of lower metabolic demand,
and carbohydrate has a greater relative contribution to energy provision in cases of higher
metabolic demand. This is illustrated by results from radioisotope tracer work of van Loon et al.
(2001), quantifying energy provision (kJ/min) from muscle and other fuel sources at various
exercise intensities from rest to 75% of maximal workload (Wmax) (Figure 1). These data show
that carbohydrate oxidation increases continually with increasing intensity, while whole-body fat
oxidation increases across low intensities of exercise (up to at least 55% Wmax), and decreases at
some point thereafter (by 75% Wmax). In addition to exercise intensity, substrate availability and
training status can influence muscle fuel selection, which will be discussed later in this chapter.
First, the enzymatic control of the major energy-producing pathways will be reviewed.
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Figure 1 – Contribution of carbohydrate and fat at various exercise intensities. Increasing absolute and relative contribution from carbohydrate stores with increasing exercise intensity. Increasing absolute and decreasing relative contribution from fat stores until ~55% Wmax; decreasing absolute contribution from fat by 75% Wmax. Wmax: maximal workload; TG: triglycerides; FFA: free fatty acids. From van Loon et al. (2001).
1.2.2 Enzymatic Control of Metabolism Energy demands at any point in time are met by three energy-yielding systems: the phosphagen
system, the glycolytic system, and the oxidative system (Spriet & Howlett, 1999). The former
two energy production pathways do not require oxygen, and occur in the sarcoplasm. These
systems contribute to energy production through substrate-level phosphorylation, and are
prominent during transitions to a higher power output and power outputs requiring rapid energy
production (Holloway & Spriet, 2009). However, the majority of daily energy provision comes
from the oxidative system (Brand & Murphy, 1987). The rate of substrate oxidation depends on a
variety of factors including the supply of substrate from carbohydrate and fat, which is subject to
extensive enzymatic control to tightly match the energy needs of the cell (Brand & Murphy,
1987). This control can follow various forms, including covalent/transformational regulation
(conversion between the active (a) and less active (b) form by other proteins, often through
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phosphorylation status), allosteric/post-transformational regulation (control of enzyme activity
through the binding of compounds to some site other than the enzyme’s active site), and
substrate/product concentrations (Meyer & Foley, 1996). The major signals involved in
increasing metabolic enzyme activity in skeletal muscle through these first two control systems
include calcium (Ca2+), metabolites related to the cytoplasmic phosphorylation potential (free
ADP [ADPf]; the fraction of ADP not bound to other proteins, inorganic phosphate [Pi], free
adenosine monophosphate [AMPf], inosinic acid [IMP]), and the redox status of nicotinamide
adenine nucleotide (NAD), all of which are altered by stressors such as exercise (Meyer & Foley,
1996).
1.2.3 Regulation of Carbohydrate Metabolism Carbohydrate metabolism begins with the breakdown of liver or muscle glycogen into
glucose 1-phosphate (G1P) molecules by glycogen phosphorylase (PHOS) to be converted to
glucose 6-phosphate (G6P) by phosphoglucomutase in a process called glycogenolysis (Katz &
Westerblad, 2014). Muscle glycogen is preferentially used at the onset of exercise due to its
proximity to the major sites of energy consumption and higher ATP yield (3 ATP through
substrate level phosphorylation versus 2 from blood glucose), while liver glycogen is used
increasingly as exercise progresses to maintain blood glucose (Cermak & van Loon, 2013).
Muscle glycogenolysis is rapidly stimulated by the rise in Ca2+ at the onset of exercise, and by
elevated epinephrine levels with continued exercise, both increasing the amount of PHOS in its
more active a form (Richter et al., 1982). This feedforward control sets the upper limit of
glycogenolytic flux, but does not regulate enzyme activity across varying exercise intensities
(Gollnick et al., 1978). Rather, the measurement of allosteric activators AMP, ADPf, IMP,
allosteric inhibitors ATP and G6P, and substrate (Pi) following electric stimulation, acute
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exercise, and exercise training, has identified allosteric control as the major regulator of PHOS
flux (Ren & Hultman, 1990; Howlett et al., 1998).
Liver PHOS, on the other hand, is mainly activated by the rise in epinephrine and
glucagon, and reductions in insulin during exercise (see Kjaer, 1998 for review). G6P is
converted to glucose in the liver to be delivered to skeletal muscle via the circulation (Kjaer,
1998). Glucose is taken up from the circulation to the muscle cell via facilitated diffusion
through glucose transport proteins (GLUT). Glucose uptake occurs through GLUT1 at rest, while
exercise-mediated glucose transport is regulated primarily by the GLUT4 isoform, which
translocates to the plasma membrane to facilitate glucose uptake in response to contraction-
related signals including Ca2+ and AMPK (reviewed in Richter & Hargreaves, 2013). Once
inside skeletal muscle, the next step in liver carbohydrate catabolism is the phosphorylation of
glucose to G6P by hexokinase, in an energy-consuming reaction. Hexokinase is stimulated by
insulin and the concentration of free glucose and may become limiting at high rates of glucose
uptake, when it is subject to feedback inhibition by G6P (Katz et al., 1986).
G6P from muscle and liver sources undergoes glycolysis to generate ATP, producing
NADH and pyruvate for mitochondrial oxidation in the process. The rate-limiting enzyme in
glycolysis is phosphofructokinase (PFK), which experiences extensive regulation. PFK is
sensitive to activation by the reaction substrate fructose 6-phosphate, and allosteric inhibition by
(required substrate) ATP (Boscá et al, 1985). Allosteric activators of PFK include ADP, Pi,
AMP, and cyclic-AMP, which attenuate ATP inhibition, as well as potassium and ammonia,
which independently increase PFK capacity during exercise (Kemp & Foe, 1983). Hydrogen
(H+) from ATP hydrolysis further enhances ATP inhibition of PFK, but is not limiting to
glycolysis except in very intense exercise due to the aforementioned allosteric activators (Spriet
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et al, 1987). Similarly, phosphoenolpyruvate, 3-phosphoglycerate, and citrate are inhibitory on
PFK and most effective in resting conditions (Krzanowski & Matchinsky, 1969; Peters & Spriet,
1995).
Pyruvate generated by glycolysis can be reduced to lactate in the cytosol, or converted to
acetyl-CoA by pyruvate dehydrogenase (PDH) to enter the citric acid cycle (CAC) for oxidation
in the mitochondria (Adeva-Andany et al., 2014). Due to the near-equilibrium nature of the
lactate production pathway, flux from pyruvate to lactate is higher during high power outputs,
when pyruvate production rates of glycolysis exceed PDH flux and the production of NADH
exceeds the capacity of the shuttle systems to move it into the mitochondria (Adeva-Andany et
al., 2014). Lactate dehydrogenase then oxidizes NADH, regenerating NAD+ to enable the
proximal steps in glycolysis to continue. Pyruvate fated for oxidation is transported into the
mitochondrial matrix by the mitochondrial pyruvate carrier on the inner mitochondrial
membrane (Halestrap, 1975). Within the matrix, pyruvate enters PDH, a complex of subunits
that are covalently regulated by phosphorylation of the E1 site to control what is the first
irreversible step in carbohydrate oxidation (Ward et al., 1982). The relative activities of PDH
kinase (PDK, phosphorylating to inactivate) and PDH phosphatase (PDP, dephosphorylating to
activate) determine the amount of PDH in the active form. Conversion of PDH to the a form
corresponds to PDH flux, indicating that transformation determines pyruvate oxidation when
carbohydrate supply is not limiting (Howlett et al., 1998). Under resting conditions, activators of
PDK are high, including ATP/ADP, NADH/NAD+, and acetyl-CoA/CoA ratios, maintaining
PDH in its b form. Furthermore, the primary activator of PDP, Ca2+, remains low. At the onset of
exercise, elevations in Ca2+ stimulate the initial activation of PDP to convert PDH to the a form.
Subsequent regulation comes from pyruvate inhibition of PDK, and a reduction in the ATP/ADP
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ratio (further reducing PDKa) to fine tune PDH activity (see Peters, 2003 for review).
Conversion of pyruvate to acetyl-CoA produces one NADH molecule while releasing one
molecule of carbon dioxide (CO2) and requiring one CoA per pyruvate. Acetyl-CoA is the final
product of carbohydrate metabolism required for entry into the CAC.
1.2.4 Regulation of Fat Metabolism Like glycogen, triglycerides must be broken down into smaller units before they can be
transported to or oxidized in skeletal muscle. Representing the majority of lipid stores, adipose
tissue triglycerides are metabolized by adipose tissue triglyceride lipase (ATGL), hormone
sensitive lipase (HSL), and monoglyceride lipase (MGL), which cleave individual fatty acids
(FA) from their glycerol backbone (Lass et al., 2011). Given that MGL is not typically limiting
to lipolysis in vivo, the rate of lipolysis falls under the combined control of ATGL and HSL.
While the precise regulation of ATGL remains unclear, there is evidence that ATGL is subject to
hormone-related control by glucocorticoids (upregulation) and insulin (downregulation), and
possesses multiple phosphorylation sites (Bartz et al., 2007; Ahmadian et al., 2009). HSL
activity, on the other hand, is fairly well characterized. At rest, adipose tissue HSL remains less
phosphorylated, and its access to the lipid droplets is physically blocked by perilipins; proteins
that surround adipocytes. At the onset of exercise, adipose tissue HSL activity is upregulated
primarily through epinephrine-related signalling (reviewed in Holm, 2003), resulting in the
phosphorylation of both HSL and perilipins predominantly by protein kinase A to increase
lipolysis.
While adipose tissue triglycerides provide the majority of FA for oxidation at rest,
IMTGs represent an important source of energy during exercise (Stellingwerff et al., 2007; van
Loon, 2004). Lipolysis of intramuscular triglycerides is also regulated by lipases. ATGL is
8
phosphorylated by AMP-activating protein kinase (AMPK) (Ahmadian et al., 2011), and
upregulated by exercise training (Alsted et al., 2009), but the role of exercise on ATGL
phosphorylation and lipolytic capacity in humans has not been established. Skeletal muscle HSL
activity appears to be primarily regulated by calcium- and epinephrine-related phosphorylation
(Talanian et al., 2006; Watt et al., 2006; Watt & Hoy, 2012), as well as through contraction- and
epinephrine-stimulated translocation to the lipid droplet (Prats et al., 2006). AMPK
phosphorylation of HSL actually reduces its activity with prolonged exercise (Watt et al., 2006)
FAs from adipose tissue are transported to skeletal muscle via the circulation, but due to
their hydrophobic nature, the majority of FAs in the blood are bound to albumin (Richieri et al.,
1993). Those not bound to albumin represent free fatty acids (FFA), and it is those FFA that are
able to cross the vascular endothelium into the interstitial space, where they are available for
transport into the muscle cell. Fatty acid uptake by the muscle occurs through passive diffusion
(Kamp & Hamilton, 1992) and protein mediated transport (Bonen et al., 1998). Major regulatory
proteins in fatty acid transport across the sarcolemma include fatty acid translocase/cluster of
differentiation 36 (FAT/CD36), plasma membrane-associated fatty acid-binding protein
(FABPpm), and the fatty acid transport protein (FATP) family (Glatz et al., 2010).
