Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (DEX) treated animals, glycogen accumulation was enhanced. We examined the influence of DEX on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. Following DEX, cardiac tissue had limited contribution towards the development of whole body insulin resistance. Measurement of GLUT4 at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated to an increased GLUT4 mR.NA. Both total and phosphorylated AMPK increased following DEX. Immunoprecipitation of AS 160 followed by Western blotting demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in DEX treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr172, which correlated well with AS 160 phosphorylation. In DEX hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas GSK-3-β phosphorylation was augmented. Our data suggest that AMPK mediated glucose entry, combined with activation of glycogen synthase and reduction in glucose oxidation (Qi, D., et al. Diabetes 53:1790, 2004), act together to promote glycogen storage. Our data suggest that in the presence of intact insulin signaling, AMPK mediated glucose entry, combined with activation of glycogen synthase and the previously reported reduction in glucose oxidation, act together to promote glycogen storage. Should these effects persist chronically, they may explain the increased morbidity and mortality observed with long term excesses in endogenous or exogenous glucocorticoids.

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (DEX) treated animals, glycogen accumulation was enhanced. We examined the influence of DEX on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. Following DEX, cardiac tissue had limited contribution towards the development of whole body insulin resistance. Measurement of GLUT4 at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated to an increased GLUT4 mR.NA. Both total and phosphorylated AMPK increased following DEX. Immunoprecipitation of AS 160 followed by Western blotting demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in DEX treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr172, which correlated well with AS 160 phosphorylation. In DEX hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas GSK-3-β phosphorylation was augmented. Our data suggest that AMPK mediated glucose entry, combined with activation of glycogen synthase and reduction in glucose oxidation (Qi, D., et al. Diabetes 53:1790, 2004), act together to promote glycogen storage. Our data suggest that in the presence of intact insulin signaling, AMPK mediated glucose entry, combined with activation of glycogen synthase and the previously reported reduction in glucose oxidation, act together to promote glycogen storage. Should these effects persist chronically, they may explain the increased morbidity and mortality observed with long term excesses in endogenous or exogenous glucocorticoids.

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (DEX) treated animals, glycogen accumulation was enhanced. We examined the influence of DEX on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. Following DEX, cardiac tissue had limited contribution towards the development of whole body insulin resistance. Measurement of GLUT4 at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated to an increased GLUT4 mR.NA. Both total and phosphorylated AMPK increased following DEX. Immunoprecipitation of AS 160 followed by Western blotting demonstrated no change in Akt phosphorylation at Ser473 and Thr308 in DEX treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr172, which correlated well with AS 160 phosphorylation. In DEX hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas GSK-3-β phosphorylation was augmented. Our data suggest that AMPK mediated glucose entry, combined with activation of glycogen synthase and reduction in glucose oxidation (Qi, D., et al. Diabetes 53:1790, 2004), act together to promote glycogen storage. Our data suggest that in the presence of intact insulin signaling, AMPK mediated glucose entry, combined with activation of glycogen synthase and the previously reported reduction in glucose oxidation, act together to promote glycogen storage. Should these effects persist chronically, they may explain the increased morbidity and mortality observed with long term excesses in endogenous or exogenous glucocorticoids. / Pharmaceutical Sciences, Faculty of / Graduate

The production of ATP is of the utmost importance to cell survival. To maintain energy homeostasis, cells regulate mitochondrial content through the control of degradative and synthetic processes. Mitochondrial biogenesis is primarily controlled through a small number of transcriptional regulators, primarily nuclear respiratory factor-1 (NRF-1), NRF-2, and peroxisome proliferator-activated receptors (PPARs). DNA-binding proteins regulate genes encoding the machinery of oxidative phosphorylation. In addition to these DNA-binding proteins, the coactivator PPAR gamma coactivator-1 alpha (PGC-1α) is central to control of mitochondrial genes, so much so that it has been dubbed a “master controller” of energy homeostasis in mammalian muscle tissues. Though well studied in mammals, previous studies suggest that this NRF-1-PGC1α axis may be disrupted in fish. The response to treatments such as temperature and diet cause reciprocal effects on NRF-1 and PGC-1α. A serine-rich insertion into the NRF-1 binding domain of PGC-1α most likely disrupts this interaction. In this study I looked at the ability for the goldfish PGC-1α gene to interact with the PGC-1α binding domain of NRF-1. I have found that goldfish PGC-1α does not physically bind NRF-1, which would suggest that the PGC-1α-NRF-1 axis in fish is disrupted. To further explore the role of PGC-1α in fish we looked at the role of AMP-activated kinase (AMPK) to phosphorylate goldfish, zebrafish, and human PGC-1α. The results from this analysis show that AMPK in a zebrafish embryonic cell line (ZEB2J) have their AMPK activated by the AMPK activator AICAR. This response was shown to be both dose and time dependent. Transcript data was generated looking at typical AMPK responsive targets in the mammalian system. The target of ramapamycin (TOR) gene responded with a decrease as is expected in mammals. Hexokinase 2 (HK2), PGC-1α, and NRF-1 all decreased which is opposite of the typical mammalian response. COX7C a downstream target of the PGC-1α-NRF-1 axis did not respond to treatment. Indicating a disruption in the AMPK- PGC-1α-NRF-1 pathway. / Thesis (Master, Biology) -- Queen's University, 2013-09-06 01:37:05.537

