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The role of AMP-activated protein kinase in initiating metabolic rate suppression in goldfish hepatocytes Lau, Gigi Yik Chee 2010

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THE ROLE OF AMP-ACTIVATED PROTEIN KINASE IN INITIATING METABOLIC RATE SUPPRESSION IN GOLDFISH HEPATOCYTES by  Gigi Yik Chee Lau  B.Sc., The University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2010  © Gigi Yik Chee Lau, 2010  ABSTRACT The ability to undergo metabolic rate suppression (MRS) markedly improves chances of survival during aquatic hypoxia. In this thesis, I specifically tested the hypothesis that AMPactivated protein kinase (AMPK) initiates MRS in hepatocytes from the common goldfish (Carassius auratus). My first goal was to investigate the responses of isolated hepatocytes to changes in O2. Goldfish hepatocytes showed a gradual decrease in cellular oxygen consumption  O2 ) as O2 was decreased from normoxia (~310 µM O2) down to the apparent P90 of 13 rate ( M  O2 . The apparent P90 for hepatocyte respiration µM, below which there was a steep decline in M   O2 in vivo matched published measurements of venous [O2], which suggests that hepatocyte M may be regulated by O2. To address the relationship between AMPK and MRS, several drugs were used to manipulate AMPK activity. I was able to activate AMPK with 5-Aminoimidazole4-carboxamide 1-β-D-ribofuranoside (AICAR) under normoxic conditions, which caused a  O2 ; this decrease was mediated through a decrease in protein synthesis rate via reduction in M  eukaryotic elongation factor 2 (eEF2) phosphorylation. Specifically, a maximal 7.5-fold  O2 , thus supporting the notion that AMPK activation of AMPK resulted in a 24% reduction in M  activation initiates MRS. We then used compound C, a general protein kinase inhibitor, in an attempt to reverse the AICAR effects on AMPK activation, but compound C did not reverse the effects of AICAR. A recently discovered specific AMPK activator, A769662, was also used to manipulate AMPK activity. However, at all doses, A769662 failed to activate AMPK. Nevertheless, whenever I was able to activate AMPK via AICAR incubation, there was a consistent lowering of metabolic rate. Thus I have provided evidence to support the hypothesis that AMPK is important in the initiation of MRS in goldfish hepatocytes.  ii  PREFACE Chapter two of this thesis is co-authored by Gigi Lau and Dr. Jeffrey G. Richards. A version of this chapter has been submitted for publication: Lau GY and Richards JG. AMP-activated protein kinase initiates metabolic rate suppression in goldfish hepatocytes. The research program was identified and designed by J.G.R. and the research and data analysis was carried out by G.L. under the supervision of J.G.R. The final manuscript preparation was conducted by G.L. in consultation with J.G.R. This research experiments performed for common goldfish Carassius auratus was approved by the UBC Animal Care Committee (AUP A09-0611; see Appendix).  iii  TABLE OF CONTENTS ABSTRACT ..................................................................................................................................................... ii PREFACE ....................................................................................................................................................... iii TABLE OF CONTENTS ................................................................................................................................... iv LIST OF TABLES............................................................................................................................................ vi LIST OF FIGURES ......................................................................................................................................... vii LIST OF ABBREVIATIONS............................................................................................................................ viii ACKNOWLEDGEMENTS ................................................................................................................................. x CHAPTER ONE: INTRODUCTION .................................................................................................................... 1 1.1 Environmental hypoxia .............................................................................................................. 1 1.2 Metabolic rate suppression as a unifying strategy for surviving environmental hypoxia .......... 1 1.3 Components and regulation of metabolic rate suppression........................................................ 2 Protein turnover ................................................................................................................... 3 Ion pumping activity ........................................................................................................... 4 1.4 Potential initiator of metabolic rate suppression—AMP-activated protein kinase .................... 5 1.5 AMP-activated protein kinase.................................................................................................... 6 Structure and regulation of AMPK ..................................................................................... 6 Downstream targets of AMPK ............................................................................................ 8 1.6 Pharmacological manipulators of AMPK activity ................................................................... 11 AICAR .............................................................................................................................. 11 Compound C ..................................................................................................................... 12 A769662 ............................................................................................................................ 12 1.7 Thesis objectives ...................................................................................................................... 13 CHAPTER TWO: AMP-ACTIVATED PROTEIN KINASE PLAYS A ROLE IN INITIATING METABOLIC RATE SUPPRESSION IN GOLDFISH HEPATOCYTES ................................................................................................ 17 2.1 Introduction .............................................................................................................................. 17 2.2 Materials and methods ............................................................................................................. 20 Animal care ....................................................................................................................... 20 Hepatocyte isolation .......................................................................................................... 20 High resolution respirometry ............................................................................................ 21 Series I: Response to [O2] ................................................................................................. 22 Series II: Responses of hepatocytes to AMPK activation .................................................. 23  iv  Biochemical analysis ......................................................................................................... 24 Calculations and statistical analysis .................................................................................. 26 2.3 Results ...................................................................................................................................... 27 Series I............................................................................................................................... 27 Series II ............................................................................................................................. 28 2.4 Discussion ................................................................................................................................ 29 CHAPTER THREE: GENERAL DISCUSSION AND CONCLUSION ..................................................................... 55 3.1 AMPK initiates metabolic rate suppression ............................................................................. 55 3.2 Responses of hepatocytes to physiological [O2] ...................................................................... 57 3.3 Future directions ...................................................................................................................... 58 3.4 Conclusion ............................................................................................................................... 59 BIBLIOGRAPHY ............................................................................................................................................ 60 APPENDIX .................................................................................................................................................... 68 Animal care certificate ...................................................................................................... 68  v  LIST OF TABLES  O2 max, P90, and P50 values of oxygen kinetic curves (obtained below 40 µM O2) in TABLE 2.1 M isolated goldfish hepatocytes from control, 15 mM cycloheximide and 3 mM ouabain treatments ..................................................................................................................................................... 54  O2 max, P90, and P50 values of oxygen kinetic curves (obtained below 40 µM O2) in TABLE 2.2 M isolated goldfish hepatocytes from control, 1 mM AICAR and 150 µM A769662 treatments .. 55  vi  LIST OF FIGURES  FIGURE 1.1 Model of AMPK activation .................................................................................. 15 FIGURE 2.1 Effect of oxygen concentrations on cellular oxygen consumption rate in isolated goldfish hepatocytes.................................................................................................................... 38 FIGURE 2.2 Oxygen kinetic curves of cellular oxygen consumption rate with progressive decrease in oxygen concentration in isolated goldfish hepatocytes incubated with 3 mM ouabain and 15 mM cycloheximide.......................................................................................................... 40 FIGURE 2.3 AMPK activity (A), phosphorylated AMPKα (B), phosphorylated eEF2 (C), and cellular oxygen consumption rate (D) in isolated goldfish hepatocytes incubated with AICAR (0, 0.25, 0.5, 1.0, and 2.0 mM) for 1 hr ............................................................................................ 42 FIGURE 2.4 Oxygen kinetic curves of cellular oxygen consumption rate with progressive decrease in oxygen concentration in isolated goldfish hepatocytes incubated with 1mM AICAR and 150 μM A769662 ................................................................................................................. 44 FIGURE 2.5 Effects of 15mM cycloheximide (Cyc) and 1mM AICAR incubations on cellular oxygen consumption rate (expressed in % relative to control) in isolated goldfish hepatocytes at normoxia (above 200 µM O2) ..................................................................................................... 