Overexpression in rodent skeletal muscle has suggested FAT/CD36 and FATP4 have the greatest
capacity to stimulate FA transport, while FABPpm and FAT/CD36 correlate best with FA
oxidation rates (Nickerson et al., 2009). Experiments using giant sarcolemmal vesicles have also
shown that FAT/CD36, FABPpm, FATP1, and FATP4 are all stimulated to translocate to the
sarcolemma following contraction (Bonen, 2000; Jain et al., 2009). Furthermore, the use of
inhibitors and knock-out models in rodent skeletal muscle support an important role of
sarcolemmal fatty acid transporters in enabling the increase in FA oxidation during exercise
9
(Bonen, 2000; Bonen et al., 2007). Once in the sarcoplasm, hydrophobic fatty acids exist bound
to cytosolic FABP (FABPc) until their conversion to fatty acyl-CoA molecules by acyl-CoA
synthetase (ACS). Fatty acyl-CoA synthesis is required prior to both oxidation and storage of
FAs within muscle.
ATP generation from FAs in skeletal muscle is predominantly aerobic, occurring within
the mitochondria. The transport of long-chain fatty acids (LCFA; fatty acids containing ≥14
carbon atoms) into the mitochondria is a coordinated process (Figure 2), beginning with carnitine
palmitoyl-transferase I (CPT-I) conversion of LCFA-CoA molecules into LCFA-carnitine
molecules to allow their diffusion through carnitine/acylcarnitine translocase (CACT) on the
inner mitochondrial membrane, into the mitochondrial matrix. Within the matrix, CPT-II
converts LCFA-carnitine molecules to LCFA-CoA (Kerner & Hoppel, 2000). LCFA-CoAs are
ultimately reduced to individual acetyl-CoA molecules via β-oxidation, yielding NADH and
flavin adenine dinucleotide (FADH2). Mitochondrial LCFA transport is rate-limiting in LCFA
oxidation, with CPT-I representing the main regulatory enzyme, sensitive to malonyl-CoA (M-
CoA) inhibition (McGarry et al., 1983). Acetyl-CoA carboxylase (ACC) catalyzes the
conversion of acetyl-CoA to M-CoA and is inhibited by AMPK phosphorylation. Therefore,
AMPK levels indirectly control CPT-I activity by altering the phosphorylation status of ACC,
which regulates M-CoA levels (Figure 2). In addition, M-CoA is broken down by M-CoA
decarboxylase, to further regulate the concentration of this inhibitor. However, experiments in
human skeletal muscle show increased FA oxidation during exercise in the absence of
appreciable changes in M-CoA levels (Odland et al., 1996; 1998), while rodent work exists to
suggest elevations in P-CoA with exercise may override the M-CoA inhibition of CPT-I (Smith
et al., 2012). Interestingly, training is associated with increased CPT-I activity as well as
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sensitivity to M-CoA (Starritt et al., 2000). It remains unclear why superior M-CoA sensitivity
would prove beneficial to the trained individual and generally what signals during exercise
regulate CPT-I activity in humans. It is possible that other regulatory mechanisms are involved;
as one example, studies have found a reduction in CPT-I activity caused by decreased pH during
exercise (Starritt et al., 2000).
Figure 2 – Mechanism of mitochondrial LCFA transport. LCFAs are converted to LCFA-CoAs for oxidation or storage, an energy-consuming process. Those destined for oxidation (depicted here) are converted to LCFA carnitine molecules by exchanging a CoA for a carnitine upon transfer to the inner mitochondrial space by CPT-I. CPT-I activity is rate-limiting, and inhibited by M-CoA; however regulation during exercise in humans is uncertain. Subsequent passage to the mitochondrial matrix is facilitated by CACT, after which LCFA carnitine molecules are reconverted to LCFA-CoAs by CPT-II. LCFA-CoAs then enter β-oxidation to yield acetyl-CoA molecules for oxidative phosphorylation. LCFA: long chain fatty acids; ACS: acyl-CoA synthetase; CPT: carnitine palmitoyl transferase; LCarn: L-carnitine; CACT: carnitine/acylcarnitine transferase; AMPK: AMP-activated protein kinase.
11
In addition to plasma membrane transport, FAT/CD36 has been identified in skeletal
muscle mitochondrial membranes, where it has been found to facilitate FA transfer into the
mitochondria (Campbell et al., 2004; Smith et al., 2011). Furthermore, exercise has been found
to induce FAT/CD36 translocation to the mitochondrial membrane (Campbell et al., 2004;
Holloway et al., 2006), while training increases mitochondrial CD36 content as well as
sarcolemmal FABPpm content (Talanian et al., 2010). This represents one example of the
potential for exercise training to improve FA oxidation and influence fuel shifts during
submaximal exercise. Ultimately, fat metabolism generates acetyl-CoA molecules and reducing
equivalents to enter the CAC and the electron transport chain (ETC); the common entry points of
carbohydrate and fat into oxidative phosphorylation. Altogether, the host of regulatory points in
both carbohydrate and fat oxidation presented above demonstrate the complexity of substrate
provision for oxidative phosphorylation and ATP homeostasis. The interaction between these
two metabolic pathways will now be considered with respect to fuel selection, an integrative
process sensitive to conditions such as training and disease.
1.2.5 Fuel Interactions While the influence of exercise intensity on the relative contributions of carbohydrate and fat to
total energy expenditure was introduced earlier in this review (Figure 1), there is substantial
evidence that carbohydrate and fat availability further influence the proportion of each utilized at
a given power output (reviewed in Spriet & Watt, 2003; Holloway & Spriet, 2012). This
highlights the intricacy of metabolism, with a host of mechanisms in place to regulate fuel
selection and rate of use. In general, increased fatty acid availability has been shown to increase
muscle reliance on fat (Randle, 1964). This has been supported by various groups using intralipid
infusions in humans to increase FFA availability for submaximal exercise, collectively showing
12
reduced glycogen depletion or muscle glucose uptake depending on exercise intensity and
modality (Dyck et al., 1993; Hargreaves et al., 1991; Odland et al., 2000; Romijn et al., 1995).
The downregulation of carbohydrate utilization in this work is primarily attributed to lower
PHOS and PDH activity, which would spare glycogen for later in an exercise session. In theory,
this would be beneficial given that carbohydrate is the more efficient, but more limited and
rapidly depleted energy store. The reduction in PHOS and PDH activity is thought to be caused
by higher NADH (which would offset the fall in cell energy status; Chesley et al.,1998; Odland
et al., 2000), and PDK upregulation (which would favour PDH b; Peters et al., 2001) in
conditions of higher FFA availability. Conversely, elevated carbohydrate availability has been
shown to decrease fat oxidation (Coyle et al., 1997; Horowitz et al., 1997), which is suggested to
occur through a combination of elevated plasma insulin and decreased FFA availability. Once
again, this reflects the complexity of metabolism, by which the body seeks efficiency and safety
through fuel selection. The balance between the two can be further elucidated by the study of
metabolic adaptations to endurance training.
1.2.6 Skeletal Muscle Metabolic Adaptations to Endurance Training In addition to enzymatic regulation of substrate provision, exercise training induces gene
transcription programs that can influence fuel selection. For instance, the induction of
mitochondrial biogenesis following training improves the capacity for both fat and carbohydrate
oxidation by increasing mitochondrial enzymatic machinery (Holloszy et al., 1970; Holloszy,
1967; Mole et al., 1971). This is realized through increased maximal activities of enzymes
involved in the CAC, the ETC, and reactions specific to FA oxidation (mitochondrial membrane
transport and β-oxidation); full review by Holloszy & Coyle (1984). Furthermore, mitochondrial
biogenesis has been found to reduce ADPf at a given workload, attenuating the exercise stimulus
13
for PHOS and PDH activation and thus shifting the reliance of skeletal muscle energy production
to fatty acid oxidation (Holloszy & Coyle, 1984; Dudley et al., 1987; depicted in Figure 3).
Increases in sarcolemmal FABPpm (Talanian et al., 2010), superior IMTG lipolysis (Enevoldsen
et al., 2001; van Loon, 2004; Stellingwerff et al., 2007), and increased IMTG stores (Phillips et
al., 1996b) also occur with training, and would likely support a shift towards fat oxidation at any
submaximal intensity given the role of FA availability in fuel selection outlined above. The
repeated observation by which trained individuals rely more on fat at a given workload (Phillips
et al., 1996b; Chesley et al., 1996; Putman et al., 1998) emphasizes the importance of FA
oxidation in whole body health, and with diseases characterized by impairments in FA oxidation
becoming more prominent, expanding our understanding of the mechanisms enhancing FA
oxidation is increasingly meaningful.
Figure 3 – Improved ADP sensitivity in the trained individual. Smaller concentrations of ADP are required to elicit a set mitochondrial respiration due to increased mitochondrial enzymatic machinery following endurance training. Figure from Holloway & Spriet (2009), adapted from Holloszy & Coyle (1984).
1.3 MITOCHONDRIAL RESPIRATION
1.3.1 Oxidative Phosphorylation As explained above, carbohydrate and fat metabolism are tightly regulated to maintain ATP
homeostasis. Because of the significant contribution of oxidative phosphorylation to overall
14
energy production, understanding of the regulation of metabolism within the mitochondria is
essential. Based on the overall equation for oxidative phosphorylation, it is clear that major
influences on this pathway include the delivery of reducing equivalents to the electron transport
chain (NADH), the supply of oxygen (O2), and the provision of ADP (Figure 4).
5ADP + 5Pi + 2NADH + O2 + 2H+ ! 5ATP + 5NAD+ + 7H2O
The delivery of fuel to the mitochondria was outlined in the previous section, and its conversion
to reducing equivalents will be explained in this section. The delivery of O2 to the mitochondrial
matrix is not usually limiting except during maximal exercise, the onset of exercise at high
intensities, exercise at altitude, and disease (Wagner, 1995). ADP provision to the mitochondria
is affected by ATP hydrolysis rates in the cytosol and ADP transport into the mitochondria. The
role of ADP as a regulator of mitochondrial respiration will be the focus of the current section.
Figure 4 – Regulation of mitochondrial respiration. The supply of reducing equivalents at various entry points in the electron transport chain provides electrons to be passed down the system towards a terminal electron acceptor (O2), generating a proton motive force by pumping protons into the intermembrane space along the way. This proton motive force is harvested by complex V to produce ATP. Altogether, major inputs for ATP production includes reducing equivalents, O2, and ADP (displayed in red). Q: Q-junction; ETF: electron-transferring flavoprotein; C: cytochrome C; ANT: adenine nucleotide transferase.
15
1.3.1.1 Mitochondria
Mitochondria are important organelles in the study of overall energy homeostasis, as well as
calcium-ion control, cell signalling, and apoptosis, placing them as a central focus in general
health and disease (Duchen, 2004). Mitochondria exist as a reticular structure, interacting with
each other and with other organelles, such as the sarcoplasmic reticulum (Ogata & Yamasaki,
1997). Mitochondrial membranes include a porous outer membrane, permeable to small
molecules, and an inner membrane, which is impermeable to most molecules without specific
transporters. The intermembrane space contains some important proteins for oxidative
phosphorylation, including mitochondrial creatine kinase (miCK) for ADP transport, and
cytochrome C of the ETC. The mitochondrial inner membrane contains other important proteins
involved in the ETC and ATP synthesis. Before this stage of energy production, a series of redox
reactions occur in the mitochondrial matrix, generating reducing equivalents from acetyl-CoA
molecules.