The enzyme adenosine monophosphate activated protein kinase (AMPK), a critical regulator of energy metabolism in the body, is activated by a rise in the cellular AMP: ATP ratio in response to metabolic stresses such as hypoxia. The work in this thesis arises from the recent characterization by Mahmoud, A. (PhD thesis, University of Edinburgh, 2015) of the response to hypoxia of mice lacking the α1 and α2 isoforms of the catalytic subunit of the AMPK molecule. Targeted conditional deletion of the genes encoding the α1 and/or α2 subunits of AMPK in catecholaminergic cells (including cells in the carotid body and the brain) was achieved by crossing mice expressing Cre-recombinase under the control of the tyrosine hydroxylase (TH) promoter, with mice in which either or both α subunits of AMPK were flanked by loxP sequences. AMPKα1/α2-/- mice showed a profoundly abnormal ventilatory response to hypoxia, compared to AMPKα1/α2fl/fl controls. Interestingly however, in vitro recordings from the CSN in isolated carotid body preparations from AMPKα1/α2-/- mice showed that the carotid body afferent response to hypoxia was completely normal in these mice. The abnormal response to hypoxia in AMPKα1/α2-/- mice appears therefore to be due to a deficit in the central, catecholaminergic brainstem neurons involved in respiratory control, where a lack of AMPK activation appears to inhibit the normal hypoxia-induced hyperventilation. While the importance of the peripheral carotid body chemoreceptors in oxygen-sensing has long been established, these findings indicate that the synergistic activation of AMPK in central brainstem neurons by the hypoxic metabolic stress, is also required for the normal response to hypoxia. In this thesis, the responses to hypoxia of AMPKα1/α2-/- mice, AMPKα2-/- mice and AMPKα1/α2fl/fl controls were studied using whole-body plethysmography. The findings showed that AMPKα1/α2-/- mice displayed a respiratory phenotype of longer and increased number of apnoeas coupled with hypoventilation in comparison to both the AMPKα2-/- mice and AMPKα1/α2fl/fl controls confirming the earlier results of Mahmoud (2015). In addition TH immunostaining in the carotid bodies of AMPKα1/α2-/- mice and AMPKα1/α2fl/fl controls was compared to determine if there was any change in the number or density of TH-positive cells in the AMPKα1/α2-/- animals, and the gross result showed a 2-fold decrease in the number of TH- immunopositive cells in the AMPKα1/α2-/- mice as compared to the AMPKα1/α2fl/fl. Intriguingly, if this observation is statistically confirm coupled with the unattenuation of the normal afferent discharge from the carotid bodies of AMPKα1/α2-/- mice then it is plausible that a certain degree of redundancy operates in the physiology of the carotid body with regards to oxygen sensing or the glomus cells type lost may be those not involved in mediating the response to hypoxia. In the main part of this work, c-fos and TH immunohistochemistry were used to investigate the activation of brainstem catecholaminergic neurons by hypoxia, in AMPKα1/α2-/- mice, AMPKα2-/- mice and AMPKα1/α2fl/fl controls. Significant differences in c-fos immunostaining of TH+ve neurons were observed in the SubP region of the NTS, and the C2 region and the A1 region of the ventral respiratory group, implicating these specific regions in the abnormal hypoxic ventilatory response in AMPKα1/α2-/- animals. Catecholaminergic neurons in these brainstem regions are known to play key role in the control of breathing as a loss of these neurons or decrease in their catecholamine content results in severe respiratory abnormalities including respiratory arrhythmias and apnoeas as seen in the Rett syndrome. A significant hyperplasia of the brainstem stem catecholaminergic neurons was also observed in both the AMPKα1/α2-/-and AMPKα2-/- mice consistent with the known inhibitory effects of AMPK activation on cell growth and proliferation. Finally a pilot study was carried out to determine if the respiratory phenotype observed in AMPKα1/α2-/- mice could be replicated using a viral vector to deliver Cre-recombinase to targeted areas in the brainstem in AMPKα1/α2fl/fl mice, to knock out AMPK in specific neuronal subgroups. Although the data obtained from this set of experiments were encouraging, the animals did not show the respiratory phenotype as observed in the conditional knockout mice maybe due to poor targeting of specific brainstem neuronal populations including the SubP region or inadequate transfection of these cells due to low viral titre. One advantage of this pilot work was that responses due to compensatory mechanisms as may be the case in the conditional knock out animals were eliminated. These findings are of interest in understanding the neural control of respiration in hypoxia and acclimatization to altitude, and may also suggest new avenues for therapeutic intervention in breathing disorders such as non-obstructive sleep apnoea. It also raises the possibility that AMPK may be a useful therapeutic tool for disease conditions whose etiology is based on cellular proliferation, such as various forms of cancer and even atherosclerosis.