46 FIGURE 2.6 Effects of 40 µM compound C (CC) and 1mM AICAR incubations on cellular oxygen consumption rate (expressed in % relative to control) in isolated goldfish hepatocytes at normoxia (above 200 µM O2) ..................................................................................................... 48 FIGURE 2.7 AMPK activity (A), phosphorylated AMPKα (B), phosphorylated eEF2 (C), and cellular oxygen consumption rate (D) in isolated goldfish hepatocytes incubated with A769662 (0, 25, 50, 100, 150, and 200 µM) for 1hr .................................................................................. 50 FIGURE 2.8 Effects of 150 µM A769662 (A769) and 15mM cycloheximide (Cyc) incubations on cellular oxygen consumption rate (expressed in % relative to control) in isolated goldfish hepatocytes at normoxia (above 200 µM O2) ............................................................................. 52 vii  LIST OF ABBREVIATIONS 4E-BP1  eIF-4E binding protein  A769  A769662  ACC-1  acetyl coA carboxylase-1  ACC-2  acetyl coA carboxylase-2  AICAR  5-aminoimidazole-4-carboxamide ribonucleoside  AID  autoinhibitory domain  AMPfree  free adenosine monophosphate  AMPK  AMP-activated protein kinase  ANOVA  analysis of variance  ATP  adenosine triphosphate  BSA  bovine serum albumin  CaMKK  Ca2+/calmodulin-dependent protein kinase kinase  CC  compound C; 6-[4-(2-Piperidin-1-ylethoxy) phenyl]-3-pyridin-4ylpyrazolo [1,5-a] pyrimidine  CPT  carnitine palmitoyl CoA acyltransferase 1  DMSO  dimethyl sulfoxide  DNA  deoxyribonucleic acid  ECL  enhanced chemiluminescence  eEF2  eukaryotic elongation factor 2  eEF2K  eukaryotic elongation factor 2 kinase  eIF2  eukaryotic initiation factor 2  g  gram  GLUT1  glucose transporter-1  GLUT4  glucose transporter-4  HIF-1  hypoxia induced factor-1  HMG-CoA  3-hydroxy-3-methylglutaryl-coenzyme A  hr  hour  HRP  horse radish peroxidase viii  L-15  Leibovitz-15  LDH  lactate dehydrogenase  LKB1  liver kinase B1  min  minute  MO2  cellular oxygen consumption rate  MO2max  maximum cellular oxygen consumption rate  mRNA  messenger ribonucleic acid  MRS  metabolic rate suppression  mTOR  mammalian target of rapamycin  N2  nitrogen  NMDA  N-methyl-d-aspartate  O2  oxygen  o  degrees Celsius  PGC1α  peroxisome proliferator-activated receptor γ coactivator 1α  s.e.m.  standard error of mean  SDS  sodium dodecyl sulfate  Thr  threonine  tRNA  transfer ribonucleic acid  TSC2  tuberous sclerosis complex 2  TTBS  Tween-20 tris-buffered saline  ZMP  5-aminoimidazole-4-carboxamide ribonucleoside monophosphate  C  ix  ACKNOWLEDGEMENTS Thank you-To my supervisor Dr. Jeffrey Richards: For his guidance, much tested but endless patience (remember when my AMPK activity assay was not working and I showed up at your office every morning at 8 AM for months?), and many words of wisdom and encouragement. To the Richards Lab Especially to Milica Mandic and Ben Speers-Roesch: For all the thesis-related and unrelated advice, laughs, calorie-sharing, and marking my thesis due date on the calendar. To my committee members Drs. Trish Schulte and David Close: For their guidance and valuable advice. To Dr. Agnes Lacombe: For always having chocolate and a hug ready. To my friends: For their understanding when I show up two hours later than I said I would because of experiments. Especially to Stella Lee: For suggesting good restaurants to hit after patiently listening to my grumbling. And Georgina Cox: For introducing me to turkey bacon, and the many productive (and not so productive) sessions at Calhoun’s. Most importantly, to my parents: For keeping their fingers crossed when my experiments were not working, always worrying about what I eat, and then of course, paying the hefty long distance phone bills after my rants.  x  CHAPTER ONE: INTRODUCTION 1.1 Environmental hypoxia Low environmental oxygen, termed hypoxia, is common in aquatic systems due to both natural and anthropogenic causes (Diaz and Rosenberg 2008). The prevalence of hypoxia in many aquatic systems has been a powerful evolutionary driving force causing the selection of adaptive traits that either serve to enhance O2 extraction from the depleted environment or to maintain metabolic energy balance when O2 uptake cannot be sustained. When there are reductions to cellular O2 supply, mitochondrial oxidative phosphorylation is reduced resulting in a large reduction in the ability of the cell to generate ATP. Under these conditions, cells attempt to increase their O2-independent ATP production by stimulating substrate-level phosphorylation (Brand 2003), but without modifications to ATP use, fermentable substrates would quickly exhaust.  1.2 Metabolic rate suppression as a unifying strategy for surviving environmental hypoxia In response to O2 lack, hypoxia-tolerant cells undergo a controlled reduction in metabolic rate to down-regulate ATP consuming processes in an attempt to match the limited capacity for ATP production from substrate-level phosphorylation. By keeping [ATP] stable, metabolic rate suppression is thought to facilitate a maintenance of cellular energy balance, which is considered by many researchers as the hallmark of hypoxia tolerance (Hochachka et al. 1996). If energy balance is not maintained, critical cellular processes fail (e.g. maintenance of membrane potential), eventually ending in cell death (Boutilier and St-Pierre 2000). Metabolic rate suppression is a common strategy used by hypoxia-tolerant fish in response to low O2. The common goldfish (Carassius auratus) is one of the champions of 1  hypoxia/anoxia tolerance. Goldfish were able to reduce metabolic rate by up to 60% when exposed to severe hypoxia and by up to 70% in complete anoxia (Van Waversveld et al. 1989; van Ginneken et al. 2004). Other species such as crucian carp (Carassius carassius), tilapia (Oreochromis mossambicus), and European eel (Anquilla anquilla) have also been shown to reduce metabolic rate by 40 to 70% when exposed to severe hypoxia or anoxia (Johansson et al. 1995; van Ginneken et al. 1999; van Ginneken et al. 2001). Isolated cells from hypoxia-tolerant animals also show the ability to undergo MRS and it appears, at least superficially, that what sets hypoxia-tolerant cells apart from those that are hypoxia-sensitive is the capacity for MRS. For example, hepatocytes isolated from the hypoxia-tolerant goldfish have been shown to suppress metabolic rate by 26% during acute hypoxia exposure (immediately exposed to ~16 µM O2) and by up to 42% during prolonged hypoxia exposure (60 min pre-exposure to ~16 µM O2 before measurement). This was in striking contrast to hepatocytes isolated from the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss), which under the same conditions did not show any reductions in metabolic rate during exposure to acute hypoxia, though some signs of MRS were observed after prolonged hypoxia exposure (Krumschnabel et al. 2000b). Clearly, goldfish hepatocytes were able to respond rapidly to hypoxia and decrease metabolic rate, whereas rainbow trout hepatocytes lacked this ability. By promptly reducing energetic demands, cells are able to effectively extend survival time in hypoxia.  1.3 Components and regulation of metabolic rate suppression At the cellular level, MRS involves the controlled down-regulation of energy consuming processes. The two main pathways that are modified during MRS are protein turnover and ion pumping activities (Hochachka et al. 1996; Hand and Hardewig 2003; Storey and Storey 2004).  2  Other ATP consuming processes are also shut down (e.g. gluconeogenesis and urea synthesis), but they only modify metabolic rate by a small amount. The effect of down-regulating these processes is a substantial reduction in cellular energy use in response to hypoxia. Protein turnover Protein turnover is an energetically expensive process that is often inhibited during MRS. In normoxia, protein synthesis accounted for 36% of total ATP turnover in hepatocytes from the western painted turtle (Chrysemys picta bellii) and was reduced by 92% in response to anoxia exposure (Land et al. 1993b). Similarly, liver protein synthesis rates were markedly reduced by up to 92% in goldfish hepatocytes and halved in heart and muscle in anoxia (Smith et al. 1996). With decreased protein generation, it is necessary for cells to simultaneously decrease protein degradation in order to preserve cellular function during MRS, which also prepares the cell for a speedy recovery when the hypoxia bout passes. Thus, there is a general reduction in protein turnover in cells employing MRS, as has been shown in turtle hepatocytes where protein half-life doubled over the first 10hrs of anoxia exposure (Land and Hochachka 1994). Both protein initiation and elongation can be modified through reversible phosphorylation of initiation or elongation factors, which brings about a reduction in protein synthesis rate. The phosphorylation of eukaryotic initiation factor 2 (eIF2), a protein which is responsible for presenting tRNA to 40S ribosomal subunit, is increased during anoxia in the snail Littorina littorea to prevent protein initiation (Larade and Storey 2002). The regulatory factor eIF-4E binding protein 1 (4E-BP1) is also regulated by reversible phosphorylation. It binds to eIF-4E when active (unphosphorylated state), which halts the initiation process (Storey and Storey 2004). Protein elongation is modified by a similar process. For instance, eukaryotic elongation factor 2 (eEF2) kinase in hibernating squirrels showed a 2-fold increase in activity, 3  concomitant with a decrease in the protein phosphatase that works against eEF2K (Chen et al. 2001). The combined effect of increased kinase activity and decreased phosphatase activity precludes eEF2 from binding to ribosomes and stops protein elongation (Wang and Proud 2006). Similarly in goldfish liver, severe hypoxia caused an increase in eEF2 phosphorylation that corresponded with a substantial reduction in protein synthesis rate (Jibb and Richards 2008). Protein synthesis can thus be closely regulated through the actions of protein kinases and phosphatases and the inhibition of protein synthesis contributes greatly to MRS. Ion pumping activity Ion pumping activity is another energetically expensive process targeted for reduction during MRS. Na+/K+ ATPase is the dominant ATP sink in the cell and plays an essential role in the maintenance of cellular membrane potential. Rainbow trout hepatocytes show a dose dependent decrease in Na+/K+ ATPase activity upon gradual decrease in O2 levels (Bogdanova et al. 2005). In turtle hepatocytes, Na+/K+ ATPase activity accounts for 28% of cellular metabolic rate under normoxic conditions, which during anoxia, is reduced by 75% without compromising the maintenance of membrane potential (Buck and Hochachka 1993). This phenomenon is termed channel arrest and is brought about not only by a large reduction in ATP consumption by Na+/K+ ATPase activity, but also by reducing channel densities, which results in a lower cell membrane permeability to ions. Turtle hepatocytes display an inherently lower permeability, and when exposed to anoxia showed further suppression of membrane permeability (Buck and Hochachka 1993), eliminating the need to use precious ATP to maintain and restore membrane potential. This phenomenon, however, is not consistently observed in all species and tissue-types. The Na+/K+ ATPase activity in the comatose turtle brain after 24 hrs of anoxia was reduced in the telencephalon and cerebellum by 31% and 34% respectively, which is in contrast with the 4  crucian carp brain where pump activity was maintained during anoxia, possibly allowing the animal to stay active (Hylland et al. 1997). While the Na+/K+ ATPase activity in the hypoxiasensitive trout hepatocytes was reduced, its activity was maintained in goldfish hepatocytes, demonstrating the importance of maintaining membrane function during hypoxia in goldfish (Krumschnabel et al. 2000b). Other channels are also modulated during O2 lack, e.g. Na+ channels and Ca2+-permeable ion channel of the N-methyl-d-aspartate (NMDA)-type glutamate receptor (Buck and Bickler 1998; Bickler and Buck 2007). Overall, ion channel activity is tightly regulated in cells during hypoxia. Channel arrest is partly achieved through phosphorylation of ion channels. During hypoxic exposure in Amazonian cichlids Astronotus ocellatus, gill Na+/K+ ATPase activity was reduced by 65% through post-translational modifications (Richards et al. 2007). The activities of voltage-gated channels are also modified by the actions of protein kinases as was evident in the turtle NMDA receptors during anoxia (Storey and Storey 2004; Bickler and Buck 2007). As a result, cells can quickly modify processes through reversible protein phosphorylation to coordinate a quick reduction in metabolic rate. Although processes that are targeted for downregulation are well known, the exact mechanisms through which MRS is initiated remains unclear.  