1.3.1.2 Citric Acid Cycle
The CAC oxidizes acetyl groups to form reduced coenzymes (NADH and FADH2) for transfer
through the electron transport chain. The rate-limiting enzymes in the CAC are isocitrate
dehydrogenase (IDH) and 2-oxoglutarate dehydrogenase (2-OGDH), which are stimulated by
elevated Ca2+ levels and ADPf. Though not rate-limiting (due to much lower maximal activity),
another regulatory enzyme in the in the CAC cycle is citrate synthase (CS), which catalyzes the
conversion of oxaloacetate to citrate with the entry of acetyl-CoA to the CAC and which is
regulated by NADH and citrate levels (allosteric inhibitors). Overall, the CAC involves several
intermediates including glutamate and malate, which supply NADH for complex I, and
succinate, which provides FADH2 for complex II. A step-by-step description of the CAC is not
16
provided in this review (full review in Akram, 2014), but important substrates and products are
depicted below.
Figure 5 – Key substrates and products of the TCA cycle. Carbohydrate and fat are reduced to acetyl-CoA to enter the TCA cycle in the mitochondrial matrix. Reducing equivalents in the form of NADH (at complex I) and FADH2 (not shown; oxidized at complex II) enter the electron transport chain to create a membrane potential that is transformed into energy at complex V. Without a continual supply of TCA cycle intermediates, oxaloacetate will accumulate and inhibit TCA cycle flux. Each cycle produces 3 NADH, 1 FADH2, 1 GTP, and releases 2 CO2. Q: Q-junction; C: cytochrome C.
1.3.1.3 Electron Transport Chain
The coupling of ADP phosphorylation to electron and hydrogen transfer was discovered by Peter
Mitchell (1961), leading to our current model of the ETC in which electrons from reducing
equivalents are passed down four protein complexes (I-IV) of increasing redox potential,
releasing free energy that is used to pump protons from the matrix into the intermembrane space
(against a concentration gradient). As shown in Figure 4 below, complex I pumps 4 H+ into the
17
intermembrane space, transferring two electrons to complex I in the process of converting
NADH to NAD+. As electrons from NADH (complex I) and FADH2 (complex II) are passed to
cytochrome c via complex III, two more H+ are pumped to the intermembrane space. Once again
there are 4 H+ pumped across the inner membrane by complex IV as electrons are transferred
there from cytochrome c. In this final step at complex IV, cytochrome c oxidase catalyzes the
conversion of ½ O2 (“oxidative” phosphorylation) to H2O using electrons pumped through the
chain, and H+ from the mitochondrial matrix. The sum of the protons pumped across the
mitochondrial inner membrane create a membrane potential, associated with a proton motive
force that is captured by complex V, or F1F0ATPase, as protons flow back into the mitochondrial
matrix down a concentration gradient. This protein uses 3H+ to catalyze the production of each
ATP molecule from ADP + Pi (coupled phosphorylation).
18
Figure 6 – Electron transport chain. Electrons are donated in the form of reducing equivalents to complex I (NADH) and complex II (FADH2), or directly to the Q cycle from G3P and ETF. Electron flow down the electron transport chain provides energy to pump protons across various sites (depicted above) into the intermembrane space. Electrons passed on to ½O2 to generate H2O at complex IV. The accumulation of protons in the intermembrane space provides the proton motive force used by complex V to generate ATP. A-CoA: acetyl-CoA; Q: Q-junction; ETF: electron-transferring flavoprotein; C: cytochrome C; G3P: glycerol-3-phosphate.
Under resting conditions, there is no shortage of NADH or O2, while ADP availability to
complex V remains low as it is tightly coupled to cytosolic ADPf (Wilson, 1994). The increase in
ADPf with exercise is therefore pivotal in coordinating not only the rise in glycogenolysis and
glycolysis via PHOS and PFK, but also oxidative phosphorylation. However, the control of ADP
availability to complex V and its effects on respiration remain poorly understood despite the
discovery of its regulated transport and responsiveness to exercise training.
19
1.3.1.4 ADP Transport
ADP transport from the cytosol into the mitochondria where it is required for oxidative ATP
production can occur via two mechanisms: miCK-independent or miCK-dependent. ADP import
independent of miCK follows passive diffusion across voltage-dependent anion channel (VDAC)
in the outer mitochondrial membrane, and adenine nucleotide transferase (ANT) on the inner
mitochondrial membrane. As portrayed in Figure 7, ATP is anti-ported with ADP in a 1/1 ratio,
linking cytosolic energy demand to mitochondrial ADP supply. Work in the 1960s revealed ADP
transport (rather than ATP synthase) as the driver of respiration (for a summary of experiments,
see Klingenberg, 2008). Furthermore, the use of ANT inhibitors bongkrekic acid and
isobongkrekic acid illustrated that ANT is essential in mitochondrial ADP transport (Fiore et al.,
1997). ADP transport can be augmented through miCK in the intermembrane space through the
transfer of a phosphate from exported ATP to creatine as depicted below (Aliev et al., 2011).
The resultant ADP is then quickly returned to the mitochondrial matrix by ANT, efficiently
maintaining matrix ADP levels, while the PCr produced in the process is exported to the cytosol
through a VDAC on the outer mitochondrial membrane to be recycled by creatine kinase.
Computer modelling of mitochondrial ADP transfer in cardiac muscle indicates that a significant
component (80%) of ADP transfer occurs through miCK (Aliev et al., 2011), additional
regulation indicating that mitochondrial ADP provision represents an important signal for
mitochondrial respiration that is coupled with cytosolic phosphorylation status (Walsh et al.,
2001b). While the recruitment of miCK in ADP transport has been found to differ between fibre
types and training statuses in PmFB (Zoll et al., 2003; Guerrero et al., 2005), its importance in
vivo across exercise intensities remains unclear, supporting the need for further study of ADP
transport regulation.
20
Figure 7 – Mechanisms of mitochondrial ADP transport. Left side: miCK-independent ADP transport occurs through passive diffusion directly through VDAC on the outer mitochondrial membrane, and ANT on the inner mitochondrial membrane. Right side: miCK-dependent ADP transport amplifies ADP signals by “recycling” exported ATP from the intermembrane space into ADP through phosphate transfer to creatine by miCK. ADP can then be immediately transported back into the matrix through ANT. miCK: mitochondrial creatine kinase; VDAC: voltage dependent anion channel; ANT: adenine nucleotide transferase. Figure from Perry et al, (2012).
1.3.2 Mitochondrial ADP Sensitivity
Given the central role of ADPf in the regulation of metabolism introduced in section 1.2 of this
review, the importance of mitochondrial ADP sensitivity represents a logical extension of the
importance of ADP signals and a basic index of mitochondrial function. The positive relationship
between cytosolic ADPf concentration and rate of oxidative phosphorylation was demonstrated
in the early 1950s (Lardy & Wellman, 1952; Chance & Williams, 1955). These studies
measured increased oxygen consumption rates upon the addition of ADP to mitochondria
isolated from rat liver with various substrates present. Subsequent work by Jacobus et al. (1982)
used ADP and ATP titrations to manipulate respiration media and found that ADP concentration
(independently of ATP/ADP ratio) consistently corresponded with respiratory rate, implicating
ADP availability as a driving factor in respiratory sensitivity. In the 1970s, Karlsson and
21
colleagues (1972) observed reduced phosphagen depletion during submaximal work following
training in man, suggesting that ADP sensitivity could be influenced by training. Dudley et al.
(1987) established a role of mitochondrial content in the regulation of ADP sensitivity using
exercise training and hypothyroidism to manipulate mitochondrial content in rats. They found
that muscle with a higher oxidative capacity also had a higher ADP sensitivity (determined from
lower calculated ADPf at a given oxygen consumption). However, ADP sensitivity has since
been shown to increase even before statistically significant increases in mitochondrial content,
suggesting mitochondrial content is not the only factor influencing ADP sensitivity (Green et al.,
1992; Phillips et al., 1996a).
The use of PmFB allows the controlled study of mitochondria in situ, with cytosolic enzymes
and metabolites removed. Of importance, experiments in PmFB have revealed differences in
miCK regulation of ADP sensitivity compared with isolated mitochondria (Saks et al., 1998),
which can be attributed to the cleavage of miCK through the isolation procedure and stresses the
importance of evaluating the mitochondrial function in their cellular milieu. An early study on
the effects of an acute bout of exercise on mitochondrial respiration in PmFB from humans
demonstrated that maximal ADP-stimulated respiration (Vmax) is not changed immediately after
exhaustive exercise (Tonkonogi et al., 1998). However, training (Walsh et al., 2001a) and
physical activity level (Zoll et al., 2002) correspond to higher Vmax. Concerning mitochondrial
sensitivity to ADP, the early work by Tonkonogi et al. (1998) revealed higher mitochondrial
respiration with a submaximal concentration of ADP and creatine following an acute exercise
session; indicative of improved sensitivity to ADP. However, respiratory sensitivity remained
unchanged following exercise if creatine was absent from the respiration medium, supporting an
important role for miCK in ADP sensitivity. Surprisingly, cross-sectional analysis of ADP-
22
stimulated mitochondrial respiration in humans has indicated lower sensitivity/higher michaelis-
menten constant (Km: the concentration required for half-maximal function) in athletic
individuals compared with sedentary counterparts (Zoll et al., 2002), and this has since been
shown following an exercise intervention in young, healthy individuals (Walsh et al., 2001a) and
in lung transplant patients in the absence of creatine only (Guerrero et al., 2005). Among the
aforementioned athletes (but not sedentary individuals), the presence of creatine in the
respiration medium lowered the apparent ADP Km value, consistent with previous analyses (Saks
et al., 1995). This points to the intricacy of the regulation of ADP transport especially in the
response to training.
The observation that PmFB spontaneously contract during respiration experiments (Perry et
al., 2011) has inspired analyses using blebbistatin (BLEB), a myosin ATPase inhibitor, to control
for this response. Perry et al. (2012) demonstrated that changes in ADP sensitivity following
acute exercise are contraction-mediated such that sensitivity is increased with creatine present
only in spontaneously contracting fibre bundles. In relaxed fibre bundles, sensitivity was
decreased (no creatine) or unchanged (with creatine). Therefore, regulation of ADP sensitivity
depends on the contractile state of the tissue in addition to the function of miCK.
Recently, Smith et al. (2013) explored the regulation of ADP sensitivity in disease using the
zucker diabetic fatty (ZDF) rat. ZDF animals were found to have lower submaximal ADP-
stimulated respiration rates and ANT2 protein content compared with lean control animals.
Interestingly, both ADP sensitivity and ANT2 protein content were recovered following
resveratrol supplementation. This occurred independently of mitochondrial content, which did
not change. This brings forth the potential for ANT to act as yet another regulator of ADP
sensitivity. Altogether, experiments in PmFB have identified a host of regulatory factors
23
affecting submaximal ADP sensitivity and metabolic control beyond mitochondrial content,
highlighting the importance of ADPf levels. Moving forward, a focus should be placed on the
ways in which models such as exercise and disease may alter the mitochondrial environment and
affect ADP transport and availability.
