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c-Myc and AMPK Control Cellular Energy Levels by Cooperatively Regulating Mitochondrial Structure and Function.

Edmunds LR, Sharma L, Wang H, Kang A, d'Souza S, Lu J, McLaughlin M, Dolezal JM, Gao X, Weintraub ST, Ding Y, Zeng X, Yates N, Prochownik EV - PLoS ONE (2015)

Bottom Line: The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes.Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state.Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS.

View Article: PubMed Central - PubMed

Affiliation: Section of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, United States of America; The University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America.

ABSTRACT
The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because Myc over-expression depletes ATP with the resultant activation of AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in ampk+/+ (WT) and ampk-/- (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs had a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox states. Transcriptional profiling of pathways pertinent to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are highly co-dependent and appear to engage in significant cross-talk across numerous pathways which support metabolic and ATP-generating functions.

No MeSH data available.


Related in: MedlinePlus

Metabolite profiling of WT and KO MEFs.(A) HPLC-ESI-MS/MS quantification of select metabolites in MEFs prior to or following MycER activation for 8 days. Each box represents the average of biological quadruplicate samples. Levels of each depicted metabolite were arbitrarily set to 1 in WT cells (white boxes). (B) Enzymatic and metabolite feedback control of PDH and PK. Note the control of the former by the stimulatory phosphatase PDP2 and the inhibitory kinase PDK1 as well as additional indirect and direct control of PDH and PK, respectively by ATP and ADP. PDH and PK enzyme activities (C and D, respectively) and acetyl CoA assays (E) were performed on whole cell extracts as previously described [23].
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pone.0134049.g006: Metabolite profiling of WT and KO MEFs.(A) HPLC-ESI-MS/MS quantification of select metabolites in MEFs prior to or following MycER activation for 8 days. Each box represents the average of biological quadruplicate samples. Levels of each depicted metabolite were arbitrarily set to 1 in WT cells (white boxes). (B) Enzymatic and metabolite feedback control of PDH and PK. Note the control of the former by the stimulatory phosphatase PDP2 and the inhibitory kinase PDK1 as well as additional indirect and direct control of PDH and PK, respectively by ATP and ADP. PDH and PK enzyme activities (C and D, respectively) and acetyl CoA assays (E) were performed on whole cell extracts as previously described [23].

Mentions: We used high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to quantify steady state levels of a select group of metabolites in WT and KO cells prior to or following MycER activation. The metabolites were chosen to reflect relevant glycolytic and TCA cycle intermediates, anabolic substrates and determinants of cellular redox and energy status. Significant differences were seen between WT and KO cells prior to MycER activation with the most striking being the generally lower levels of glycolytic substrates and higher levels of TCA cycle substrates in KO cells (Fig 6A). This suggested that the higher glycolytic rate of KO cells (Fig 1A) might be responsible for depleting some of the intermediates in this pathway whereas defects in the ETC might allow a buildup of TCA intermediates. Higher levels of nucleosides and deoxynucleosides in KO cells were consistent with the previously report of AMPK being a negative regulator of the Warburg effect [75], which could also explain the relative depletion of glycolytic substrates if they were being shunted into Warburg-related pathways. The modestly higher levels of AMP and ADP in WT cells in response to MycER activation, together with reduced ATP levels (Fig 1D and S1B Fig), is likely sufficient to account for any activation of AMPK that is not otherwise attributable to ROS (Fig 1E). In contrast, the even higher levels of AMP in KO cells, both prior to and following MycER activation, can likely be explained by the inability to normalize ATP and AMP in AMPK’s absence.


c-Myc and AMPK Control Cellular Energy Levels by Cooperatively Regulating Mitochondrial Structure and Function.