1.4 Potential initiator of metabolic rate suppression—AMP-activated protein kinase AMP-activated protein kinase (AMPK) has been hypothesized to initiate MRS in response to hypoxia exposure (Jibb and Richards 2008; Stenslokken et al. 2008). AMPK is sensitive to changes in cellular energy status as it is activated by an increase in [AMPfree]/ [ATP]. Activation of AMPK results in the phosphorylation of downstream targets that bring about a decrease in anabolic processes, primarily protein synthesis, and an increase in catabolic 5  processes, mainly through effects on glucose metabolism. In fish, a tight association has been observed between AMPK activation and the onset of MRS during hypoxia exposure. In response to severe hypoxia exposure, AMPK in the liver of the common goldfish was activated within 30 min, concurrent with an increase in [AMPfree]/[ATP] and the phosphorylation of eEF2, which resulted in a strong reduction in protein synthesis rates (Jibb and Richards 2008). Likewise, AMPK was phosphorylated and hence activated in heart, brain, and liver of crucian carp exposed to 1 and 7 days of anoxia. When a general protein kinase inhibitor, compound C, was injected into anoxic crucian carp, there was an elevation of metabolic rate, measured as an increase in ethanol production (Bain et al. 2007; Stenslokken et al. 2008), suggesting that an inhibition of AMPK (or other kinases) reduces the capacity for MRS. Undoubtedly, a strong association exists between AMPK and MRS, but evidence for a direct relationship between AMPK activation and the onset of MRS is lacking.  1.5 AMP-activated protein kinase Structure and regulation of AMPK The structure and regulation of AMPK reflect its role as a sensitive cellular energy sensor. AMPK is a highly conserved protein with close counterparts present in plants and yeast (Hardie and Carling 1997). It is a heterotrimeric serine/threonine protein kinase comprised of a catalytic α subunit, and two regulatory subunits- β and γ. The γ subunit contains the AMP/ATP binding domains (Bateman domains) where either AMP or ATP molecules bind, but with a higher affinity for AMP (Hardie 2006). The β subunit joins the entire complex together, such that the α and γ subunits are in close proximity and that the binding of AMP to the γ subunit induces conformational changes at the α subunit. The α catalytic subunit contains the kinase domain at which the enzyme can be phosphorylated at Thr172 by its upstream kinases and an 6  autoinhibitory domain (AID) which plays a pivotal role in the activation process (Chen et al. 2009). In normoxia, when [AMPfree]/[ATP] is low, the AID keeps the enzyme in an open and inactive conformation. This open conformation prevents the kinase domain from interacting with the phosphorylation site (Thr172) on the α subunit. Continual dephosphorylation of Thr172 on the α subunit by protein phosphatases results in low AMPK activation. During hypoxia, when [AMPfree]/[ATP] is elevated, AMP outcompetes ATP for binding to the Bateman domains on the γ subunit. Binding of AMP to the Bateman domain induces an allosteric activation of the enzyme and prompts the AID to release the kinase domain on the α subunit, thus transitioning AMPK into its active confirmation. At the same time, dephosphorylation at Thr172 is prevented through an inhibition of protein phophatases, resulting in the activation of AMPK (Fig.1.1; Xiao et al. 2007; Chen et al. 2009; Young 2009). The tumor suppressor liver kinase B1 (LKB1) and Ca2+/ calmodulin dependent protein kinase kinase (CaMKK) are the two mammalian upstream kinases of AMPK. Though their relative importance in different tissues has not been determined, LKB1 seems to have a broader tissue distribution than CaMKK, which seems to be more prominent in neural tissues (Witters et al. 2006). Recently, the roles of LKB1 and CaMKK in the regulation of AMPK in aestivating snails Otala lactea were investigated by Ramnanan et al. (2010) who showed that LKB1 was a more important upstream regulator of AMPK than CaMKK. The overall function and regulation of AMPK depends on the subunit isoforms expressed (α1 and α2, β1 and β2, γ1, γ2 and γ3). Certain combinations of AMPK complexes are more prevalent in some tissues than others, which could have a profound influence over the sensitivity and activity of AMPK. There is evidence that variations in each the α and γ subunit isoforms can 7  influence AMP dependence of the complex (Cheung et al. 2000). It also appears that complexes with different α and β subunit isoforms show a preference for different upstream kinases (McCartney et al. 2005; Sakamoto et al. 2006). Different α isoforms in mice demonstrated varying sensitivities to energy availability as well (Karagounis and Hawley 2009). Intriguingly, the expression of isoforms responds to changes in physiological conditions. For instance, the expression of two regulatory γ subunit isoforms increased in the heart of crucian carp during anoxia (Stenslokken et al. 2008). Although the actual functional significance of different AMPK complexes has not been determined, these potential changes to the enzyme can significantly alter its sensitivity to cellular energy charge and perhaps even its target of action during exposure to environmental stresses. Downstream targets of AMPK Once activated, AMPK coordinates an inhibition of anabolic pathways and enhancement of catabolic pathways. It interacts with all the major pathways of energy use and generation, including protein synthesis, fatty acid and glucose metabolism. This coordination is achieved through reversible protein phosphorylation in the short-term, and during longer term exposures to metabolic stress, AMPK is known to also regulate the expression of genes through reversible phosphorylation of transcription factors responsible for helping cells return to a state of energy balance. AMPK facilitates a down-regulation of protein synthesis in two ways. Firstly, AMPK directly phosphorylates eukaryotic elongation factor 2 (eEF2)-kinase (eEF2K), which activates it, causing it to phosphorylate eEF2 and slow protein elongation (Wang and Proud 2006). Secondly, AMPK regulates the mammalian target of rapamycin (mTOR) pathway. When AMPK is 8  activated, it stimulates tuberous sclerosis complex 2 (TSC2) that causes the inhibition of mTOR, a protein that is involved in both the initiation and elongation steps of mRNA translation. mTOR prevents 4E-BP1 from binding to eIF4E, and thus stalls the formation of an active translation initiation complex. Additionally, mTOR phosphorylates eEF2K at sites that are distinct from the AMPK direct phosphorylation site, inhibiting it from phosphorylating eEF2. Through its influences on mTOR and eEF2K, AMPK activation effectively slows protein synthesis (Hardie and Sakamoto 2006). Fatty acid metabolism is also regulated closely by AMPK. 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase is phosphorylated by AMPK, which leads to inhibition of cholesterol synthesis. Acetyl-CoA carboxylase 1 (ACC1) and 2 (ACC2) both convert acetyl CoA into malonyl CoA. The malonyl CoA pool generated from ACC1 contributes to fatty acid synthesis, whereas that from ACC2 inhibits carnitine palmitoyl CoA acyltransferase 1 (CPT1) and prevents the transport of fatty acid into the mitochondria, slowing fatty acid oxidation. The phosphorylation of ACC1 and ACC2 by AMPK stops malonyl CoA generation and consequently slows fatty acid synthesis and encourages fatty acid oxidation (Hardie and Pan 2002; Munday 2002). However, in situations of low O2, fatty acid oxidation is likely blocked by the general inhibition of mitochondrial oxidative phosphorylation (Whitmer et al. 1978; Speers-Roesch et al. 2010). AMPK regulates glucose metabolism to, on one hand, increase glycolytic capacity, which includes transporting glucose into the cell and modifying glycolytic enzymes, and on the other hand to lower gluconeogenesis and glycogen production to decrease energy usage. Glucose uptake is stimulated through an activation of glucose transporter GLUT1 and by increasing GLUT4 translocation to the plasma membrane (Hardie and Hawley 2001; Hardie et al. 2006). 9  Hexokinase and cardiac 6-phosphofructo-2-kinase activities are increased to stimulate glycolysis (Marsin et al. 2000). Glucose production is reduced in the liver when AMPK is stimulated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR; Bergeron et al. 2001; Zhou et al. 2001). Also, glycogen synthesis is blocked by AMPK through a decrease in glycogen synthase activity (Carling and Hardie 1989). Together, numerous mammalian studies have provided strong support that AMPK is important for whole-animal glucose homeostasis. AMPK also regulates other processes during exposure to metabolic stress in an effort to maintain energy balance. To reduce energy use, AMPK induces cell cycle arrest which halts DNA replication (Jones et al. 2005). Also, mitochondrial biogenesis is under the regulation of AMPK through its effects on peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). An activation of AMPK normally stimulates mitochondrial biogenesis, though this would only be useful in aerobic situations; this response has been shown to be muted via an unknown mechanism during exposure to hypoxia (Ramnanan et al. 2010). Chronic activation of AMPK changes gene expression patterns via regulation of transcription factors. Prolonged activation of AMPK has been shown to suppress the expression of glucose-activated genes, such as those involved in gluconeogenesis, e.g. phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (Lochhead et al. 2000; Ferre et al. 2003). Furthermore, AMPK activation has also been shown to decrease the expression of lipogenic genes ACC and fatty acid synthase (Woods et al. 2000). In short, AMPK exerts broad effects on cellular metabolism such that it helps the cell to achieve energy homeostasis.  10  1.6 Pharmacological manipulators of AMPK activity Pharmacological agents have long been used to study the effects of the AMPK cascade on metabolism. Unfortunately, using drugs for manipulation studies is not without disadvantages; drugs often have off-target effects which could lead to a misinterpretation of study results. Of the different AMPK manipulators, I used two activators, AICAR and A769662, and a general protein kinase inhibitor, compound C, for my manipulation experiments. AICAR 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is frequently used as a pharmacological activator of AMPK. Once administered, AICAR is transported into the cell by adenosine transporters and converted to 5-aminoimidazole-4-carboxamide ribonucleoside monophosphate (ZMP) by adenosine kinase. ZMP is an AMP analogue and allosterically activates AMPK and decreases dephosphorylation of the catalytic α subunit, much like an accumulation of cellular AMP. Unfortunately, the addition of an AMP analogue implies that any AMP-dependent cellular processes inside the cell would be affected at the same time, through AMPK-independent mechanisms. These potential off-target effects include a direct inhibition on complex I of the mitochondrial electron transport chain (Guigas et al. 2007) and an inhibition of gluconeogenesis and glycolysis (through inhibitory effects on fructose-1,6,-bisphosphatase , 6phosphofructo-2-kinase, and glucokinase; Vincent et al. 1991; Vincent et al. 1992; Viollet et al. 2006). ZMP also stimulates glycogen phosphorylase to enhance glycogenolysis (Young et al. 1996; Longnus et al. 2003). Despite these caveats, AICAR is the most well characterized pharmacological activator of AMPK and probably for that reason, remains the most used.  11  Compound C When first discovered, 6-[4-(2-Piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine (Compound C) was thought to be a specific AMPK antagonist that would allow better isolation of the effects of AMPK cascade. Initially, it was shown to be a potent inhibitor of AMPK that effectively reversed the inhibition of ACC via AICAR without affecting several structurally related protein kinases (Zhou et al. 2001). This was confirmed in several other studies with human cell lines where compound C blocked AICAR-induced AMPK (Lee et al. 2003; Meley et al. 2006). More recently, administration of compound C to anoxic crucian carp reversed the decrease in metabolic rate and elevated ethanol (Stenslokken et al. 2008). Similar effects of compound C in blocking hypoxic AMPK activation were also observed in rat hepatocytes and aestivating snail hepatopancreas and foot tissues, where the drug reversed the inhibition of ACC phosphorylation through AMPK activation (Ramnanan et al. 2010). Recently, a more detailed study of compound C revealed that it did affect other protein kinases and it was subsequently concluded that compound C functioned as a general protein kinase inhibitor (Bain et al. 2007). As such, it was pointed out that compound C could be used as a protein kinase inhibitor to reverse AICAR actions on AMPK. To isolate AMPK effects, AICAR can first be used to activate AMPK in a cell system and presumably if AMPK was the only protein kinase activated by AICAR, the addition of compound C would reverse any AMPK specific cellular effects (Hardie 2007). This has led to the use of compound C in conjunction with AICAR in order to isolate specific AMPK effects. A769662 Recent research screening through a library of 700,000 compounds led to the discovery of the thienopyridones, which showed high specificity for AMPK activation (Cool et al. 2006). 12  Without affecting [AMP] or [ATP] inside the cell, A769662 works through a mechanism dependent on the β subunit of AMPK (Scott et al. 2008). Similar to in vivo AMPK activation, the binding of A769662 appeared to protect AMPK from dephosphorylation by protein phosphatases at Thr172 (Treebak et al. 2009), and thus, A769662 was deemed more specific than AICAR for activating AMPK. Since then, many studies have replaced AICAR with A769662 to investigate AMPK specific effects though recent evidence have led to questions about the specificity of A769662. For example, it has been shown to inhibit Na+/K+ ATPase activity in a dose-dependent fashion and also reduced pump abundance at the surface of skeletal muscle cells (Benziane et al. 2009). Glucose uptake was induced in intact muscles upon A769662 addition which was independent of AMPK effects (Treebak et al. 2009). Though A769662 seems to offer a more specific mechanism in activating AMPK, it does not come without off target effects. Studies using AICAR, compound C and A769662 have determined that AMPK activation coordinates a metabolic reorganization in response to changes in cellular energy status. In fish, even though a close association has been observed between AMPK activation and the downregulation of energetically expensive processes (Jibb and Richards 2008; Stenslokken et al. 2008), direct evidence is necessary to establish the role of AMPK in coordinating MRS.  1.7 Thesis objectives The goal of this thesis was to ascertain whether there is a direct relationship between AMPK activation and the onset of MRS as suggested by earlier studies (Jibb and Richards 2008). To accomplish this goal, I first characterized the response of isolated common goldfish   O2 ) in response to changes hepatocytes to O2, where I measured cellular O2 consumption rate ( M  O2 associated with the maintenance of protein in O2 levels. I also determined the proportion of M  13  synthesis and Na+/K+ ATPase activity, by selectively manipulating these two processes with cycloheximide and ouabain, respectively. Next, I specifically addressed the hypothesis that an activation of AMPK in goldfish hepatocytes under normoxic conditions would decrease cellular Mo2. This was accomplished by selectively manipulating AMPK activity with pharmacological agents (AICAR, compound C, and A769662) and studying the relationship between AMPK  O2 . activation state, its downstream target, eEF2, and its effect on protein synthesis, and M  14  Figure 1.1 Model of AMPK activation. The enzyme complex consists of a α subunit that has a kinase domain (KD) containing Thr172 in the activation loop and an autoinhibitory domain (AID), a β subunit that contains a glycogen binding domain (GBD), and a γ subunit that contains the Bateman domains for AMP/ATP binding. When AMP/ATP is low during normoxia, less AMP is bound and the enzyme is in an inactive configuration. Protein phosphatase 2C (PP2C) thus continually dephosphorylate Thr172 that gets phosphorylated by upstream kinases LKB1 and CaMKK, keeping AMPK inactive. When AMP/ATP increases during O2 lack, more AMP binds to the γ subunit, the AID releases the KD to form an active configuration. PP2C access to Thr172 is denied, thus keeping the complex phosphorylated and active. (Taken from Young 2009).  15  Figure 1.1  16  CHAPTER TWO: AMP-ACTIVATED PROTEIN KINASE PLAYS A ROLE IN INITIATING METABOLIC RATE SUPPRESSION IN GOLDFISH HEPATOCYTES1 2.1 Introduction Hypoxia survival requires a reorganization of cellular energy metabolism to bring the rate of ATP consumption in line with the reduced capacity for O2-dependent ATP production (Richards, 2009). The ability to suppress metabolic rate in response to hypoxia exposure has been demonstrated in many hypoxia and anoxia-tolerant vertebrates (e.g. freshwater turtles and fish from the family Cyprinidae), including the common goldfish, Carassius auratus, which suppresses whole-animal metabolic rate by up to 60% in response to severe hypoxia exposure (Van Waversveld et al. 1989; van Ginneken et al. 2004). At the cellular level, metabolic rate suppression is primarily mediated by the post-translational modification and inhibition of proteins involved in protein synthesis (e.g. eukaryotic elongation factor-2; eEF2; Smith et al., 1996; Krumschnabel et al., 2000), the maintenance of cellular membrane potential (e.g. Na+/K+ ATPase; Buck and Hochachka 1993), and pathways involved in macromolecular synthesis (e.g. gluconeogenesis; Hochachka et al., 1996). In goldfish hepatocytes, the down-regulation of these processes during 90 min of hypoxia exposure resulted in a 42% decrease in metabolic rate (calculated from ATP turnover; Krumschnabel et al. 2000b). Although metabolic rate suppression has been put forward as a unifying theory explaining the variation in hypoxia tolerance among vertebrates (Hochachka et al. 1996; Staples and Buck 2009), little is known about the cellular mechanisms that initiate this process.  A version of this chapter has been submitted for publication: Lau GY and Richards JG. AMP-activated protein kinase initiates metabolic rate suppression in goldfish hepatocytes.  17  Recent evidence suggests that AMP-activated protein kinase (AMPK) plays an important role in initiating the metabolic responses of fish to hypoxia (Jibb and Richards 2008; Stenslokken et al. 2008). During severe hypoxia exposure, AMPK activity increased rapidly (within 30 min) in the liver of goldfish and the activation of AMPK occurred in close association with an increase in [AMPfree]/[ATP] and the phosphorylation and inhibition of eEF2, resulting in a severe reduction in the rate of protein synthesis (Jibb and Richards 2008). An activation of AMPK was also observed during anoxia exposure in the heart and brain of the crucian carp, Carassius carassius (Stenslokken et al. 2008) and in developing embryos of zebrafish, Danio rerio (Mendelsohn et al. 2008). Furthermore, when crucian carp were exposed to anoxia and injected with a general protein kinase inhibitor, compound C, there was an elevation in ethanol production rate and an increase in energy charge, suggesting that the inhibition of AMPK restricted metabolic rate suppression (Bain et al. 2007; Stenslokken et al. 2008). Overall, these studies suggest that there is a tight association between the activation of AMPK and metabolic rate suppression in fish during hypoxia and anoxia exposure. AMPK is well known to respond to changes in cellular energy balance and as a result is aptly named the cellular energy gauge (Kahn et al. 2005). Increases in cellular [AMPfree]/[ATP] result in AMP binding to the regulatory γ subunit of AMPK (Carling et al. 1989), which both allosterically activates and limits the dephosphorylation and inactivation of the catalytic α subunit by protein phosphatase (Suter et al. 2006; Sanders et al. 2007a). The regulatory β subunit of AMPK contains a specific glycogen-binding domain which makes the enzyme sensitive to cellular glycogen content (McBride and Hardie 2009). Once activated, AMPK inhibits pathways that consume energy while simultaneously activating pathways that boost metabolic energy production. For example, protein synthesis is inhibited through an increase in the 18  phosphorylation of eEF2, which halts protein elongation (Horman et al. 2002) and also through a suppression of mammalian target of rapamycin (mTOR) pathway, which stops mRNA translation (Wang and Proud 2006). Cholesterol and fatty acid synthesis are inhibited via phosphorylation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and acetyl CoA carboxylase (ACC) respectively (Henin et al. 1995) and glycogen synthesis is inhibited through phosphorylation of glycogen synthase (Carling and Hardie 1989). On the other hand, stimulation of AMPK increases cellular glucose uptake through an activation of GLUT1 and increased GLUT4 translocation to the membrane and increases hexokinase activity enhancing glycolytic capacity (Hardie and Hawley 2001; Hardie et al. 2006). An activation of AMPK in mammals has also been shown to result in mitochondrial biogenesis (Reznick and Shulman 2006) and enhanced fatty acid oxidation (Merrill et al. 1997) but these processes are likely down-regulated through other mechanisms during hypoxia exposure (Whitmer et al. 1978; Hochachka and Lutz 2001; Zhang et al. 2007; Ramnanan et al. 2010; Speers-Roesch et al. 2010). Although there is accumulating evidence to suggest that AMPK may initiate metabolic rate suppression during hypoxia exposure, this contention has not been explicitly tested. The objective of the present study was to test the hypothesis that pharmacological activation of AMPK in goldfish hepatocytes during normoxia would initiate metabolic rate  O2 ). In order to activate suppression (measured as a reduction in cellular respiration rate; M  AMPK, I used 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone drug, A769662, and investigated the effects of these agents on AMPK phosphorylation and activity, the phosphorylation state of eEF2, protein synthesis and cellular  O2 in isolated goldfish hepatocytes. The interactive effects of [O2] and AMPK activation were M  also examined. AICAR has long been the agonist of choice when studying the AMPK cascade as 19  it is readily taken up by cells via adenosine transporters and once inside the cell, it is converted into 5-aminoimidazole-4-carboxamide ribonucleoside monophosphate (ZMP) by adenosine kinase. ZMP acts as an AMP analogue stimulating AMPK (Corton et al. 1995; Hardie 2007; Guigas et al. 2009). However, AICAR has been shown to affect other AMP-dependent cellular processes, thus I used the general protein kinase inhibitor 6-[4-(2-Piperidin-1-ylethoxy) phenyl]3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine (compound C) to block AICAR effects to isolate AMPK specific actions (Zhou et al. 2001; Meley et al. 2006; Hardie 2007). In search of a more specific AMPK agonist, Cool et al. (2006) described the identification of A769662, which was shown to be a small molecule AMPK activator that was more specific and potent than AICAR in activating AMPK, although evidence of its specificity is conflicting (Moreno et al. 2008; Benziane et al. 2009). If AMPK is involved in initiating metabolic rate suppression, I predict that  O2 . pharmacological activation of AMPK under normoxia will reduce M  2.2 Materials and methods Animal care Adult goldfish were purchased from a local supplier (~30 g; Delta Aquatics, Richmond BC, Canada) and held at the University of British Columbia in flow-through dechlorinated City of Vancouver tap water at 12oC. Fish were fed to satiation every other day with commercial flakes (Nutrafin Max). All experimental procedures were approved by the University of British Columbia animal care committee under AUP A09-0611. Hepatocyte isolation Hepatocytes were isolated from individual goldfish using a standard collagenase technique (Mommsen et al. 1994). Briefly, fish were anaesthetized with benzocaine (250 mg/mL; Sigma-Aldrich, Oakville ON, Canada) and the spine and caudal artery were severed close to the 20  gills and the tail was excised. A polyethylene catheter was then inserted into the caudal vein and the fish was perfused with heparinised Hanks’s saline (in mM: 136.9 NaCl; 5.4 KCl; 0.8 MgSO4·7H2O; 0.33 Na2HPO4·7H2O; 0.44 KH2PO4; 5 HEPES; 5 HEPES Na; pH 7.6 with 0.3 mg/mL heparin) at a rate of ~1.6 mL/min for 20 min. Following the infusion, the liver was removed and chopped into small pieces with a razor blade and digested in 0.4% collagenase (w/v; Sigma-Aldrich) in Hanks’s saline for 20 min. The digested liver was then filtered through two stacked layers of polyethylene mesh (202 µm and 72 µm) to rid the suspension of cell clumps. The filtrate was then diluted with Hanks’s saline and subjected to centrifugation at 400 g. The supernatant was discarded and the cell pellet was resuspended in Hanks’s saline. This procedure was repeated a total of three times to thoroughly wash the cells. Afterwards, the cell pellet was washed with modified Hanks’s saline containing 1.5 mM CaCl2 and 1% BSA (w/v; Sigma Aldrich) at pH 7.6 followed by centrifugation at 400 g. The pellet was washed twice more with Hanks’s saline to remove Ca2+ and finally the cell pellet was resuspended in modified Leibovitz L-15 culture media containing 10 mM HEPES, 14 mM NaHCO3, and 1% BSA (w/v) at pH 7.6. Cells were allowed to recover in a shaking water bath at 12oC for at least 1 hr before experiments were performed. Cell yield was determined by counting the cells on a hemocytometer (done in triplicate) and cell viability was determined by measuring trypan blue exclusion and lactate dehydrogenase (LDH) liberation as described in Vassault (1983). High resolution respirometry To characterise the responses of goldfish hepatocytes to [O2] and pharmacological manipulation of AMPK, I ran two series of experiments. In series I, I examined the effects of [O2]  O2 and the relative contributions of protein synthesis (inhibited with on hepatocyte M  cycloheximide; Sigma-Aldrich) and Na+/K+ ATPase activity (inhibited with ouabain; Sigma21   O2 . In series II, I attempted to pharmacologically activate AMPK with Aldrich) to cellular M  AICAR (Sigma-Aldrich) and A769662 (purchased from Dr. Kei Sakamoto; University of Dundee, Scotland), while monitoring AMPK activation status, inhibition of downstream target  O2 . Compound C (Sigma-Aldrich) was also incubated with AICAR-treated cells to eEF2 and M  determine specific effects of AICAR on the AMPK cascade. Cycloheximide was used in conjunction with AICAR and A769662 in an attempt to isolate the interactions of these drugs with protein synthesis. Cell viability was assessed before and after each pharmacological manipulation.  O2 was determined using high resolution respirometry (Oroboros ® Hepatocyte M  Instruments GmbH, Innsbruck, Austria). All calibrations and analyses of hepatocyte respiration rates were carried out in modified L-15 media at 12oC. The O2 sensors were calibrated daily. Data were recorded at 2 Hz with DatLab software version 4.3.2.7 (Oroboros ® Instruments GmbH). At the start of each experiment, cells were added to fully air saturated medium in the respirometer and the signal was allowed to stabilize for at least 20 min before measurements were made. For series I, I added 4 x 106 to 6 x 106 cells per chamber (~2 mL chamber volume) whereas for series II, 10 x 106 to 12 x 106 cells were added to each chamber. More cells were used in series II so that samples could be taken for biochemical analysis (see below). Series I: Response to [O2] I investigated the response of hepatocytes to different [O2] levels. To accomplish this,  O2 was measured over the following O2 ranges 300 to 230, 200 to 130, 100 to 60, and 50 to 40 M  μM, which corresponded to mean [O2] exposures of 258 ± 4, 148 ± 3, 70 ± 5, and 43 ± 1 µM,  22  respectively. To adjust [O2] in the respirometer, N2 was blown over the medium inside the chamber.  O2 at physiological In order to gain a finer time scale of the effects of [O2] on cellular M  levels, I used N2 to reduce [O2] to roughly 80 µM, at which point the respirometer was sealed and the cells were allowed to consume the O2 to anoxia; this procedure is referred to as an O2 kinetic trial. A cell sample was taken at the end of these O2 kinetic trials to assess cell number and viability. The medium was then reoxygenated to ~300 µM by opening the chamber and the trial was repeated a second time. The two O2 kinetic trials were done sequentially with the first serving as control. The second trial was done either in the presence of 3 mM ouabain, 15 mM cycloheximide (predetermined effective dosages) or equal volumes of the carrier (sham control). The sham control trials were done to control for the possible effects of the carrier (DMSO or L15 media) and the running of two sequential trials. Series II: Responses of hepatocytes to AMPK activation  O2 , To assess the effects of AICAR and A769662 on AMPK activation and hepatocyte M  I performed paired trials on a single set of cells. These paired trials consisted of monitoring an   O2 with no added drug, then a single dose of AICAR or A769662 was added (25, initial control M 50, 100, 150, and 200 µM of A769662 and 0.25, 0.5, 1, and 2 mM AICAR) to the oxygraph chamber. The cells were incubated in the presence of the drug dose for 1 hr in an open chamber,  O2 measurement. Throughout these and the chamber was sealed after the incubation to obtain M  O2 , [O2] within the chamber was always maintained >200 manipulations and measurements of M  µM (normoxia). At the end of the drug manipulation trials, two samples were taken from the chamber. The first sample was used for cell counting and the second sample was processed for 23  biochemical analyses. In a separate set of experiments, cells were also taken after the first and second control (no drug) trials for biochemical analysis. Briefly, the cell samples were centrifuged at 300 g for 2 min at 4oC, after which the supernatant from each sample was transferred into a separate tube for LDH analysis, and the resulting cell pellet was frozen in liquid N2 and stored at -80oC.  O2 In order to determine whether the effects of AICAR and A769662 on hepatocyte M  were mediated by an inhibition of protein synthesis, I performed the following experiment.  O2 was taken, followed by an injection of either 1 mM AICAR or 150 µM A769662. Control M  O2 was measured. 15 mM After 1 hr incubation at normoxia, the chamber was sealed and M  O2 was cycloheximide was then injected into the chamber and after the signal stabilised, M  measured again. In a separate experiment, the same protocol was employed, but the order of the drug incubation was reversed (i.e. cycloheximide injection first, followed by 1 hr with AICAR/A769662). The same approach was used for experiments with AICAR and 40 µM compound C (Meley et al. 2006; Bain et al. 2007). The potential interactive effects of [O2],  O2 were determined following the O2 kinetic protocol AICAR and A769662 on hepatocyte M  described above under series I. Biochemical analysis AMPK activity Hepatocyte pellets (~20 mg) were disrupted with a Kontes sonicator for five, 5 sec bursts in a 1:5 ratio with homogenization buffer. AMPK activity was measured using the SAMS peptide [32P]-ATP method outlined in Jibb and Richards (2008) except that the AMPK activity  24  was assayed during a 10 min incubation at 22°C rather than a 5 min incubation at 20oC as reported. Western blotting Western blots were carried out following the protocol described in Jibb and Richards (2008) with modifications. Two separate blots were prepared for each sample. One blot was probed sequentially for both phospho-eEF2 and total eEF2 and the second blot was probed sequentially for phospho-AMPKα and total AMPKα. The blots were stripped of antibody between the phospho and non-phospho antibody incubations. Briefly, cell pellets (~20 mg) were sonicated in 1:5 ratio with buffer containing 100 mM Tris pH 7.5, 10% SDS, and 1x Halt Protease Inhibitor Cocktail Kit (Thermo Scientific, Rockford IL, USA). Samples were subjected to centrifugation at 13,000 g for 3 min at 4oC. A portion of the supernatant was used to quantify total protein according to Bradford (1976) while the remaining was added to an equal volume of SDS-sample buffer (Laemmli 1970), and boiled for 3 min. The protein sample (20 µg) was loaded on each lane of a denaturing SDS-polyacrylamide gel and electrophoresed at 75 V for 15 min, followed by 75 min at 150 V. A common sample was loaded onto each gel to account for gel-to-gel variation. The separated proteins were then transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules CA, USA) using a Trans-Blot semi-dry transfer cell (Bio-Rad Laboratories). Blots for AMPKα were blocked with 5% BSA whereas other blots were blocked with Tween-20 Tris-buffered saline (TTBS: 17.4 mM Tris-HCl, 2.64 mM Tris, 0.5 mM NaCl, and 0.05% Tween-20) with 2% (w/v) non-fat powered milk. Membranes were then incubated overnight with 1:1000 dilutions of phosphorylated protein primary antibody (rabbit IgG anti-phospho-Thr172 AMPKα or rabbit IgG anti-phospho-Thr56 eEF2; Cell-Signaling Technology, Danvers MA, USA) with gentle agitation. The membranes 25  were washed once with TTBS for 15 min, followed by four additional 5 min washes. This was followed by an hour’s incubation with 1:1000 dilutions of anti-rabbit IgG (goat)-HRP conjugated secondary antibody (Perkin Elmer, Woodbridge ON, Canada) with gentle agitation. After a 15 min and four 5 min TTBS washes, proteins are visualised with the Western Lightning-Enhanced Chemiluminescence (ECL) kit (Perkin Elmer) following the manufacturer’s instructions. Bands were visualised using FluorChem 8800 imager (Alpha Innotech, San Leandro CA, USA) and quantified with AlphaEase FC software (v.3.1.2; Alpha Innotech). Following visualisation, the blots were stripped of primary antibody and reprobed with total eEF2 and AMPKα antibodies according to the protocol outlined in the ECL kit. Briefly, after four 5 min washes with TTBS, membranes were incubated for 30 min in stripping buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 100 mM mercaptoethanol) at 50oC with gentle agitation. Blots were washed six times for 5 min with TTBS, then blocked and reprobed with the primary antibodies of total protein (rabbit IgG anti-AMPKα or rabbit IgG anti-eEF2; Cell Signaling). In separate experiments, I verified that the stripping procedure was effective at removing all primary and secondary antibodies. Calculations and statistical analysis For oxygen kinetic curves, a double rectangular hyperbola ( y   ax cx ) was fit  b x d  x   O2 max, P90, and P50. The equations were solved for M  O2 max at through the data and solved for M  100 µM O2, which is viewed to be well beyond physiological O2 concentrations. The P90 and P50  O2 was 90% and 50% of values were then calculated from the equation as the [O2] at which M  O2 max respectively. Data shown in Fig.2.3D and 2.7D was analysed using students t-test where M  each experimental group was compared directly to its own control set to 100%. All remaining data were analysed using a one-way analysis of variance (ANOVA), and when significant 26  differences were detected, Holm-Sidak post hoc all pairwise multiple comparison analysis was used to determine where significant differences occurred. For all statistical analysis, p< 0.05 was accepted as significant.  2.3 Results Cell characterization Hepatocyte yield from a ~30 g goldfish ranged from 10 x106 to 80 x106 cells with less than 8% red blood cell contamination. LDH activity was never detected in the incubation media neither before nor after any experimental manipulation (data not shown). There was also no effect of any pharmacological agent or experimental manipulation on trypan blue dye exclusion  O2 compared (data not shown). The addition of up to 1% DMSO (carrier control) did not affect M  with controls where no DMSO was added, therefore in all cases the data from the carrier and non-carrier controls were combined. Series I  O2 between 258 to 43 µM There was no significant effect of [O2] on hepatocyte M  O2 , but as (Fig.2.1). At [O2] below ~40 µM, goldfish hepatocytes maintained a relatively stable M  [O2] fell to roughly 13 µM (P90; Table 2.1), respiration rate conformed to the depleting [O2]   O2 was elevated and (Fig.2.2, Table 2.1). When reoxygenated after reaching complete anoxia, M  O2 at the beginning of the experiment (data not shown). not significantly different than the M  To examine the relative contributions of Na+/K+ ATPase activity and protein synthesis to  O2 , I added ouabain to inhibit Na+/K+ ATPase activity and cycloheximide to overall hepatocyte M  inhibit protein synthesis. At >200 µM O2, the addition of 3 mM ouabain resulted in only a slight   O2 compared with controls whereas the (5 ± 2%; n = 4), non-significant decrease in hepatocyte M 27   O2 by 47 ± 4% (n = 4) addition of 15 mM cycloheximide significantly decreased hepatocyte M  relative to control values. At [O2] below 40 µM, the addition of 15 mM cycloheximide caused a  O2 max compared with controls, while the addition of 3 mM ouabain did not 44% decrease in M  O2 max (Fig.2.2, Table 2.1). There was no effect of cycloheximide or ouabain on P90 or P50 affect M  (Table 2.1). Series II AICAR Above 200 µM O2, there was a dose-dependent increase in AMPK activity in isolated hepatocytes with the addition of AICAR up to 1 mM. However, AMPK activity declined to levels that were not significantly different from controls at 2 mM AICAR (Fig.2.3A). The relative proportion of phosphorylated AMPK increased at 0.25 mM AICAR compared with the controls and remained elevated at 1 and 2 mM AICAR (Fig.2.3B). Phosphorylated eEF2 was  O2 decreased in a dosealso significantly elevated at 1 and 2 mM AICAR (Fig.2.3C). Cellular M  O2 at 1 and 2 dependent fashion with increasing [AICAR] giving a 24 and 33% reduction in M  mM AICAR respectively (Fig.2.3D). At [O2] below 40 µM, there was no significant effect of 1  O2 max, P90 and P50 (Fig.2.4, Table 2.2). In a separate experiment, above 200 mM AICAR on M  µM O2, the addition of 1 mM AICAR alone caused a statistically insignificant 29% decrease in  O2 max, which was lowered further by an additional 22% with a subsequent addition of 15 mM M  cycloheximide (n= 3). The addition of 15 mM cycloheximide caused a significant 52% decrease  O2 max which was further decreased, but not significantly, by 10% with 1 mM AICAR in M  (Fig.2.5).  28  There was no effect of the addition of 40 µM compound C either before or after 1 mM  O2 at >200 µM O2 (Fig.2.6). However, the addition of 1 mM AICAR alone AICAR on cellular M  O2 , which is consistent with the results to goldfish hepatocytes did result in a 20% decrease in M  shown in Fig.2.3D. The addition of 40 µM compound C following AICAR caused a further,  O2 (Fig.2.6). albeit non-significant decrease in M  A769662 There were no effects of A769662 on AMPK activity in isolated goldfish hepatocytes incubated above 200 µM O2 (Fig.2.7A). Furthermore, increases in [A769662] did not alter the  O2 amount of phosphorylated AMPKα (Fig.2.7B), phosphorylated eEF2 (Fig.2.7C), or cellular M  O2 max, P90, (Fig.2.7D). At [O2] below 40 µM, the addition of 150 µM A769662 did not impact M  or P50 (Fig.2.4, Table 2.2). Similarly, addition of 150 µM A769662 and/or 15 mM cycloheximide  O2 (Fig.2.8). did not show significant changes in M  2.4 Discussion In this thesis, I attempted to pharmacologically activate AMPK in goldfish hepatocytes under high [O2] conditions to test the hypothesis that AMPK initiates metabolic rate suppression. Of the two pharmacological activators tested, AICAR and A769662, only the addition of AICAR up to 1 mM resulted in a dose-dependent increase in AMPK activity (Fig.2.3A) and a corresponding dose-dependent decrease in cellular MO2 (Fig.2.3D). Analysis of the phosphorylation state of eEF2 (Fig.2.3C) and the differential effects of adding cycloheximide and/or AICAR (Fig.2.5) suggest strongly that the observed AICAR-mediated decrease in MO2 was due primarily to a reduction in protein synthesis rate. These results are consistent with my hypothesis that AMPK initiates metabolic rate suppression in goldfish hepatocytes. However, the 29  addition of compound C, a general protein kinase inhibitor, after the administration of AICAR did not reverse the AICAR-specific effects on cellular MO2 as expected (Fig. 2.6). The addition of the “specific” AMPK activator, A769662, to goldfish hepatocytes had no measurable effects on AMPK activity, phosphorylation state of AMPK or eEF2, and no effect on cellular MO2 (Fig.2.7). In goldfish hepatocytes, it seems that A769662 is not an effective pharmacological agent for investigating AMPK. AICAR is transported into the cell via adenosine transporters where it is phosphorylated by adenosine kinase forming ZMP, which is an AMP analogue. AMPK is highly sensitive to changes in [AMPfree]/[ATP] and Jibb and Richards (2008) have previously shown that hypoxia exposure causes an 11-fold increase in [AMPfree]/[ATP] and a concomitant 5.5-fold activation of AMPK. In rat hepatocytes, ZMP accumulates in a dose-dependent manner with AICAR administration, causing a subsequent dose-dependent activation of AMPK (Corton et al. 1995). At high concentrations however, ZMP has been shown to inhibit AMPK which was not observed at similar concentrations of AMP (Corton et al. 1995). In this study, there was a dose-dependent increase in AMPK activity up to 1 mM AICAR, giving a 24% reduction in MO2 , but not a similar dose-dependent increase in AMPK phosphorylation (Fig.2.3A, B, and D), suggesting that other regulators may be acting upon AMPK. At 2 mM AICAR, I observed the highest level of AMPK phosphorylation (Fig.2.3B), but in this case AMPK activity was not elevated relative to controls, possibly because of the inhibitory effects of ZMP mentioned earlier. Even without a significant elevation in AMPK activity at 2 mM AICAR, I still observed high levels of eEF2 phosphorylation (Fig.2.3C) and a large 33% decrease in cellular MO2 relative to controls (Fig.2.3D). Although the results seen with 2 mM AICAR draw into question the relationship  30  between AMPK activity and the decrease in MO2 , these results must be viewed cautiously since any non-specific effects of AICAR will likely be accentuated at high concentrations. At the cellular level, metabolic rate suppression involves the selective inhibition of energy consuming processes to reduce ATP demand. The two major energy consuming processes targeted during metabolic rate suppression are typically protein synthesis and Na+/K+ ATPase (Hochachka et al. 1996; Wieser and Krumschnabel 2001). In goldfish hepatocytes,  O2 , thus it is a likely target protein synthesis is by far the most important contributor to cellular M  for inhibition during the onset of metabolic rate suppression. The selective inhibition of protein  O2 , while the selective inhibition synthesis with cycloheximide resulted in a 44% reduction in M  O2 (Fig.2.2, Table 2.1). of Na+/K+ ATPase with ouabain resulted in only a 9% decline in cellular M  These results are in reasonable agreement with the work of Krumschnabel et al. (1994b; 1994a), who also demonstrated that protein synthesis is the predominant cellular process contributing to  O2 in goldfish hepatocytes. Therefore, the suppression in protein synthesis could routine M  potentially translate into substantial energy savings during O2 lack. Indeed, hypoxic exposure in both turtles and crucian carp caused a significant reduction in protein synthesis rates (Land et al. 1993a; Smith et al. 1996). The activation of AMPK is well-known to be associated with an inhibition of protein synthesis through its interactions with eEF2 kinase which phosphorylates eEF2 (Wang and Proud 2006). The phosphorylation of eEF2 prevents it from binding to ribosomes, halting protein elongation (Horman et al. 2002; McLeod and Proud 2002; Browne et al. 2004). Several investigators have demonstrated a tight association between increased AMPK phosphorylation, increased eEF2 phosphorylation, and reduced protein synthesis rates (Bartrons et al. 2004; Jibb and Richards 2008; Rider et al. 2009). I obtained similar results, where for the most part, an 31  activation of AMPK increased the phosphorylation of eEF2 (Fig.2.3C). Furthermore, by examining the interactive effects of 1 mM AICAR and 15 mM cycloheximide, I have provided indirect evidence that the majority of AICAR-mediated reduction in MO2 is due to an inhibition of protein synthesis (Fig. 2.5). The addition of AICAR on its own resulted in a 29% decrease in  O2 , which was further decreased by an additional 22% following the addition of cycloheximide M  (maximal inhibition of protein synthesis). The addition of AICAR after cycloheximide resulted  O2 relative to cycloheximide on its own, indicating that roughly one-third in a 10% decrease in M  O2 is due to interactions with metabolic processes other than of AICAR-specific decrease in M  protein synthesis (e.g. gluconeogenesis). As a result, the remaining two-thirds of the AICAR O2 is likely mediated by decreases in protein synthesis. specific decrease in M  Although my results with AICAR are consistent with the hypothesis that AMPK initiates metabolic rate suppression, AICAR is known to have off-target effects that could, at least partially, explain the results shown here. For example, ZMP has been shown to compete with adenosine for transporters to enter the cell (Corton et al. 1995) and resulted in adenosine accumulation in myocardium of dogs (Gruber et al. 1989). The application of supraphysiological doses of adenosine (100 µM) to isolated goldfish hepatocytes has been shown to cause a rapid (within 15 min) and significant 50% reduction in protein synthesis rate (Krumschnabel et al.   O2 observed in the present study (Fig. 2000a), which could contribute to reduction in cellular M 2.3D, 2.5). Furthermore, AICAR has also been shown to inhibit aerobic respiration through direct effects on complex I of the mitochondrial electron transport chain, which in rat  O2 caused by AICAR (Guigas et al. hepatocytes explained the majority of the reduction in M  2007). However, these direct effects of AICAR on complex I are unlikely to manifest through 32  changes in protein synthesis as shown in the present study. AICAR has also been shown to inhibit gluconeogenesis and glycolysis independent of AMPK effects (Vincent et al. 1991; Vincent et al. 1992), likely because fructose-1,6-bisphosphatase, 6-phosphofructo-2-kinase, and glucokinase are all influenced by [AMPfree] and thus [ZMP] (Viollet et al. 2006). As with many pharmacological agents, AICAR has both specific and non-specific effects on cellular metabolism and therefore results from pharmacological studies must always be interpreted with caution. Despite the fact that AICAR is known to have off-target effects, it remains the most frequently used pharmacological agent for studying AMPK function (Hardie 2007). To help resolve the issue of AICAR’s off-target effects, several authors have proposed using compound C in conjunction with AICAR to help isolate AMPK-specific effects. When first described, compound C was thought to be specific because it reversed the effects of AICAR on the AMPK target acetyl CoA carboxylase in rat hepatocytes (Zhou et al. 2001). Compound C also reversed the decrease in metabolic rate due to anoxic exposure in crucian carp, suggesting that inhibition of AMPK limits the ability of carp to utilize metabolic rate suppression (Stenslokken et al. 2008). The specificity of compound C for AMPK was recently drawn into question when it was shown to inhibit other structurally-related protein kinases (Bain et al. 2007). However, it is still believed that if compound C reverses the effects of AICAR (Hardie 2007), as demonstrated by Zhou et al. (2001), then those effects could be attributed to AMPK. In my study, compound C did not reverse the AICAR-specific decrease in MO2 , but instead compound C appeared, albeit non-significantly, to result in a suppression of MO2 on its own (Fig.2.6). These results are in agreement with reports suggesting that compound C may directly inhibit mitochondrial  33  respiration and prevent the activation of HIF-1 during hypoxia, independent of AMPK (Emerling et al. 2007). A769662 was identified as a new, highly specific activator of AMPK which was extremely potent in rat liver cell free assays (Cool et al. 2006; Goransson et al. 2007). The mechanism of action of A769662 is not known, but it mimicked AMP effects on AMPK without changing [AMPfree]/[ATP] ratios (Goransson et al. 2007; Sanders et al. 2007b). Furthermore, it has been shown to have no effect on the activity of other protein kinases (Goransson et al. 2007; Sanders et al. 2007b). In this study, I did not observe an effect of A769662 on AMPK activity, AMPK phosphorylation, nor cellular MO2 (Fig.2.7). Also, there was no effect of A769662 on the phosphorylation state of eEF2 (Fig.2.7C) and there were no additive effects of cycloheximide and A769662 in isolated goldfish hepatocytes (Fig.2.8). The lack of effect of A769662 on goldfish hepatocytes may be related to species differences in AMPK structure, but given that the mechanisms of action of A769662 is unclear, no reasonable conjecture can be made. In addition, like most pharmacological agents, there is emerging evidence to suggest that A769662 may not be a specific activator of AMPK. Benziane et al. (2009) showed that A769662 directly inhibited the α subunit of Na+/K+ ATPase and caused a dose-dependent inhibition of Na+/K+ ATPase activity along with a decrease in pump abundance in the cell membrane of cultured skeletal muscle cells. In goldfish hepatocytes, Na+/K+ ATPase activity only contributes a small amount to cellular MO2 (Fig.2.2, Table 2.1), further explaining why I did not observe any effect of A769662 on cellular MO2 . Additionally, A769662 has also been reported to cause an elevation of glucose uptake in mice skeletal muscles (Treebak et al. 2009). Along with my results, there is accumulating evidence to suggest that A769662 may not be specific for AMPK nor superior to AICAR in studying the metabolic effects of the AMPK cascade in goldfish hepatocytes. 34  Jibb and Richards (2008) demonstrated that AMPK was activated in response to an O2 limitation. In the present study, I showed that the respiration rate of isolated goldfish hepatocytes was highly dependent on available O2 below ~13 µM O2 (P90; Fig.2.2 & Table 2.1) but above this level there were only minor effects of [O2] on MO2 (Fig.2.1). These results are in agreement with the biphasic O2 kinetics described by Gnaiger (2003), which show that as [O2] decreased from normoxia, cells exhibit a gradual decrease in respiration rate down to roughly 10 µM O2 below which MO2 decrease markedly with falling [O2]. Measurements of venous PO2 in goldfish and common carp (Cyprinus carpio) vary between 2.2 torr (measured of blood from caudal vein) and 9 torr (measured of mixed venous blood taken from ventral aorta) (~4-17 µM O2; Burggren, 1982; Takeda, 1990). Since the liver receives a mixed blood supply from both the hepatic portal vein and the hepatic artery, it is likely that the blood O2 levels of goldfish hepatocytes experience in vivo span these venous PO2 measurements. This places the hepatocytes in the precise range on the O2 kinetic curve where it would be extremely sensitive to any alterations in available O2 (P90 of 13 µM; Fig.2.2). Therefore, it seems reasonable to speculate that the metabolic rate of goldfish hepatocytes in vivo may be primarily regulated by O2 availability. Since AMPK is activated in the liver of hypoxia-exposed goldfish, I had originally predicted that the effects of AICAR and A769662 would be augmented at low [O2]   O2 compared with (more activation of AMPK under low [O2]) to cause a greater reduction in M  O2 observed with what I observed under high O2 conditions. In fact, the 24% reduction in M  AICAR at high [O2] was diminished to only 14% at physiological O2 conditions (Fig. 2.3, 2.4). Curiously, at low [O2], it seems that the sensitivity of cellular MO2 to AICAR, as assessed by changes in P90 and P50 (Table 2.2), did not differ from control hepatocytes. Further, maximal inhibition of protein synthesis or Na+/K+ ATPase had no effect on cellular O2 sensitivity as 35  measured by P90 or P50 (Fig.2.2, Table 2.1). These results prevents me from quantitatively translating results obtained at high [O2] to actual cellular responses in vivo, but it must be emphasized that it does not change the qualitative observation that activation of AMPK under high O2 conditions caused a decrease in MO2 . In summary, using AICAR, I provide evidence to support the hypothesis that an activation of AMPK plays a role in the initiation of metabolic rate suppression in isolated goldfish hepatocytes. However, the limitations of the pharmacological agents used prevent me from concretely ascribing the changes in cellular MO2 observed in this study solely to AMPK. Clearly, further investigations using techniques that are more specific for manipulating AMPK are required (e.g. gene manipulation studies) to assess the direct role of AMPK in initiating metabolic rate suppression in goldfish hepatocytes. As a result, I add to the growing list of evidence supporting the notion that AMPK is involved in the initiation of metabolic rate suppression, but AMPK is most likely one of the many processes contributing to the regulation of this complex process to enhance survival during environmental hypoxia exposure.  36  FIGURE 2.1 Effect of oxygen concentrations on cellular oxygen consumption rate in isolated goldfish hepatocytes (n= 12, 13, 7, and 13 from left to right)  37  FIGURE 2.1  38  FIGURE 2.2 Oxygen kinetic curves of cellular oxygen consumption rate with progressive decrease in oxygen concentration in isolated goldfish hepatocytes incubated with 3 mM ouabain and 15 mM cycloheximide (Closed circles: control; open circles: ouabain; closed triangles: cycloheximide). Data are means ± s.e.m. (n=7). Each drug treatment was analysed relative to individual control treatments; the controls are not statistically significant and have been combined in this figure  39  FIGURE 2.2  40  FIGURE 2.3 AMPK activity (A), phosphorylated AMPKα (B), phosphorylated eEF2 (C), and cellular oxygen consumption rate (D) in isolated goldfish hepatocytes incubated with AICAR (0, 0.25, 0.5, 1.0, and 2.0 mM) for 1 hr. In panel D, the open symbols represent the controls (before AICAR added) for each AICAR dose (closed symbols). Data are means ± s.e.m. (n= 6, 9, 7, and 6 from 0.5 to 2 mM AICAR) and * indicate significant differences relative to paired controls (p<0.05)  41  FIGURE 2.3  42  FIGURE 2.4 Oxygen kinetic curves of cellular oxygen consumption rate with progressive decrease in oxygen concentration in isolated goldfish hepatocytes incubated with 1mM AICAR and 150 μM A769662 (Closed circles: control; open circles: AICAR; closed triangles: A769662). Data are means ± s.e.m. (n= 5 for AICAR; n= 3 for A769662). Each drug treatment was analysed relative to individual control treatments; the controls were not statistically significant and have been combined in this figure  43  FIGURE 2.4  44  FIGURE 2.5 Effects of 15mM cycloheximide (Cyc) and 1mM AICAR incubations on cellular oxygen consumption rate (expressed in % relative to control) in isolated goldfish hepatocytes at normoxia (above 200 µM O2). On the left portion of the figure, cells were first incubated with AICAR and then Cyc was added (referred to as AICAR+Cyc). On the right portion of the figure, are the results of a separate experiment where the order of the drugs was reversed. Data are means ± s.e.m. (n= 3). Values that do not share letters are significantly different from each other (p<0.05)  45  Figure 2.5  46  FIGURE 2.6 Effects of 40 µM compound C (CC) and 1mM AICAR incubations on cellular oxygen consumption rate (expressed in % relative to control) in isolated goldfish hepatocytes at normoxia (above 200 µM O2). On the left portion of the figure, cells were first incubated with CC and then AICAR was added (referred to as CC + AICAR). On the right portion of the figure, are the results of a separate experiment where the order of the drugs was reversed. Data are means ± s.e.m. (n= 3). Values that do not share letters are significantly different from each other (p<0.05)  47  FIGURE 2.6  48  FIGURE 2.7 AMPK activity (A), phosphorylated AMPKα (B), phosphorylated eEF2 (C), and cellular oxygen consumption rate (D) in isolated goldfish hepatocytes incubated with A769662 (0, 25, 50, 100, 150, and 200 µM) for 1hr. In panel D, the open symbols represent the controls (before A769662 added) for each A769662 dose (closed symbols). Data are means ± s.e.m. (n = 7, 6, 6, 8, and 3 from 25 to 200 µM A769662) and * indicates significant differences relative to paired control (p<0.05)  49  FIGURE 2.7  50  FIGURE 2.8 Effects of 150 µM A769662 (A769) and 15mM cycloheximide (Cyc) incubations on cellular oxygen consumption rate (expressed in % relative to control) in isolated goldfish hepatocytes at normoxia (above 200 µM O2). On the left portion of the figure, cells were first incubated with A769 and then Cyc was added (referred to as A769 + Cyc). On the right portion of the figure are the results of a separate experiment where the order of the drugs was reversed. Data are means ± s.e.m. (n= 3). Values that do not share letters are significantly different from each other (p<0.05)  51  FIGURE 2.8  52   O2 max, P90, and P50 values of oxygen kinetic curves (obtained below 40 µM O2) TABLE 2.1 M in isolated goldfish hepatocytes from control, 15 mM cycloheximide and 3 mM ouabain treatments  Treatments   O2 max M 6  Control (n=7) 15 mM Cycloheximide (n=7)  (pmol O2/10 cells/sec) 9.54 ± 1.03 5.35 ± 1.15*  Control (n=7) 9.84 ± 1.06 3 mM Ouabain (n=7) 8.93 ± 0.91 *indicates significant difference from control (p<0.001)  P90 (µM)  P50 (µM)  13.44 ± 3.31 14.59 ± 2.14  0.49 ± 0.09 0.32 ± 0.08  12.31 ± 2.91 12.42 ± 0.95  0.44 ± 0.10 0.48 ± 0.11  53   O2 max, P90, and P50 values of oxygen kinetic curves (obtained below 40 µM O2) in TABLE 2.2 M isolated goldfish hepatocytes from control, 1 mM AICAR and 150 µM A769662 treatments  Treatments   O2 max M (pmol O2/10 cells/sec) 7.04 ± 0.63 6.05 ± 0.33  P90 (µM)  P50 (µM)  18.99 ± 4.05 26.11 ± 5.84  1.49 ± 0.38 2.31 ± 0.67  7.59 ± 1.41 6.22 ± 0.40  31.23 ± 7.29 27.86 ± 4.04  3.40 ± 1.78 2.69 ± 1.02  6  Control (n=5) 1 mM AICAR (n=5) Control (n=3) 150 µM A769662 (n=3)  54  CHAPTER THREE: GENERAL DISCUSSION AND CONCLUSION  Given its unique ability to both sense changes in cellular energy levels and regulate energy use and production, AMPK has been proposed to be an important initiator of MRS in fish during hypoxia. The goal of this thesis was to test the hypothesis that AMPK initiates MRS in isolated goldfish hepatocytes. I am able to provide additional evidence with my MSc thesis that AMPK plays a role in initiating MRS in goldfish hepatocytes, though it is likely that other regulatory processes are also involved in initiating MRS.  3.1 AMPK initiates metabolic rate suppression It is not difficult to comprehend why AMPK was deemed the prime candidate to act as an initiator of MRS during hypoxia as it provides an obvious link between energy sensing and cellular reorganization to restore energy balance. In fish, this relationship was first explored by Jibb and Richards (2008) who observed a large and rapid activation of AMPK in the liver of goldfish exposed to severe hypoxia. Accompanying the activation of AMPK in goldfish liver was an increase in eEF2 phosphorylation and a massive reduction in protein synthesis rate. The same pathway has been investigated in anoxic crucian carp and zebrafish embryos (Stenslokken et al. 2008; Mendelsohn et al. 2008) and in each those studies, AMPK was shown to be a likely candidate for initiating MRS. In order to test the hypothesis that AMPK initiates MRS in goldfish hepatocytes, I attempted to pharmacologically activate AMPK under high O2 conditions and observed  O2 . To manipulate AMPK activity, I used three different subsequent changes to cellular M  pharmacological agents, each with varying modes of action. AICAR is the most popular drug 55  activator for manipulating AMPK, but there are limitations to the drug due to its role as an AMP analogue. As an AMP analogue, it is possible that any AMP-dependent cellular pathway would be affected by the addition of AICAR. To overcome this obvious side effect, I used two promising new specific manipulators of AMPK activity- A769662 and compound C (Zhou et al. 2001; Cool et al. 2006). A769662 was thought to be a selective activator of AMPK that worked through a more specific pathway (Cool et al. 2006; Scott et al. 2008), but it failed to activate AMPK in goldfish hepatocytes as evidenced by the fact that there was no increase in AMPK phosphorylation or activity (Fig.2.7). Compound C was originally thought to be a specific inhibitor of AMPK, but is now viewed to be a general protein kinase inhibitor and only through the use of compound C in combination with AICAR can conclusions about AMPK be made  O2 and (Bain et al. 2007). However, compound C alone caused a non-significant reduction in M  the addition of compound C after the administration of AICAR did not reverse the effects of  O2 (Fig.2.6). AICAR on M  The limitations of pharmacological agents used in this study prevent me from ascribing  O2 solely to AMPK effects; however, I still gathered support for the notion that changes in M  AMPK is involved in initiating MRS in goldfish hepatocytes. Where I was able to pharmacologically activate AMPK with AICAR, I observed a significant decrease in AMPK  O2 . In fact, a maximal 7.5-fold stimulation of activity, there was a consistent decrease in M   O2 under normoxic conditions (Fig.2.3). The AMPK activity resulted in a 24% decrease in M  O2 occurred concurrently with an increase in activation of AMPK and subsequent decrease in M  the phosphorylation of eEF2 causing a suppression of protein synthesis (Fig.2.3C). Thus, my thesis lends support to the contention that AMPK partakes in the initiation of MRS. 56  It is evident that whole body MRS is not due to changes in liver metabolism through effects of AMPK alone. Goldfish have the ability to lower whole-body metabolic rate by up to 60% during hypoxia (Van Waversveld et al. 1989). Jibb and Richards (2008) previously showed that only liver display a significant increase in AMPK activation out of all the tissues evaluated in the goldfish. With AICAR, I have shown that activation of AMPK in hepatocytes lead to a  O2 . Taken together, it seems unlikely that a reduction in liver metabolism ~24% reduction in M  regulated via AMPK alone can explain whole-body metabolic rate reductions. Clearly, AMPK is not the sole initiator of MRS. When exposed to hypoxia, the AMPK cascade likely acts as the first line of defence, quickly modifying pathways through reversible protein phosphorylation. The activation of other mechanisms is potentially important in inducing necessary changes for long term hypoxic survival. One such mechanism could be hypoxia inducible factor (HIF-1); though the role of HIF-1 during O2 lack is not very well characterized in fish, it plays a crucial role in regulating molecular changes in response to hypoxia in mammalian studies (Richards 2009).  3.2 Responses of hepatocytes to physiological [O2] The vast majority of published studies using isolated cells held under high O2 concentrations, which clearly represent O2 levels never encountered in vivo. I show that as [O2] was gradually lowered from normoxia (~310 µM O2) to more physiologically relevant levels,  O2 down to approximately 13 µM (P90), below there was a gradual decrease in hepatocyte M  O2 . There are few published measurements of which there was a rapid decline in hepatocyte M  liver O2 tensions in fish, but from comparing P90 values in this study against available values from goldfish and common carp of 4 to 17 µM O2 (Burggren 1982; Takeda 1990), it suggests 57  that the liver would normally encounter O2 levels that make cellular respiration rate extremely sensitive to any changes in O2 in vivo. The essentially “hyperoxic” conditions used in many in vitro studies, including some of the work in this thesis, must be interpreted carefully in order to translate in vitro results to intact tissue or whole organism responses. Even in the present study,  O2 with the addition of AICAR under high O2 the significant reductions in hepatocyte M  conditions were diminished at more physiologically relevant O2 levels. Though this prevents me from directly translating my results to what actually occurs in vivo, this does not affect the conclusion drawn in this thesis that the activation of AMPK causes a decrease in metabolic rate in isolated goldfish hepatocytes. Taken together, the results of my experiments suggest that physiologically relevant O2 conditions must be considered in order to truly understand in vivo cellular responses.  3.3 Future directions Genetic manipulations in mammalian systems have provided more specific methods of studying the AMPK cascade. For example, Woods et al. (2000) worked with a constitutively active AMPK complex, where the catalytic α subunit was dissociated from the regulatory β and γ subunits. This altered AMPK complex no longer responded to changes in energy status or glycogen availability. The differential responses between a cell that contains a complete AMPK complex and one that is no longer responsive to energy status could be useful in addressing the actual role of AMPK in regulating MRS, without problems of non-specificity as in pharmacological manipulations. To gain a better understanding of whether AMPK activation is actually important in determining hypoxia tolerance, it would be illuminating to observe the activation pattern of 58  AMPK in hepatocytes from closely-related fish species with varying hypoxia tolerance. This would address the question of whether AMPK activation during O2 lack is adaptive and determines hypoxia tolerance.  3.4 Conclusion In this thesis, I have confirmed that AMPK plays an important role in the initiation of MRS; however, it is not likely the only signal transduction pathway involved. 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