1.4 PALMITOYL-COA INTERACTIONS The inhibitory effects of long chain acyl-CoA esters on mitochondrial respiration were
identified in 1971 in rat heart mitochondria. In this work, Pande and colleagues (1971) observed
a high rate of oxidation of P-CoA and carnitine in the presence of saturating ADP. However, P-
CoA was found to inhibit respiration in the presence of submaximal ADP, malate, and pyruvate,
an effect that was alleviated upon the addition of carnitine to promote fat oxidation. Furthermore,
increasing ADP concentrations in the experimental media relieved the P-CoA inhibition and
kinetic analysis identified P-CoA as a competitive inhibitor of ADP-stimulated respiration,
causing an increase in the apparent ADP Km. Follow-up experiments included the addition of the
uncoupler DNP, which recovered the impairment in respiration caused by P-CoA. For several
reasons, these experiments suggested ANT as the site of inhibition of P-CoA: the degree of
inhibition was proportional to the number of mitochondria; inhibition was instantaneous upon the
addition of P-CoA; inhibition was reversible; inhibition was overcome by the addition of ADP
but not other adenine nucleotides (AMP, ATP, GDP); and inhibition was prevented by DNP.
Since this early work, the direct inhibition of ANT by P-CoA and other LCFA-CoA molecules
has become well-established (Shrago et al., 1974; Morel et al., 1974; Ho & Pande, 1974),
suggesting a regulatory role of LCFA-CoA molecules in fatty acid oxidation through control of
ADP availability. Of note, reduced mitochondrial ADP sensitivity (increased apparent Km) has
been found with acute exercise and diabetes, two cases associated with elevated P-CoA levels
24
(Ellis et al., 2000; Watt et al., 2003). As P-CoA is known to inhibit the ADP transport protein
ANT, the potential for P-CoA to regulate ADP transport and mitochondrial respiratory
sensitivity may identify it as a previously unexplored regulator of oxidative energy production
with important implications for health and disease. The effects of exercise training on this
interaction have not been investigated, but may rectify the inconsistency between the increased
whole muscle ADP sensitivity in vivo and the seemingly decreased mitochondrial ADP
sensitivity in situ following training.
1.5 CONCLUSIONS AND FUTURE DIRECTIONS As presented in this review, carbohydrate and lipid metabolism are highly regulated to
maintain ATP homeostasis in skeletal muscle. Exploration of aerobic metabolism, responsible
for the majority of energy production, has revealed an adaptable system primarily limited by
mitochondrial content. However, the use of saponin-PmFB has facilitated the exploration of a
host of additional regulators of mitochondrial respiration, including ADP transport. While the
control of ADP transport is shown to change following exercise training and acute exercise, the
effects on respiratory sensitivity are divergent. With no mechanism to explain this discrepancy, it
is clear that our understanding of the regulation of mitochondrial ADP sensitivity is lacking.
Given the ability of the lipid metabolism intermediate P-CoA to interact with the ADP transport
protein ANT, the effect of this molecule on mitochondrial function may provide important
insights into the control of respiration. Importantly, P-CoA and ANT levels have been shown to
change in conditions of exercise and disease, providing more support for their potential role in
regulating metabolic control. Therefore, this thesis explored the relationship between
P-CoA, ANT, and mitochondrial respiratory sensitivity using chronic exercise training to
elucidate its role in training-induced improvements in mitochondrial function.
25
CHAPTER 2: AIMS OF THESIS The PmFB technique presents a useful tool for the analysis of mitochondrial respiration,
allowing manipulation of the regulation of respiration in situ. Investigation into the regulation of
mitochondrial ADP sensitivity by acute and chronic exercise has yielded mixed results. The
consensus is an increased apparent ADP Km following exercise training, which is perplexing
given the well-established capacity for training to improve metabolic control.
While lipid-supported respiration has been evaluated in PmFB, the effects of palmitoyl-
CoA on ADP kinetics have not. P-CoA is a known inhibitor of ANT, which is tightly linked to
metabolism and energy balance.
Therefore, the purposes of this thesis were to:
1) Confirm the effects of exercise training on mitochondrial ADP sensitivity in a group of
inactive, middle aged males.
2) Establish a methodology to evaluate the effects of P-CoA on mitochondrial respiration in
PmFB.
3) Evaluate the role of P-CoA on mitochondrial ADP sensitivity and the capacity for exercise to
modulate this interaction.
It was hypothesized that:
1) Six weeks of combined endurance and interval training would improve maximum rates of
mitochondrial ADP stimulated respiration but reduce the sensitivity to ADP.
2) The addition of physiological amounts of P-CoA to standard respiration media would acutely
inhibit state III respiration of PmFB.
3) P-CoA would impair ADP kinetics (apparent Km) in PmFB and exercise training would
recover this impairment.
26
CHAPTER 3: PALMITOYL-COA INHIBITION OF MITOCHONDRIAL ADP SENSITIVITY IS ATTENUATED WITH EXERCISE TRAINING IN HUMAN SKELETAL MUSCLE
3.1 METHODS
3.1.1 Subjects Fourteen inactive middle-aged males (BMI 33.4 ± 1.2 kg/m2; ≤2 hr light-moderate physical
activity/week, 51.3 ± 1.7 years of age) were recruited from the Guelph community with posters
in public locations. Participants were screened using a medical questionnaire and a fasting blood
sample to ensure fasting blood glucose levels <7 mM (confirming the absence of diabetes).
Subjects provided written informed consent prior to experiments, all of which conformed to the
declaration of Helsinki, and were approved by the University of Guelph and Hamilton Integrated
Research Ethics Boards.
3.1.2 Blood Samples Oral glucose tolerance tests (OGTTs) were performed before and after 6 weeks of supervised
exercise training. Participants reported to the Human Nutraceutical Research Unit at the
University of Guelph following a 12-hour overnight fast. Weight and height measures were taken
at this time. Pre-training only, fasting blood glucose was measured from a finger prick with a
glucometer (Abbott Laboratories, Abbott Park, Illinois, USA) prior to the 2-hour oral glucose
tolerance test. If participants met the inclusion criteria of fasting blood glucose levels <7 mM,
trained phlebotomists inserted a catheter into the antecubital vein for all subsequent samples.
Blood was collected before (T = 0), and 15, 30, 60, 90, and 120 minutes following the ingestion
of a 75 g glucose drink (TRUTOL; Thermo Scientific) for a fasting blood profile, and a time
27
course of plasma glucose levels. Post-training OGTTs were performed ~72 hours following the
last exercise session (Figure 8).
Figure 8 – Overview of experimental design. OGTT: oral glucose tolerance test; END: endurance training session; HIT: high-intensity interval training session.
3.1.3 VO2peak Testing VO2peak was measured before and after training using a MOXUS metabolic cart (AEI
technologies). Exercise intensity was increased every two minutes on a case-by-case basis with
participants on an electronically braked cycle ergometer (Lode). Tests began after a two-minute
warm up at a power output of 50 watts. All tests lasted between 8-14 minutes and were
concluded when participants reached volitional fatigue (indicated by cadence dropping below 60
rpm). Reported VO2peak values were averaged over 20-40 seconds. Participants performed the
tests between 6-10AM and were instructed not to consume food or drink (other than water)
within two hours of performing the test.
3.1.4 Muscle Samples Muscle samples were obtained in the Department of Medicine at McMaster University.
Participants arrived at 8 AM following an overnight fast and provided diet logs for the 3 days
preceding the procedure to be replicated post-training. Incisions were made while under a local
anaesthetic (2% lidocaine without epinephrine), and samples were obtained from the vastus
lateralis using a Bergstrom needle. A first sample was placed in ice-cold BIOPS for the
28
preparation of PmFB, described below. A second sample was snap frozen by immediately
plunging the biopsy needle in liquid nitrogen, and stored for subsequent analyses (Western
blotting).
3.1.5 Exercise Training Subjects began training one week following muscle biopsy procedures. Training sessions were
supervised, 5 days/week for 6 weeks. Participants completed endurance (END) sessions on
Monday/Wednesday/Friday, and high-intensity interval training (HIT) sessions on
Tuesday/Thursday to elicit a strong training stimulus. Sessions were completed on Monark
bicycles, and included a 5-minute warm up and cool down with very low resistance. Endurance
sessions began at 30 minutes at 65% VO2peak and progressed to 45 minutes at 65% (pre-
training) VO2peak. HIT sessions began at 10x1 minute at 80% VO2peak with 2 minutes recovery
between each repetition and progressed to 10x1.5 minutes at 90% VO2peak with 1 minute
recovery between each repetition. Full details on the training progression are outlined in
Appendix A.
3.1.6 Animals Red gastrocnemius muscle obtained under resting conditions from male wild-type mice (8-10
weeks, 22.3 ± 0.7 g) was used for acute experiments characterizing the respiratory response to P-
CoA in PmFB and establishing a protocol for kinetic analyses in human tissue. Mice were stored
in cages of 3-4 and exposed to a 12h/12h light/dark cycle. Animal housing and experiments
conformed to the policies set forth by the University of Guelph Animal Care Committee.
3.1.7 Preparation of Permeabilized Fibre Bundles Permeabilized fibre preparation was performed as described previously by our lab (Perry et al.,
2012; Smith et al. 2011); for full details, see Appendix B. Muscle samples for PmFB preparation
29
were placed in BIOPS (50 mM MES, 7.23 mM K2EGTA, 2.77 mM CaK2EGTA, 20 mM
imidazole, 0.5 mM DTT, 20 mM taurine, 5.77 mM ATP, 15 mM PCr, and 6.56 mM
MgCL2.H2O; pH 7.1) on ice and separated into small (0.15-0.5 mg) bundles with fibres separated
along their longitudinal axis. Individual fibre bundles were then incubated in 30 µg/ml saponin in
BIOPS while turning in 4 °C for 30 minutes. PmFBs were subsequently washed in respiration
medium MiR05 (0.5 mM EGTA, 10 mM KH2PO4, 110 mM sucrose, and 1 mg/ml fatty acid free
BSA; pH 7.1) for 15-30 minutes.
3.1.8 Mitochondrial Respiration in Permeabilized Fibres Mitochondrial respiration (pmol"s-1
"mg dry weight-1) was calculated from high-resolution O2
consumption measures in the Oxygraph-2 k (Oroboros, Innsbruck, Austria). PmFB were placed
in 2 ml chambers containing MiR05 and 25 µM BLEB (myosin II inhibitor) to prevent
spontaneous contraction.
For chronic training responses in human tissue, mitochondrial sensitivity and capacity
was determined through standard ADP titrations with 0 mM versus 20 mM creatine to vary the
recruitment of mitochondrial creatine kinase as per Perry et al. (2012). Additionally, respiratory
kinetics were measured with 0 mM Cr after incubating PmFB in 50 µM P-CoA for 15 minutes.
All titrations were performed with saturating pyruvate and malate concentrations to provide
complex I electron transport chain substrates. Glutamate and succinate were added to
experiments following titrations to establish maximal complex I- and II-supported respiration.
Finally, 10 µM cytochrome C was added and a <10% increase in respiration across all
experiments indicated outer mitochondrial membrane integrity. A detailed outline of respiration
experiments is provided in Appendix B.
30
For acute experiments in rodent tissue, state III respiration (ADP-stimulated respiration)
was induced in the presence of saturating concentrations of malate + glutamate + pyruvate +
succinate or malate + G3P. Physiological concentrations of P-CoA (60µM) were then added to
the chambers and the real-time response was observed. Finally, dinitrophenol (DNP) was titrated
into those same chambers to elicit maximal uncoupled respiration.