Type 2 diabetes, as well as other metabolic diseases, is an increasing global health concern and many of the mechanisms of both the disease and its current drug treatments have not been fully described. It has been shown that the anti-diabetic class of drugs, the thiazolidinediones, work via both a known PPARγ-dependent, and a lesser known PPARγ-independent mechanism of action. This PPARγ-independent mechanism likely involves the metabolic regulatory molecule AMPK, which has a newly elucidated inhibitory site of phosphorylation at Ser485/Ser491. In this study we sought to determine if the thiazolidinedione troglitazone affects AMPK in HepG2 liver cells via phosphorylation at both the known Thr172 site as well as the letter understood Ser485 site. We also looked for potential upstream kinases of the Ser485 site by comparing our results to recently proposed mechanisms of phosphorylation here. HepG2 cells were cultured in the lab and treated with troglitazone to determine time- and dose- dependent effects on AMPK. We also treated cultured HepG2 cells with PMA as well as troglitazone and PMA in order to compare mechanisms of action of troglitazone on AMPK. Results were analyzed using common western blot techniques and statistical analysis. Our data found that troglitazone increased AMPK activity by increasing phosphorylation at Thr172 in a time- and dose- dependent manner. The inhibitory site Ser485 was also increasingly phosphorylated with troglitazone treatments, although the net result of troglitazone treatment remained AMPK activation. The recently elucidated results from our laboratory showing the mechanism of p-AMPK Ser485 phosphorylation via PKD after PMA treatment also occurred in HepG2 cells, although this did not appear to be the mechanism by which troglitazone phosphorylated AMPK at Ser485. These data support the current research that there is an AMPK mediated PPARγ-independent mechanism of troglitazone treatment for type 2 diabetes and other metabolic diseases. The results do however bring into question the full effects of the drug on AMPK at a molecular level and leaves room for new research in this area, specifically the exact mechanism by which troglitazone phosphorylates AMPK at Ser485. Our data also brings up new questions as to the simultaneous phosphorylation of AMPK at both Thr172 and Ser485 and what this means for the activity of the molecule as a whole, a current area of critical research. Lastly our data support the newly elucidated mechanism of AMPK phosphorylation at Ser485 via PKD1, an exciting and novel discovery and potential target for therapeutic intervention.