Edmunds LR, Sharma L, Wang H, Kang A, d'Souza S, Lu J, McLaughlin M, Dolezal JM, Gao X, Weintraub ST, Ding Y, Zeng X, Yates N, Prochownik EV - PLoS ONE (2015)

Metabolite profiling of WT and KO MEFs.(A) HPLC-ESI-MS/MS quantification of select metabolites in MEFs prior to or following MycER activation for 8 days. Each box represents the average of biological quadruplicate samples. Levels of each depicted metabolite were arbitrarily set to 1 in WT cells (white boxes). (B) Enzymatic and metabolite feedback control of PDH and PK. Note the control of the former by the stimulatory phosphatase PDP2 and the inhibitory kinase PDK1 as well as additional indirect and direct control of PDH and PK, respectively by ATP and ADP. PDH and PK enzyme activities (C and D, respectively) and acetyl CoA assays (E) were performed on whole cell extracts as previously described [23].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521957&req=5

pone.0134049.g006: Metabolite profiling of WT and KO MEFs.(A) HPLC-ESI-MS/MS quantification of select metabolites in MEFs prior to or following MycER activation for 8 days. Each box represents the average of biological quadruplicate samples. Levels of each depicted metabolite were arbitrarily set to 1 in WT cells (white boxes). (B) Enzymatic and metabolite feedback control of PDH and PK. Note the control of the former by the stimulatory phosphatase PDP2 and the inhibitory kinase PDK1 as well as additional indirect and direct control of PDH and PK, respectively by ATP and ADP. PDH and PK enzyme activities (C and D, respectively) and acetyl CoA assays (E) were performed on whole cell extracts as previously described [23].
Mentions: We used high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to quantify steady state levels of a select group of metabolites in WT and KO cells prior to or following MycER activation. The metabolites were chosen to reflect relevant glycolytic and TCA cycle intermediates, anabolic substrates and determinants of cellular redox and energy status. Significant differences were seen between WT and KO cells prior to MycER activation with the most striking being the generally lower levels of glycolytic substrates and higher levels of TCA cycle substrates in KO cells (Fig 6A). This suggested that the higher glycolytic rate of KO cells (Fig 1A) might be responsible for depleting some of the intermediates in this pathway whereas defects in the ETC might allow a buildup of TCA intermediates. Higher levels of nucleosides and deoxynucleosides in KO cells were consistent with the previously report of AMPK being a negative regulator of the Warburg effect [75], which could also explain the relative depletion of glycolytic substrates if they were being shunted into Warburg-related pathways. The modestly higher levels of AMP and ADP in WT cells in response to MycER activation, together with reduced ATP levels (Fig 1D and S1B Fig), is likely sufficient to account for any activation of AMPK that is not otherwise attributable to ROS (Fig 1E). In contrast, the even higher levels of AMP in KO cells, both prior to and following MycER activation, can likely be explained by the inability to normalize ATP and AMP in AMPK’s absence.

Bottom Line: The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes.Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state.Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS.

View Article: PubMed Central - PubMed

Affiliation: Section of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, United States of America; The University of Pittsburgh School of Medicine, Pittsburgh, PA, United States of America.

ABSTRACT
The c-Myc (Myc) oncoprotein and AMP-activated protein kinase (AMPK) regulate glycolysis and oxidative phosphorylation (Oxphos) although often for different purposes. Because Myc over-expression depletes ATP with the resultant activation of AMPK, we explored the potential co-dependency of and cross-talk between these proteins by comparing the consequences of acute Myc induction in ampk+/+ (WT) and ampk-/- (KO) murine embryo fibroblasts (MEFs). KO MEFs showed a higher basal rate of glycolysis than WT MEFs and an appropriate increase in response to activation of a Myc-estrogen receptor (MycER) fusion protein. However, KO MEFs had a diminished ability to increase Oxphos, mitochondrial mass and reactive oxygen species in response to MycER activation. Other differences between WT and KO MEFs, either in the basal state or following MycER induction, included abnormalities in electron transport chain function, levels of TCA cycle-related oxidoreductases and cytoplasmic and mitochondrial redox states. Transcriptional profiling of pathways pertinent to glycolysis, Oxphos and mitochondrial structure and function also uncovered significant differences between WT and KO MEFs and their response to MycER activation. Finally, an unbiased mass-spectrometry (MS)-based survey capable of quantifying ~40% of all mitochondrial proteins, showed about 15% of them to be AMPK- and/or Myc-dependent in their steady state. Significant differences in the activities of the rate-limiting enzymes pyruvate kinase and pyruvate dehydrogenase, which dictate pyruvate and acetyl coenzyme A abundance, were also differentially responsive to Myc and AMPK and could account for some of the differences in basal metabolite levels that were also detected by MS. Thus, Myc and AMPK are highly co-dependent and appear to engage in significant cross-talk across numerous pathways which support metabolic and ATP-generating functions.

No MeSH data available.


Related in: MedlinePlus