A separate set of PmFB from rodents were incubated in Oxygraph chambers with MiR05
and various lipids (250 µM palmitate, 250 µM palmitate+1mM CoA, 60 µM P-CoA) in the
presence or absence of 5 mM ADP + 2 mM L-carnitine for 15 minutes. Fibres were then washed
in a chamber with standard MiR05 for 10 minutes before determining maximum complex II and
complex II-supported state III respiration. Respiration of control fibres following the incubation
and transfer protocol in standard respiration medium was similar to previous publications (Smith
et al., 2013), confirming that the pre-incubation process itself did not affect state III respiration
(control medium, Figure 14). Cytochrome C was again added after state III respiration was
achieved to ensure outer mitochondrial membrane integrity.
3.1.9 Blood Analyses Full details on blood handling and analysis can be found in Appendix C. Of note, a fasting lipid
profile including high-density lipoprotein (HDL) cholesterol, total cholesterol, serum
triglycerides (TG), and high-sensitivity C-reactive protein (HS-CRP) was measured from serum
samples processed by LifeLabs, Guelph. Fasting plasma samples were analyzed in-house for
non-esterified FA (NEFA) and insulin using commercially available kits (Wako Diagnostics,
Millipore ELISA). Plasma glucose was measured at all time points during the OGTT using a
standard plate assay.
31
3.1.10 Western Blotting Changes in protein content from training were measured in the same PmFB used for respiration
experiments to ensure measures were not influenced by slight fibre type differences. PmFB were
recovered from respiration chambers, freeze-dried, and weighed. PmFB were then digested in 5
ug/ul lysis buffer (10% glycerol [w/v], 5% beta-mercapoethanol [v/v], 2.3% SDS [w/v] in 62.5
mM Tris HCl pH 6.8, 1% bromophenol blue [v/v]) by shaking for 15 minutes at 65 oC. Samples
were then loaded in equal volumes (10 µl) onto a 12% SDS-PAGE gel and transferred to PVDF
membranes as previously described (Lally et al., 2013). Commercially available antibodies were
used to detect α-tubulin (Abcam, Cambridge, MA, USA), ANT1 (Mitosciences, Eugene, OR,
USA), ANT2 (Abcam), COXIV (Invitrogen, Eugene, OR, USA), and OXPHOS protein subunits
(Mitosciences). All samples pre- and post-training for a given protein were detected on the same
membrane using chemiluminescence and the FluorChem HD imaging system (Alpha Innotech,
Santa Clara, CA, USA). Changes measured from digested PmFB were confirmed in standard
muscle homogenate, presented in Appendix D.
3.1.11 Statistics All values are presented as means ± SEM. Apparent Km values were determined using Prism
(GraphPad Software, Inc. La Jolla, CA, USA) using Michaelis-Menten kinetics for standard
ADP titrations, or one-phase associations with P-CoA present in the media. Vmax is presented as
the highest respiration rate directly measured in titrations. Pre- versus post-training measures
were compared using a paired t test with P < 0.05 considered statistically significant. Training
characterization was analyzed with a one-way ANOVA across the 6 weeks of training, using a
Newmann-Kewls post-hoc test.
32
3.2 RESULTS
3.2.1 Six Weeks of Combined HIT and Endurance Training Significantly Improves Muscle Oxidative Capacity, Glucose Tolerance, and Cardiorespiratory Fitness To confirm the effects of exercise training on mitochondrial respiration and cardiorespiratory
fitness, muscle and blood samples were obtained from previously inactive, middle aged males
before and after 6 weeks of combined HIT and endurance training characterized in Table 1. The
duration of endurance sessions increased over the first three weeks and while intensity was
clamped at 65% VO2peak, the corresponding power output increased each week (104 ± 7W in
week one to 128 ± 7W in week 6). The intensity of the HIT sessions increased over the 6 weeks
from 79.5 ± 1.5% max HR for the first two weeks, to 84.5 ± 1% max HR for the next two weeks,
to 88 ± 1.5% max HR for the final two weeks of training. Of the 30 exercise sessions per 14
participants, only 1 session was missed by 1 participant due to a work-related emergency. This
attendance yielded robust training responses, displayed in Table 2.
33
Tabl
e 1
– C
hara
cter
izat
ion
of T
rain
ing
Prot
ocol
.
For
END
ses
sion
s, du
ratio
n an
d po
wer
out
put w
as in
crea
sed
acro
ss th
e 6
wee
ks. F
or H
IT s
essi
ons,
inte
rval
dur
atio
ns in
crea
sed
and
reco
very
be
twee
n in
terv
als
decr
ease
d ov
er th
e 6
wee
ks, r
efle
cted
with
var
iabl
e po
wer
out
puts
but
incr
easi
ng in
tens
ities
(he
art r
ate
resp
onse
s). V
alue
s re
pres
ent m
eans
± S
EM. n
=14.
*=S
igni
fican
tly (p
<0.0
5) d
iffer
ent f
rom
wee
k 1;
†=d
iffer
ent f
rom
wee
k 2;
‡=d
iffer
ent f
rom
wee
k 3;
¥=d
iffer
ent
from
wee
k 4.
EN
D: e
ndur
ance
trai
ning
sess
ion;
HIT
: hig
h-in
tens
ity in
terv
al tr
aini
ng s
essi
on.
34
Table 2 – Subject characteristics and training responses.
Values represent means ± SEM. n=12-14. *=Significantly (p < 0.05) different from Pre. BMI: body mass index; HOMA: homeostatic model assessment insulin sensitivity; NEFA: non-esterified fatty acids; TG: triglycerides; LDL: low-density lipoprotein; HDL: high-density lipoprotein; HS-CRP: high-sensitivity C-reactive protein; OXPHOS: oxidative phosphorylation protein subunits; Com: complex.
35
Training caused a significant reduction in BMI (-1.7 ± 0.8%) and a significant
improvement in VO2peak (13.8 ± 3.4%) and peak power during a VO2peak test (21.6 ± 3.3%)
(Table 2). Changes in fasting blood profile from training included reduced fasting blood glucose,
LDL cholesterol, LDL/HDL cholesterol, and HS-CRP (Table 2). Insulin sensitivity (HOMA)
was increased measured 72 hours after the last training session. Response to a glucose challenge
was also improved following training, in a 2 hour OGTT (Figure 9A). This corresponded to a
16% reduction in glucose area under the curve (AUC, Figure 9B).
Figure 9 – Training improves the OGTT response 72 hours following the last exercise bout. Area under the curve (B) was reduced 16% following training. Values represent means ± SEM. n=10. *=Significantly (p<0.05) different from Pre. Several subunits of electron transport chain protein complexes I-V were measured by
Western blotting as markers of mitochondrial content and all significantly increased (17.9-
32.1%) with training. This occurred in the absence of a change in α-tubulin content. Values as
arbitrary OD units/µg protein are shown in Table 2, and representative blots are presented in
Appendix D.
A B
0 20 40 60 80 100 120
4
6
8
10
Time (min)
Blo
od G
luco
se (m
M)
PrePost
*
*
*
p=0.06 p=0.07
Pre Post0
200
400
600
AUC
*
36
3.2.2 Improved Mitochondrial Capacity and Reduced Sensitivity Following Exercise Training Having confirmed the expected increase in mitochondrial content from the current training
protocol, the effects of chronic exercise on mitochondrial respiration were investigated in PmFB.
First, changes in maximal ADP-stimulated respiration with saturating concentrations of
substrates (state III respiration, Figure 10) were characterized. The present training protocol
increased state III respiration by an average of 63%, an expected increase based on the change in
mitochondrial content. Furthermore, respiratory control ratios (RCR) were calculated as the ratio
of state III/state IV respiration, with state IV representing respiration with pyruvate + malate in
the absence of ADP (“basal” respiration). Values of 6.0 pre-training and 7.1 post-training
indicate that mitochondria remained coupled throughout the preparation and experiments.
Figure 10 – Training improves mitochondrial capacity in permeabilized muscle fibres. StateIV: leak respiration with 2mM malate + 10mM pyruvate; State III complex 1 linked: 2mM malate + 10mM pyruvate + 5mM ADP; State III max complex 1 linked: 2mM malate + 10mM pyruvate + 5mM ADP + 10mM glutamate; State III max complex 1 and 2 linked: 2mM malate + 10mM pyruvate + 5mM ADP + 10mM glutamate + 10mM succinate. Values represent means ± SEM. n=11. *=Significantly (p<0.05) different from Pre.
37
Respiratory kinetics in response to ADP titrations were evaluated to measure the effects
of training on mitochondrial ADP sensitivity. Representative responses are presented in Figure
11 in the presence (A-C) and absence (D-F) of creatine in the respiration media. Confirming
previous observations (Walsh et al., 2001a; Guerrero et al., 2005), the apparent ADP Km values
(concentration of substrate required to reach half-maximum respiration rate) were increased
following training, which reflects a reduced sensitivity to ADP. The magnitude of the increase
was 38% with creatine (p<0.05) and 17% without creatine (p=0.08). Maximal respiration was
taken as the highest respiration achieved at the end of the titration and increased by an average of
6 7% across participants, regardless of the presence of creatine.
Figure 11 – Mitochondrial ADP sensitivity is reduced following exercise training. ADP titrations in the absence (A-C) and presence (D-F) of creatine are shown with pyruvate and malate present in the respiration media. Corresponding Vmax (B, E) indicate improved mitochondrial capacity, while an increase in apparent ADP Km (C, F) suggests submaximal sensitivity is reduced. Values represent means ± SEM. n=11-13. *=Significantly (p<0.05) different from Pre.
38
3.2.3 Increased ANT1 Protein Content Following Training Given that the present increase in apparent ADP Km was observed despite an increase in
mitochondrial content, the role of mitochondrial transport proteins on ADP sensitivity was
examined. Because the trend in apparent Km occurred in both the presence and absence of
creatine (suggesting regulation by factors other than miCK), changes in ANT1 and 2 isoforms
were determined (Figure 12).
Figure 12 – Increase in ANT1 and no change in ANT2 isoform content following 6 weeks of training. Values represent means ± SEM. n=12-14. *=Significantly (p<0.05) different from Pre.
ANT1 content increased 16% with training (Figure 12). There was no change in ANT2
content with training. Importantly, samples were prepared directly from PmFB recovered from
respiration experiments to ensure trends observed in respiration experiments were not biased by
the individual fibres arbitrarily selected for use in Oxygraphs. Furthermore, changes were
confirmed in Western blots from muscle homogenate to ensure changes were not biased by the
fibre digestion process, and also yielded an increase in ANT1 exclusively (Appendix D).
39
3.2.4 Palmitoyl-CoA Rapidly Inhibits Mitochondrial Respiration in Rodent PmFB With the knowledge that acyl-CoA molecules inhibit ADP transport in isolated mitochondria by
binding to ANT, the nature of this interaction was examined in PmFB from rodent red
gastrocnemius. The provision of 60 µM P-CoA to State III respiring fibre bundles (with complex
I-linked substrates and ADP present) rapidly repressed respiration by ~50% (Figure 13).
Subsequent provision of the uncoupling molecule dinitrophenol (DNP) recovered respiration to
~90% of the previous state III rate. These data demonstrated the acute nature of the inhibition of
mitochondrial function by P-CoA molecules and implicated ADP transport in this process.
Figure 13 – Respiration is acutely inhibited by P-CoA and recovered by DNP. Representative Oxygraph traces show real-time changes in state III mitochondrial respiration achieved with saturating glutamate+malate (A) or pyruvate+malate (B) and ADP. Smith, 2013. Investigations into the regulation of skeletal muscle mitochondrial metabolism. Unpublished doctoral dissertation, University of Guelph, Guelph, Canada.
60 65 70 75 80 85 90 95 1000
100
200
300
400
500
Time (min)
JO2
(pm
ol⋅s
ec-1⋅m
g-1 d
ry w
t)
50 55 60 65 70 75 80 85 900
100
200
300
400
500
Time (min)
JO2
(pm
ol⋅s
ec-1⋅m
g-1 d
ry w
t)
Glutamate(+(malate(
+(ADP(
+(P-CoA(+(DNP(
Pyruvate(+(malate(
+(ADP(+(P-CoA(
+(DNP(
A(
B(
40
Given the rapid nature of this inhibition, the effects of acute P-CoA exposure through
pre-incubation on standard mitochondrial bioenergetics were determined again in mouse red
gastrocnemius. Incubation with 250 µM palmitate alone had no effect on state III respiration.
However, incubation with 250 µM palmitate + 1 mM CoA impaired respiration by 55%. This
illustrates the requirement for the conversion of LCFA to LCFA-CoA for inhibition of
respiration. This was further confirmed by the finding that incubation with 60 µM P-CoA
inhibited state III respiration to a similar degree (49%).
Figure 14 – ADP and L-carnitine prevent inhibition of mitochondrial respiration by P-CoA. Middle bars indicate state III respiration is reduced following incubation with lipids. This effect is absent if ADP + L-carnitine are present in the incubation media. Legend indicates presence (+) or absence (-) of various lipids in the incubation medium. Values represent means±SEM. n=3-6. *=Significantly (p<0.05) different from control.
Pre-incubation was then performed with 5mM ADP and 2mM L-carnitine in the same
experimental incubation media. This prevented any detriment in respiration, suggesting that
increased metabolic flux (oxidation of P-CoA) blunts these inhibitory effects.
41
3.2.5 Exercise Training Improves Mitochondrial ADP Sensitivity and Capacity in the Presence of Palmitoyl-CoA
Given the capacity for exercise to promote fat oxidation, the effects of exercise training
on the P-CoA inhibition of mitochondrial respiration were examined. This approach
demonstrates that state III respiration in the presence of P-CoA was 70-75% lower than standard
state III respiration rates (Vmax Figure 15B vs. Figure 11B). In stark contrast to typical
assessments of ADP sensitivity (Figure 11), training decreased the apparent ADP Km in the
presence of P-CoA by 47% (p=0.05), suggesting an exercise training-mediated improvement in
ADP transport. This is not withstanding a >100% increase in Vmax following training (Figure
15B), making the training effect particularly striking. In addition to the apparent Km differences,
on an absolute level, six weeks of cycling training increased the ability of 250 µM ADP to
stimulate respiration ~ 4-fold (Pre; 7.7 ± 1.9, Post; 34 ± 11.3, pmol/sec/mg dry weight, P<0.05).
This suggests a reduced sensitivity to P-CoA inhibition.
42
Figure 15 – Mitochondrial ADP sensitivity in the presence of P-CoA is improved with training. ADP titrations (A) following a 15 minute incubation in medium with P-CoA demonstrate that both Vmax (B) and apparent Km (C) are improved with exercise training. Values represent means ± SEM. n=8-10. *=Significantly (p<0.05) different from Pre.
43
CHAPTER 4: DISCUSSION This thesis presents a novel view of P-CoA as a regulator of mitochondrial ADP sensitivity with
a capacity to respond to exercise training. Specifically, the present experiments 1) established an
increase in the apparent ADP Km as classically assessed following 6 weeks of combined
endurance and interval training in a group of previously inactive middle aged males, 2)
determined a methodology to evaluate ADP kinetics in PmFB in the presence of P-CoA, and 3)
demonstrated that exercise training decreases the apparent ADP Km in the presence of P-CoA.
These findings place P-CoA as a regulator of mitochondrial function through its interaction with
ADP transport via ANT.
4.1 MITOCHONDRIAL ADP TRANSPORT AND REGULATION OF ENERGY BALANCE
Attention to the role of mitochondrial ADP transport as a regulator of mitochondrial
respiration is relatively new despite its identification in the 1960s (Heldt et al., 1965), and has
been facilitated by the combination of the PmFB technique with high resolution respirometry.
Recent literature has focused on the regulation of mitochondrial ADP transport by miCK (Saks et
al., 1995; Walsh et al., 2001), uncovering its role in acute and chronic changes in ADP
sensitivity following exercise (Guerrero et al., 2005; Perry et al., 2012). To date, only two
studies have evaluated the change in the apparent ADP Km of PmFB following training in
humans. The first was performed before and after six weeks of endurance exercise in young,
healthy males and females (Walsh et al., 2001), and the second was performed before and after
12 weeks of endurance exercise in lung transplant versus control participants across a wide age
range (Guerrero et al., 2005). Therefore, this study provides important confirmation that the
apparent ADP Km increases with training in a group of previously inactive 40-60-year-old males
performing six weeks of combined endurance and interval work. This reduction in respiratory
44
sensitivity following training is perplexing. Considering in vivo analyses yield improved ADPf
sensitivity and FA oxidation at the whole muscle level, it is plausible that additional regulators of
respiration may be missing from the in situ environment, limiting conclusions on physiological
Km shifts. Based on its central role in ADP transport and the response to exercise, P-CoA
represents a likely candidate. Especially given the observation by Perry et al. (2012) of mi-CK
independent shifts in ADP sensitivity following acute exercise (a perturbation known to increase
P-CoA levels), the role of ANT in regulating ADP transport deserves attention moving forward.
Therefore, standard PmFB respiration conditions were expanded to evaluate mitochondrial ADP
sensitivity in the presence of P-CoA.
4.2 PALMITOYL-COA AS A REGULATOR OF MITOCHONDRIAL FUNCTION The acute experiments presented here confirmed the inhibitory effects of palmitoyl-CoA on
mitochondrial respiration in the PmFB preparation. The concentration of P-CoA used (60µM) is
representative of that in muscle during low-intensity exercise, and therefore this concentration is
likely to model the regulation of ADP sensitivity in conditions surrounding half-maximal
respiration (Watt et al., 2003). The recovery of respiration following the addition of DNP, which
uncouples respiration from ADP phosphorylation, attributes this inhibition to mitochondrial ADP
transport. This is consistent with previous work in isolated mitochondria, which also identified
site-specific binding of P-CoA to ANT as competitive with ADP (Ho & Pande, 1974; Shrago et
al., 1974; Woldegiorgis et al., 1982). This information points to P-CoA as an important regulator
of mitochondrial respiration via ANT, which could influence ADP sensitivity. Furthermore, the
addition of ADP- and Lcarn to the respiration medium is found to prevent P-CoA inhibition of
mitochondrial respiration, likely by supporting P-CoA oxidation. This would suggest that
conditions enhancing fatty acid oxidation, such as exercise training, may be beneficial in
45
mitigating P-CoA inhibition of ANT thereby improving ADP sensitivity. The promotion of FA
oxidation remains a controversial issue in the context of whole body health and will be discussed
later in this chapter. Altogether, real-time responses of mitochondrial respiration to P-CoA and
uncoupling in PmFB line up with experiments in isolated mitochondria, confirming the
inhibition of ADP transport by P-CoA and enabling its study in PmFB.
4.3 EXERCISE IMPROVES APPARENT ADP Km IN THE PRESENCE OF P-COA The major finding of the current thesis is a lower apparent ADP Km (improved sensitivity)
following exercise training with P-CoA present. This provides a possible explanation for the
unexpected decrease in mitochondrial ADP sensitivity previously found following training
interventions, supporting the importance of the respiration medium in evaluating mitochondrial
function. The magnitude of the increase in respiration in the presence of P-CoA following
training underscores the dramatic effects of exercise on the sensitivity of mitochondrial
respiration to P-CoA inhibition. The interaction between P-CoA and ANT supports a model
(Figure 16) placing P-CoA-inhibition of ADP transport as a regulator of mitochondrial
respiration via ANT inhibition. As depicted below (left side), it is proposed that in the untrained
state, P-CoA interacts with ANT to inhibit ADP transport into the mitochondria. This reduces
mitochondrial ADP availability and mitochondrial respiration at a given ADPf, keeping
mitochondrial respiration low. The observed Km shift in the presence of P-CoA following
training suggests that training (right side) reduces the sensitivity of ANT to P-CoA, allowing
greater mitochondrial ADP availability and respiration at a given ADPf. Altogether, this would
offer alterations in ANT sensitivity to P-CoA as a mechanism for shifts in fuel selection whereby
exercise-induced reductions in P-CoA sensitivity improve ADP sensitivity, contributing to
46
glycogen sparing. On the other hand, increased P-CoA inhibition in the untrained state could
contribute to the metabolic inflexibility typical of diseases such as obesity and insulin resistance.
Figure 16 – Exercise training reduces mitochondrial ADP sensitivity to P-CoA – a model. Left side: P-CoA represents a prominent inhibitor of mitochondrial ADP transport in untrained individuals, regulating ADP availability for complex V. Right side: proposed model suggesting exercise training increases mitochondrial ADP sensitivity in the presence of P-CoA by reducing ANT sensitivity to P-CoA inhibition, allowing superior mitochondrial ADP availability at a given workload. Working model supported under resting conditions. V: complex V.
At this stage, this model is limited by several factors, including the absolute Km values in
the presence of P-CoA ranging from ~2800µM ADP pre- to 1500µM ADP post-training. This is
significantly higher than the in vivo ADPf values of ~100-250µM (varying with energy
47
expenditure), suggesting the amount of inhibition observed exceeds physiological levels (Phillips
et al., 1996). It can be also noted that apparent Km values in the presence of Cr do shift around
the in vivo concentrations of ADPf calculated for submaximal exercise (160µM to 225µM in this
case), despite the unexpected direction of the shift. This would suggest that the majority of the
ADP provided to the PmFB is available as ADPf and that the system withholds a physiological
sensitivity to ADP in situ when miCK is maximally recruited in tissue obtained from resting
conditions. While P-CoA-influenced respiration measures in this work were made with creatine
absent from the respiration medium to isolate the role of ANT in ADP transport, this observation
suggests that its extrapolation to the in vivo situation should (naturally) include its coupling to
miCK. Furthermore, the absence of Lcarn from the respiration medium prevents FA transport
into the mitochondria and therefore matrix-side interactions between P-CoA and ANT. While the
presence of Lcarn in the PmFB preparation would alter standard respirometry measures (which
were the focus of this thesis), this interaction as it exists in humans should be considered in
future work modelling the effects of P-CoA on ANT. Respiratory changes (ADP kinetics) should
also be measured at varying P-CoA concentrations and in muscle samples obtained following
exercise at varying intensities to elucidate a more complete role of P-CoA in regulating ADP
sensitivity. Finally, while data from rodents shown here illustrates the uncoupling effects of DNP
to relieve P-CoA-induced inhibition of respiration support the role of ANT as the intermediary in
P-CoA effects on respiration, experiments directly measuring this interaction with exercise and
training are required to support this model. That is, the proposed conclusion also requires a direct
measurement of ANT sensitivity (rather than general mitochondrial ADP sensitivity) with
alterations in P-CoA and health status.
48
4.4 MITOCHONDRIAL ADP AVAILABILITY AND THE REGULATION OF MEMBRANE POTENTIAL Membrane potential is the electrical and chemical protonmotive force across the inner
mitochondrial membrane generated by the pumping of protons into the intermembrane space as
electrons flow down the electron transport chain (Divakaruni & Brand, 2011). While this is
primarily harvested for ATP production by complex V (“coupled” respiration), an imbalance in
energy supply and cellular demand for ATP (supply in excess of demand) can raise the
mitochondrial membrane potential by supplying electrons in excess of the demand for H+ to
produce ATP (Fisher-Wellman & Neufer, 2012). While reactive oxygen species (ROS) are a
normal byproduct of mitochondrial respiration, elevated ROS levels are also associated with
mitochondrial dysfunction, aging, and pathologies including type 2 diabetes (Anderson et al.,
2009; Brand, 2010). ROS are produced as electrons slip onto O2 during electron flow down the
electron transport chain, and the delay of electron flow associated with metabolic disease can
exacerbate this slippage and raise ROS to unhealthy levels. Interventions that improve the
availability of ADP for complex V could “relieve the brake” on the final stage of the electron
transport chain, and help dissipate mitochondrial membrane potential while lowering
mitochondrial ROS, a theory that has been supported using capacity measurements in isolated
mitochondria and PmFB (Anderson et al., 2006; Korshunov et al., 1997; Smith et al., 2013).
Given that ADP transport controls ADP availability in the mitochondrial matrix, it is directly
related to mitochondrial membrane potential. Therefore, it is logical that P-CoA-induced
inhibition of ADP transport as measured in this work would have implications for ROS emission
and related pathologies. Furthermore, there is evidence that ANT mediates fatty acid-induced
uncoupling (Schönfeld et al., 2000; 1997), which in itself is an important cellular defense
mechanism from ROS-induced oxidative stress. Altogether, the clear relationship between P-
49
CoA, ANT, and membrane potential should emphasize avenues exploring the regulation of ANT
as it applies to ROS production and oxidative stress.
Despite the strong support for a role of mitochondrial ROS in diet-induced insulin
resistance (Anderson et al., 2009; Boden et al., 2012), the effects of exercise on the capacity for
ROS emission are largely unexplored. The standard measure of mitochondrial ROS is performed
in the presence of saturating succinate and oligomycin, inducing reverse electron flow to
measure the propensity for ROS production through complex I. While we did not observe
changes in maximal succinate-supported H2O2 emission following the current training protocol
(Figure 17), we did find reductions in markers of oxidative stress (Figure18).
Figure 17 – Propensity for succinate-supported mitochondrial ROS emission before and after exercise training. Measured in the presence of 25 uM BLEB and 20 mM succinate. Values represent means ± SEM. n=12.
State IV +100uM ADP +500uM ADP0
10
20
30
Mito
chon
dria
l H2O
2 em
issi
on(p
mol
H2O
2/min
/mg
dw) Pre
Post
*
* †
State IV +100uM ADP +500uM ADP0
10
20
30
Mito
chon
dria
l H2O
2 em
issi
on(p
mol
H2O
2/min
/mg
dw) Pre
Post
*
* †
50
Figure 18 – Reductions in markers of oxidative stress following 6 weeks exercise training. Lower 4HNE measured post-training indicates decreased lipid peroxidation (A), while a reduction in protein carbonylation can be seen post-training in (B). Values represent means ± SEM. n=8 *=Significantly (p<0.05) different from Pre.
In light of the changes in ADP sensitivity noted in the present respiration experiments, an
extension to the current work would be to evaluate the effects of P-CoA on ROS emission and
membrane potential. Indeed, we have pilot data illustrating the ability of P-CoA to increase H2O2
emission in the presence of ADP (Figure 19), suggesting that mechanisms improving ADP
transport would provide beneficial therapies for ROS-induced oxidative stress. Similar
experiments following chronic exercise could determine the effects of training on this interaction
and could link the observed training-induced improvement in ADP sensitivity in the presence of
P-CoA to reductions in ROS emission and membrane potential.
Pre Post0
20
40
60
80
100
120
4HN
E(a
rbitr
ary
OD
uni
ts/µ
g pr
otei
n)*
Pre Post0
20
40
60
80
100
120
Pro
tein
car
bony
latio
n(a
rbitr
ary
units
/µg
prot
ein) *
Pre Post Pre Post
Pre Post Pre Post
A
B
51
Figure 19 – P-CoA inhibits ADP attenuation of ROS emission. Presence (+) and absence (-) of substrates is identified below. PmFB were incubated in 60 uM P-CoA prior to measurement with 25 uM BLEB and 25 mM succinate. Right side shows a dramatic reduction in ROS emission upon providing submaximal ADP to PmFB. Furthermore, P-CoA blunts this effect. Values represent means ± SEM. n=6 *=Significantly (p<0.05) different from P-CoA absence.
4.5 FATTY ACID OXIDATION IN METABOLIC DISEASE The role of P-CoA as an inhibitor of mitochondrial respiration brings with it a host of questions
regarding the place of fatty acid oxidation in the development of metabolic disease.
Mitochondrial dysfunction has been linked to metabolic disease, although its role as a cause or
consequence is under debate (Hoeks & Schrauwen, 2012; Lowell & Shulman, 2005). Of note,
mitochondrial function has been traditionally evaluated in the presence of saturating ADP, which
is not representative of conditions in vivo (Boushel et al., 2007; Phielix et al., 2008). Work by
Smith et al. (2013) has in fact found impairments in submaximal ADP-stimulated mitochondrial
respiration in tissue from ZDF rats compared with lean control animals. Given that P-CoA levels
are elevated with insulin resistance (Ellis et al., 2000), its role in the etiology of this disease
should be considered. As suggested in Figure 20, conditions with elevated P-CoA (e.g. insulin
resistance) would likely (i) increase fatty acid uptake into the mitochondria via reduced M-CoA
sensitivity (Smith et al., 2012) and (ii) inhibit ANT (Pande & Blanchaer, 1971). This
52
combination of events threatens the balance between mitochondrial fatty acid supply and
oxidation, a hallmark of metabolic disease and insulin resistance. Manipulation of M-CoA
activity has produced compelling evidence that increased FA uptake into the mitochondria in
situations of high lipid availability increases FA oxidation intermediates and insulin resistance,
implicating mitochondrial overload in the disease (Koves et al., 2008). Given the role of P-CoA
presented here, the combined mitochondrial FA import and reduced ADP sensitivity could
contribute to insulin resistance through reduced FA oxidation compounded by elevated ROS
production. While the mechanisms for improved FA oxidation following training remain unclear,
its implications for metabolic disease are ever more apparent.
Figure 20 – Effects of P-CoA on mitochondrial respiration. Elevated P-CoA corresponds to reduced M-CoA inhibition of CPT-I, facilitating transport into the mitochondrial matrix. Within the mitochondria, P-CoA inhibits ANT from binding ADP, reducing ADP availability to complex V.
53
4.6 FUTURE DIRECTIONS While improved ADP sensitivity is an established training adaptation at the whole muscle level,
the regulation of mitochondrial ADP sensitivity in health and disease remains unclear. This is
curious considering that mitochondrial respiration is the major source of ATP, meeting cellular
demands by anti-port with ADP through ANT. This makes ADP transport a key regulator of
mitochondrial respiration and ATP homeostasis. While it has been reported that ADP sensitivity
is decreased following training (i.e. increased apparent ADP Km), advances in our awareness of
the regulation of ADP transport across the mitochondrial matrix can offer some explanation.
Focus on the regulation of mitochondrial ADP transport has been placed on miCK
recruitment given its potential to amplify ADP signals to the mitochondria. While this provides a
fundamental difference between fibre types and training statuses (greater regulation by miCK in
oxidative fibres and trained individuals; Guerrero et al., 2005; Zoll et al., 2003), its relationship
to mitochondrial ADP sensitivity is somewhat surprising. Specifically, the increase in regulation
by miCK that occurs with training corresponds to an increased apparent ADP Km. This would
suggest that our understanding of the regulation of ADP availability to the mitochondria is
lacking. The exploration of the role of P-CoA as a regulator of ADP transport and sensitivity in
the current work may begin to explain this discrepancy. What remains to be determined is the
mechanism of action.
Considering the observed changes in ADP kinetics following training cannot be
attributed solely to increased ANT content (>200% increased sensitivity well exceeds the ~16%
increased ANT1 protein content), further investigation into the mechanism by which training
reduces P-CoA sensitivity is required. Post-translational modifications of ANT following
training would provide a good starting point for insight into this improved ADP affinity,
54
especially given findings such as reduced protein carbonylation following the present training
protocol. Since the use of X-ray crystallography to characterize the structure of mitochondrial
ANT, experiments have identified various carbonylation, acetylation, nitrosylation,
glutathionylation, and phosphorylation sites on ANT that are subject to modification by
increased substrate concentrations, exercise, oxidative stress, and aging (Yan & Sohal, 1998;
Feng et al., 2008; Queiroga et al., 2010; Kim et al., 2010; Chang et al., 2014). Concerning the
control of respiration, Mielke et al (2014) recently linked ANT acetylation status to its affinity
for ADP. Furthermore, this group linked exercise-induced ANT deacetylation at Lysine (Lys) 23
to glucose disposal (greater deacetylation corresponded to greater glucose disposal). This
provides compelling evidence that exercise can regulate ANT sensitivity to ADP through post-
translational modifications, a potential mechanism for the hypothesized reduction in sensitivity
to P-CoA following training. Future experiments should seek to elucidate the post-translational
modifications of ANT following acute and chronic exercise to establish a link to P-CoA
regulation of ADP sensitivity.
A starting point would be to characterize these changes in samples from the current
training study, with a focus on acetylation status. Immunoprecipitation of ANT was attempted on
the tissue, but the available antibodies were not sufficient to purify ANT from other integral
membrane proteins (data not shown). Therefore, liquid chromatography techniques should be
used to purify ANT for further analyses. Following characterization, experimental manipulation
of acetylation status could be performed and corresponding changes in apparent ADP Km
(respirometry in PmFB) and ADP transport (3H ADP in isolated mitochondria) could be
measured with various concentrations of P-CoA as proof of principle that acetylation status alters
mitochondrial sensitivity to P-CoA inhibition of ADP transport and sensitivity. Pending insights
55
from basic characterization experiments, this could be done through single point mutations of
ANT e.g. Lys 23 substitution in rodents, to achieve permanent deacetylation of ANT in tissue
obtained before and after acute and chronic exercise for comparison with control samples.
The current data also has implications for insulin resistance as analysis of pre-training
fasting blood samples obtained from 9 participants in this cohort (Figure 21) shows that insulin
sensitivity correlated negatively with apparent ADP Km (r= -0.73). Given that exercise training
typically improves insulin sensitivity (Holloszy, 2005), its divergent relationship with ADP
sensitivity indicates some discrepancy in the regulation of ADP availability to the mitochondria
within health.
Figure 21 – Lower apparent ADP Km corresponds to higher insulin sensitivity (HOMA). n=9 due to availability of blood samples. R2=0.53.
Conclusions on the effects of P-CoA levels on ADP sensitivity with metabolic disease
require longitudinal experiments as well as direct manipulation of ANT in models of insulin
resistance. Insights into the role of P-CoA sensitivity in the development of insulin resistance
would be gleaned from ANT siRNA and overexpression in rodents fed chow versus a high-fat
diet to induce insulin resistance. Changes in respiratory sensitivity to ADP in the presence of
0 2 4 6 80
100
200
300
ISI (HOMA)
App
aren
t AD
P K
m(μ
M A
DP)
r= -0.73
56
various P-CoA concentrations from rest and exercise muscle obtained before and after a high-fat
dietary intervention could also support a role for changes in P-CoA inhibition of ADP sensitivity
in the development of insulin resistance. This should be followed-up by characterization of ANT
acetylation status, apparent ADP Km following P-CoA incubation, and insulin sensitivity from
samples obtained before and after an acute exercise session pre- and post-training in healthy
versus insulin resistant individuals to extend the data from the current thesis to the diseased state.
Also important for in vivo conclusions is the development of a reliable method of measuring P-
CoA content and subcellular localization in muscle. This would allow the comparison of P-CoA
content across disease states, measurement of changes in P-CoA content with training, and
insight into the dynamics of its inhibitory effects on ANT (e.g. interaction with the cytosolic or
matrix side of ANT).
Overall, the present work supports a theory implicating P-CoA as an important regulator
of mitochondrial ADP sensitivity through inhibition of ANT. A combination of techniques is
required to validate this theory, including chromatography, respirometry, transport assays, and
genetic manipulation. This could expand our understanding of the mechanisms responsible for
training-induced improvements in metabolic control.
4.7 CONCLUSION In summary, this thesis identifies P-CoA as a regulator of mitochondrial ADP sensitivity,
a process that is poorly understood. The data presented here suggest that, 1) standard apparent
ADP Km is increased (ADP sensitivity is decreased) in middle-aged males following 6 weeks of
combined endurance and interval training; 2) P-CoA acutely inhibits mitochondrial respiration in
PmFB and uncoupling with DNP recovers this inhibition, implicating ADP transport in this
process; 3) apparent ADP Km is decreased (ADP sensitivity is increased) following training
57
when evaluated in the presence of P-CoA. Altogether, this thesis highlights the intricacy of the
regulation of mitochondrial ADP sensitivity and its adaptability to perturbations such as exercise.
Future work should seek to elucidate mechanisms responsible for the regulation of ADP
transport with a focus on the post-translational modification of ANT and its role in health and
disease.
58
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APPENDICES
Appendix A: Exercise Training Progression
Week END HIT 1 30 minutes, 65% VO2peak 10x1 minute, 80% VO2peak
2 minutes recovery between 2 35 minutes, 65% VO2peak 10x1 minute, 80-85% VO2peak
2 minutes recovery between 3 40 minutes, 65% VO2peak 10x1 minute, 85-90% VO2peak
1.5 minutes recovery between 4 45 minutes, 65% VO2peak 10x1.5 minutes, 85-90% VO2peak
1.5 minutes recovery between 5 45 minutes, 65% VO2peak 10x1.5 minutes, 90% VO2peak
1.5 minutes recovery between 6 45 minutes, 65% VO2peak 10x1.5 minutes, 90% VO2peak
1 minute recovery between
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Appendix B: Mitochondrial Respiration Experiments Permeabilized Muscle Fibre Bundle Preparation
Muscle samples were immediately transferred to relaxing biopsy preservation solution (BIOPS),
gently opened by eye using fine-tipped forceps, and kept on ice in 5ml transport tubes. BIOPS
was prepared as follows (for 1L):
Dissolve in 750ml H2O:
Chemical Formula Weight Addition to 1L CaK2EGTA Recipe below 27.7ml
K2EGTA Recipe below 72.3ml Na2ATP 551.1 3.141g
MgCl26H2O 203.3 1.334g Taurine 125.1 2.502g Na2PCr 372.2 4.908g
Imidazole 68.1 1.362g Dithiothreitol 154.2 0.077g
MES 213.2 10.66g pH to 7.1 at room temperature with pH meter
Bring final volume to 1L in graduated cylinder
Filter with Watman filter paper
Store frozen (-20°C) in 45ml aliquots
K2EGTA: 7.608g EGTA + 2.3g KOH in 200ml H2O; pH to 7.0
Ca K2EGTA: 2.002g CaCO3 + 2.3g KOH in 100mM hot EGTA (7.608g/200ml H2O); pH to 7.0
Upon return to the University of Guelph, tissue was immediately viewed under the microscope
(on ice, A below) and 0.15-0.5mg fibre bundles (B below) were prepared with individual fibres
teased apart as shown. Fibres were incubated in 30ug/ml saponin in H2O for 30 minutes.
Incubation was performed in 1.5ml eppendorf tubes turning in 4°C. Following treatment in
saponin, PmFB were transferred to a different 1.5ml tubes and washed in 1ml Mitochondrial
70
Respiration medium (MiR05 – recipe below) for 15 minutes or until experiments were
performed (<30 minutes).
Mitochondrial Respiration in Permeabilized Fibres
Mitochondrial respiration was determined from Oxygraph-2 k high resolution respirometers
(Oroborus, Innsbruck, Austria). While fibres were being prepared, Oxygraphs were rinsed
several times in ethanol and water with stir bars spinning. Oxygraphs were then filled with 2ml
assay medium and connected to their software to reach 37°C and allow equilibration chambers.
Assay medium consisted of MiR05 or MiR05 with creatine. MiR05 was prepared as follows (for
1L):
Dissolve in 750ml H2O:
Chemical Formula Weight Addition to 1L EGTA 380.4 0.190g
MgCl26H2O 203.3 0.610g K-lactobionate Recipe below 120ml
KH2PO4 136.1 1.361g HEPES 238.3 4.77g Sucrose 342.3 37.65
BSA, fatty acid free 1g pH to 7.1 at room temperature
71
Bring final volume to 1L in graduated cylinder
Filter with Watman filter paper
Store frozen (-20°C) in 45ml aliquots
K-lactobionate: 0.5M stock from 35.83g lactobionate/100ml H2O; pH to 7.0 with KOH before
bringing the final volume to 200ml
MiR05 with 20mM creatine was prepared fresh each day from 149.15g/mol creatine (29.8mg
creatine/10ml MiR05). Preparation of other chemicals required for respiration experiments is
outlined below:
Blebbistatin, 10mM: 5mg in 1.71ml DMSO – stored at -20°C in 70ul aliquots in black
eppendorf tubes
Malate, 0.8M: 1.08g in 10ml H2O; pH to 7.1 – stored at -20°C in 1ml aliquots or at 4°C between
days
Glutamate, 2M: 3.38g in 10ml H2O; pH to 7.1 – stored at -20°C in 1ml aliquots or at 4°C
between days
Succinate, 1M: 2.70g in 10ml H2O; pH to 7.1 – stored at -20°C in 1ml aliquots or at 4°C
between days
Cytochrome C, 4mM: 50mg in 1ml H2O - stored at -20°C in 75ul aliquots in black eppendorf
tubes or at 4°C between days
Palmitoyl-CoA, 10mM (thaw fresh each day): 32 mg in 3.1842ml H2O – stored at
-20°C in 60ul aliquots
Pyruvate (make fresh each day):
• 0.5ml of 2M Pyr – dilute down into:
• 0.4ml 50mM Pyr (40-fold dilution – 10ul 2M Pyr in 350ul H2O)
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• 100ul 5mM Pyr (10-fold dilution – 10ul 50mM Pyr in 90ul H2O)
K-ADP titration kit (thaw fresh each day):
• 500mM ADP from 2 stock bottles: 2g in 7.979 ml H2O: add 6ml H2O, pH to 7.0 with 5N
KOH (~300ul at a time) and pH strips, top off volume to 7.979 ml
• Add 0.6ml 500mM ADP to 5.4ml H2O!6ml 50mM ADP
• Add 0.85ml 50mM ADP to 0.85ml H2O!1.7ml 25mM ADP
• Store 25mM stock in 0.05ml aliquots
• Store 50mM stock in 0.13ml aliquots
• Store 500mM stock in 0.225 ml aliquots (for 3 ADP titrations + 1 pyruvate titration x 2
participants/day)
• Store all aliquots at -20°C
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Respiration Experiments on Human Tissue Due to equipment availability and tissue viability, the following traces were performed in
singlicate for 2 participants per day (sample traces below):
Oxygraph 1 - ADP titration in MiR05 with 20mM Cr: 5ul 10mM BLEB
Fibre 12.5ul 0.8M Malate + 10ul 2M Pyruvate
ADP titration
10ul 2M Glutamate
20ul 1M Succinate
5ul 4mM Cytochrome C
Oxygraph 2 - ADP titration in MiR05 without Cr: 5ul 10mM BLEB
Fibre 12.5ul 0.8M Malate + 10ul 2M Pyruvate
ADP titration
10ul 2M Glutamate
20ul 1M Succinate
5ul 4mM Cytochrome C
Oxygraph 3 - ADP titration in MiR05 following pCoA incubation: 5ul 10mM BLEB
Fibre 10ul 10mM pCoA ≥15’ inc
12.5ul 0.8M Malate + 10ul 2M Pyruvate
ADP titration
10ul 2M Glutamate
20ul 1M Succinate
5ul 4mM Cytochrome C
ADP Titration:
Stock Concn (mM) Volume Added (µl) Final Chamber Concentration
50 1 25uM 50 3 100uM 50 3 175uM 50 3 250uM 50 10 500uM 500 2 1mM 500 4 2mM 500 8 4mM 500 8 6mM
!Continue until saturated
74
Sam
ple
Trac
e: A
DP
titra
tion
in M
iR05
follo
win
g P-
CoA
incu
batio
n
75
Appendix C: Blood Handling Procedures Three fasting blood samples were taken prior to ingestion of the glucose drink. Two 5ml samples
were collected in serum separator tubes for fasting lipid profile analysis according to the Client
Specimen Requirement Chart by LifeLabs. One 6ml sample was then collected in a sodium
heparin tube for analysis of plasma glucose and insulin in-house. Immediately upon collection,
tubes were gently inverted 8-12 times.
After sitting to clot for 30 minutes at room temperature, serum separator tubes were
centrifuged at 1200G for 15 minutes (20°C, ACC/DEC 9). They were then stored at 4°C until
LifeLabs courier pickup. One sample was used for lipid assessment (cholesterol, triglycerides,
total HDL, LDL) while a second sample was used for measurement of high sensitivity C-reactive
protein.
After fasting blood collection, participants consumed the standardized 75g glucose
beverage, and additional samples were collected in sodium heparin tubes 15, 30, 60, 90, and 120
minutes following completion of the drink. All samples obtained in sodium heparin tubes were
stored on ice and centrifuged for plasma separation within 2 hours of collection (1734G, 15
minutes, 4-10°C, ACC/DEC 9). Plasma was stored in 500ul aliquots at -80°C until analysis.
76
Appendix D: Supplementary Figures OXPHOS Protein Content from Digested PmFB
Pre Pre Post Post
CV
CIII
CII CI
αtubulin
CIV
Complex ISubunit NDUFB8
Complex IISubunit 30kDa
Complex IIISubunit Core 2
Complex IVSubunit 4
Complex VSubunit Alpha
0
50
100
150
OXP
HO
S(a
rbitr
ary
OD
uni
ts/μ
g pr
otei
n) * * * * *
77
ANT Protein Content from Muscle Homogenate