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy stress that, once activated, promotes ATP-producing process while it switches off ATP-consuming pathways, in order to restore the cellular energetic balance under conditions of stress. Activation of AMPK is dependent on the phosphorylation of the residue Thr172 in its α subunit. This phosphorylation is generally mediated by the known tumour suppressor LKB1, but also CaMKKβ has been shown to phosphorylate AMPK. As its name suggests, AMPK is also activated by the binding of AMP to its γ subunit. This binding causes a >10 fold allosteric stimulation, promotes phosphorylation of Thr172 by upstream kinases and protects AMPK from dephosphorylation of Thr172 by protein phosphatase(s). In 2010 it was reported that oxidative stress mediated by H₂O₂ activated AMPK by increasing the cellular AMP:ATP and ADP:ATP ratios (Hawley et al, 2010). However, the same year another work suggested that the mechanism of activation of AMPK by H₂O₂was direct, independent of AMP and involved the oxidation of two cysteine residues in the α subunit of AMPK (Zmijewski et al, 2010). Given this discrepancy, here we provided evidence that H₂O₂, generated by addition of glucose oxidase in the cell medium, activates AMPK mostly through an increase of AMP:ATP and ADP:ATP ratios, as previously suggested in our laboratory. However, it seems that there might be a second, minor mechanism of activation that is independent of the changes in cellular nucleotides. This second mechanism was not identified in our previous work because we were not aware of how rapidly a single bolus of H₂O₂ can be metabolized by the antioxidant defences of the cell. We could not identify the alternative mechanism of activation by H₂O₂but showed that H₂O₂ could protect Thr172 from dephosphorylation, which might suggest a direct effect of H₂O₂ on the phosphatase(s) dephosphorylating AMPK. However, since the identity of this phosphatase(s) remains unclear, we could not rule out the possibility that the protection from dephosphorylation that we observed could still be mediated by the increase in AMP:ATP and ADP:ATP ratios. Moreover, it remains still possible that a direct effect of H₂O₂ on AMPK might be responsible for the small but significant activation we detected in cell expressing a nucleotides-insensitive mutant of AMPK. Recently, a new crystal structure of AMPK obtained by Xiao et al (2013) provided new insights about AMPK structure and regulation. In particular, the authors identified a new binding pocket located at the interface between the N-lobe of the α-kinase domain and the β-CBM of AMPK, which appeared to be the binding site for two direct activators of AMPK: A769662 and 991. Here we confirm that this novel binding pocket is indeed the binding site for both A769662 and 991, and provide evidence that another direct activator of AMPK, MT63-78, also binds at the same site. Mutation of two important residues in this pocket (Lys29 and Lys31 of the α2 subunit) abolished the allosteric stimulation of AMPK by A769662, 991 and MT63-78 while it had no effect on allosteric stimulation by AMP. However, we also showed that the same mutation abolished protection against Thr172 dephosphorylation not only by A769662, 991 and MT63-78, but also by phenformin and H2O2, which are known to activate AMPK by increasing the AMP:ATP and ADP:ATP ratios. These data show that the integrity of this pocket is important for the effect of AMP to protect against Thr172 dephosphorylation, but not for its ability to cause allosteric stimulation. Moreover, in HEK-293 cell stably expressing an α2 subunit carrying the mutation of both Lys29 and Lys31, the basal activity of AMPK due to Thr172 phosphorylation was almost 6-fold less than in cells expressing wild-type α2. This result pointed out for the first time that there might be a natural ligand binding in the newly discovered binding pocket that is not able to bind to the double mutant, explaining the difference in activity observed. However the identity of this possible natural ligand remains unclear and more studies will be necessary to uncover it.

Introduction: Cardiac hypertrophy is an adaptive response to increased myocardial workload that becomes maladaptive when hypertrophied hearts are exposed to an acute metabolic stress, such as ischemia/reperfusion. Acceleration of glycolysis occurs as part of the hypertrophic response and may be maladaptive because it enhances glycolytic metabolite accumulation and proton production. Activation of AMP-activated protein kinase (AMPK), a kinase involved in the regulation of energy metabolism, is proposed as a mechanism for the acceleration of glycolysis in hypertrophied hearts. However, this concept has not yet been proven conclusively. Additionally, several studies suggest that AMPK is involved in hypertrophic remodeling of the heart by influencing cardiac myocyte growth, a suggestion that remains controversial. Hypothesis: AMPK mediates hypertrophic remodeling in response to pressure overload. Specifically, AMPK activation is a cellular signal responsible for accelerated rates of glycolysis in hypertrophied hearts. Additionally, AMPK influences myocardial structural remodeling and gene expression by limiting hypertrophic growth. Experimental Approach: To test this hypothesis, H9c2 cells, derived from embryonic rat hearts, were treated with (1 µM) arginine vasopressin (AVP) to induce hypertrophy. Substrate utilization was measured and the effects of AMPK inhibition by either Compound C or by adenovirus-mediated transfer of dominant negative AMPK were determined. Subsequently, adenovirus-mediated transfer of constitutively active form of AMPK (CA-AMPK) was expressed in H9c2 to specifically increase AMPK activity and, thereby, further characterize the role of AMPK in hypertrophic remodeling. Results: AVP induced a metabolic profile in hypertrophied H9c2 cells similar to that in intact hypertrophied hearts. Glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation. These changes were associated with AMPK activation, and inhibition of AMPK ameliorated but did not normalize the hypertrophy-associated increase in glycolysis. CA-AMPK stimulated both glycolysis and fatty acid oxidation, and also increased protein synthesis and content. Howver, CA-AMPK did not induce a pathological hypertrophic phenotype as assessed by atrial natriuretic peptide expression. Conclusion: Acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK. AMPK is a positive regulator of cell growth in these cells, but does not induce pathological hypertrophy when acting alone.

Introduction: Cardiac hypertrophy is an adaptive response to increased myocardial workload that becomes maladaptive when hypertrophied hearts are exposed to an acute metabolic stress, such as ischemia/reperfusion. Acceleration of glycolysis occurs as part of the hypertrophic response and may be maladaptive because it enhances glycolytic metabolite accumulation and proton production. Activation of AMP-activated protein kinase (AMPK), a kinase involved in the regulation of energy metabolism, is proposed as a mechanism for the acceleration of glycolysis in hypertrophied hearts. However, this concept has not yet been proven conclusively. Additionally, several studies suggest that AMPK is involved in hypertrophic remodeling of the heart by influencing cardiac myocyte growth, a suggestion that remains controversial. Hypothesis: AMPK mediates hypertrophic remodeling in response to pressure overload. Specifically, AMPK activation is a cellular signal responsible for accelerated rates of glycolysis in hypertrophied hearts. Additionally, AMPK influences myocardial structural remodeling and gene expression by limiting hypertrophic growth. Experimental Approach: To test this hypothesis, H9c2 cells, derived from embryonic rat hearts, were treated with (1 µM) arginine vasopressin (AVP) to induce hypertrophy. Substrate utilization was measured and the effects of AMPK inhibition by either Compound C or by adenovirus-mediated transfer of dominant negative AMPK were determined. Subsequently, adenovirus-mediated transfer of constitutively active form of AMPK (CA-AMPK) was expressed in H9c2 to specifically increase AMPK activity and, thereby, further characterize the role of AMPK in hypertrophic remodeling. Results: AVP induced a metabolic profile in hypertrophied H9c2 cells similar to that in intact hypertrophied hearts. Glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation. These changes were associated with AMPK activation, and inhibition of AMPK ameliorated but did not normalize the hypertrophy-associated increase in glycolysis. CA-AMPK stimulated both glycolysis and fatty acid oxidation, and also increased protein synthesis and content. Howver, CA-AMPK did not induce a pathological hypertrophic phenotype as assessed by atrial natriuretic peptide expression. Conclusion: Acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK. AMPK is a positive regulator of cell growth in these cells, but does not induce pathological hypertrophy when acting alone.

Introduction: Cardiac hypertrophy is an adaptive response to increased myocardial workload that becomes maladaptive when hypertrophied hearts are exposed to an acute metabolic stress, such as ischemia/reperfusion. Acceleration of glycolysis occurs as part of the hypertrophic response and may be maladaptive because it enhances glycolytic metabolite accumulation and proton production. Activation of AMP-activated protein kinase (AMPK), a kinase involved in the regulation of energy metabolism, is proposed as a mechanism for the acceleration of glycolysis in hypertrophied hearts. However, this concept has not yet been proven conclusively. Additionally, several studies suggest that AMPK is involved in hypertrophic remodeling of the heart by influencing cardiac myocyte growth, a suggestion that remains controversial. Hypothesis: AMPK mediates hypertrophic remodeling in response to pressure overload. Specifically, AMPK activation is a cellular signal responsible for accelerated rates of glycolysis in hypertrophied hearts. Additionally, AMPK influences myocardial structural remodeling and gene expression by limiting hypertrophic growth. Experimental Approach: To test this hypothesis, H9c2 cells, derived from embryonic rat hearts, were treated with (1 µM) arginine vasopressin (AVP) to induce hypertrophy. Substrate utilization was measured and the effects of AMPK inhibition by either Compound C or by adenovirus-mediated transfer of dominant negative AMPK were determined. Subsequently, adenovirus-mediated transfer of constitutively active form of AMPK (CA-AMPK) was expressed in H9c2 to specifically increase AMPK activity and, thereby, further characterize the role of AMPK in hypertrophic remodeling. Results: AVP induced a metabolic profile in hypertrophied H9c2 cells similar to that in intact hypertrophied hearts. Glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation. These changes were associated with AMPK activation, and inhibition of AMPK ameliorated but did not normalize the hypertrophy-associated increase in glycolysis. CA-AMPK stimulated both glycolysis and fatty acid oxidation, and also increased protein synthesis and content. Howver, CA-AMPK did not induce a pathological hypertrophic phenotype as assessed by atrial natriuretic peptide expression. Conclusion: Acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK. AMPK is a positive regulator of cell growth in these cells, but does not induce pathological hypertrophy when acting alone. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate