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. 2015 Jul 31;10(7):e0134049.
doi: 10.1371/journal.pone.0134049. eCollection 2015.

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

Lia R Edmunds  1 , Lokendra Sharma  2 , Huabo Wang  2 , Audry Kang  3 , Sonia d'Souza  2 , Jie Lu  2 , Michael McLaughlin  3 , James M Dolezal  3 , Xiaoli Gao  4 , Susan T Weintraub  4 , Ying Ding  5 , Xuemei Zeng  6 , Nathan Yates  6 , Edward V Prochownik  7
Affiliations

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

Lia R Edmunds et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Energy-generating pathway responses to MycER activation.
(A) Baseline glycolysis measurements normalized to cell number at conclusion of the experiment. WT and KO MEFs were either untreated or exposed to 4HT for 7 days to activate MycER (+Myc). Glycolysis was quantified by measuring extracellular acidification rates (ECAR). Bars represent the mean of quadruplicate measurements ± 1 standard error of the mean (SEM). (B) Baseline Oxphos measurements. Oxygen consumption rates (OCR) were simultaneously quantified on the same samples described in A. See S2 Fig for additional details regarding the glycolytic and Oxphos responses of these cells. (C) Changes in mitochondrial mass and ROS production. Untreated or 4HT-treated (7 days) WT and KO cells were stained with MitoTracker Green or NAO to independently assess mitochondrial mass. In parallel experiments, cells were also stained with CM-H2-DCFDA to quantify total ROS production or with MitoSOX to specifically quantify the production of mitochondrial-derived superoxide. In all cases, typical flow diagrams are depicted. The numbers in the upper left portion of each panel indicate the mean ratios of fluorescence intensities between 4HT-treated and control, untreated cells from at least 4 independent experiments ± 1 SEM (D) ATP levels in response to MycER activation and inactivation. ATP levels were measured at baseline (S1B Fig), after the indicated periods of exposure to 4HT (+) and its subsequent removal (-). Each value shown depicts the mean of quadruplicate samples ± 1 SEM. Basal (day 0) ATP levels in untreated KO cells were routinely found to be 30–40% lower than those of their WT counterparts (S1B Fig) but are normalized here to 100% in both cell types for easier comparison. After taking the day 0 differences into account, KO MEFs also showed significantly lower ATP levels on days +1–4 compared to their WT counterparts (P < 0.02) (S1B Fig). (E) AMPK response to MycER activation/inactivation in WT MEFs. WT cells were exposed to 4HT for the indicated times. On day 4, 4HT was removed (-4HT) and cells continued to be cultured in its absence for an additional 6 days. Total AMPK and pAMPK were assessed by immunoblotting at the time points shown. (F) MitoSox staining as described in Fig 1C after 2 days of 4HT treatment. In the concurrent presence of 1 mM NAC, no significant change in ROS was observed. (G) AMPK activation is suppressed by NAC. WT cells were exposed to 4HT in the absence or presence of 1 mM NAC. Total and phospho-AMPK were assessed by immuno-blotting as described for panel E. (H) Persistence of ROS following MycER inactivation. WT cells were exposed to 4HT for 4 days at which point ROS were quantified using MitoSox as described in (C). 4HT was then removed with ROS levels again being determined 2 and 4 days later (days 6 and 8). Numbers in upper left corner indicate the ratio of mean fluorescence intensity of 4HT-treated (red curves) to non-4HT-treated MEFs (black curves).
Fig 2
Fig 2. Structural and functional properties of ETCs complexes in WT and KO cells.
(A) Analysis of ETC complexes by non-denaturing BNGE. Mitochondria were purified from triplicate cultures of the indicated cell types and resolved by BNGE. At least 5 individual supercomplexes (SCa-e) could be resolved [20]. Note that Complex V (ATP synthase) is composed of both monomers (Vm) and dimers (Vd). (B) In situ enzymatic assays for Complex I (NADH ubiquinone oxidoreductase) plus supercomplexes, Complex Vm and Vd (ATPase), Complex III (decylubiquinol cytochrome c oxidoreductase) and Complex IV (Cytochrome c oxidase). Each assay was performed in triplicate on at least 2 occasions, with representative results being shown here (also see S2 Fig). (C) Complex II (succinate dehydrogenase) activity was quantified on lysates prepared from purified mitochondria as its in situ assessment was found to be unreliable. The results shown in the histogram represent the mean of triplicate enzymatic determinations ± 1 SEM. See S3 Fig for quantification of all enzymatic measurements.
Fig 3
Fig 3. Transcriptional and enzymatic differences between WT and KO MEFs.
(A) Transcriptional profiling. Real-time qRT-PCR was performed in triplicate for each of the indicated transcripts. The levels of each transcript determined in WT cells were arbitrarily set to 1 as indicated by the white boxes. See S4 Fig for the actual numerical values and p values for individual determinations. (B) Functional assays for oxidoreductases. Each bar represents the mean of biological triplicate determinations ± 1 SEM performed on purified mitochondrial lysates. Note that the results for SDH are the same as those presented in Fig 2C. (C) qRT-PCR profiling of transcripts involved in the regulation of mitochondrial DNA replication and maintenance, transcription and function. Analyses were performed as described for (A). See S5 Fig for the actual numerical differences and p values for individual determinations.
Fig 4
Fig 4. Mitochondrial proteomic profiling.
LC-MS/MS reliably identified and quantified 345 proteins with mitochondrial localization from representative peptides in each of the 4 experimental groups (S3 Table). (A) Differential protein expression between WT and KO MEFs prior to MycER activation. (B) Differential protein expression in WT MEFs prior to or following an 8 day period of MycER activation. (C) Venn diagram of protein overlap between WT and KO MEFs, WT vs. WT+Myc and KO versus KO+Myc. Each of the protein subsets within these 3 groups are enclosed by red, green and blue circles and correspond to the proteins denoted in panels A, B and D, respectively. All proteins were selected by a conservative q-value of <0.05 by 2-way analysis of variance (two way ANOVA). (D) Differential protein expression in KO MEFs prior to or following 8 days of Myc over-expression. (E) Quantification of 8 proteins that showed a significant interaction effect by both AMPK and Myc overexpression.
Fig 5
Fig 5. Redox states in cytoplasmic and mitochondrial compartments of WT and KO MEFs.
(A) Live cell confocal images of WT MEFs stably expressing roGFP-mito and roGFP-cyto demonstrating specific mitochondrial and cytoplasmic localization, respectively. Nuclear counter-staining was with Hoechst 3334. (B) Live cell confocal microscopy of KO MEFs expressing roGFP-mito. Cells were untreated or exposed to 1 mM H2O2 or 10 mM DTT for 30 min prior to obtaining images. (C) Quantification of redox differences between WT and KO MEFs. Monolayer cultures were grown for 24 hr. in the absence or presence of 4HT. Flow cytometry was then used to quantify the mean fluorescence ratios of oxidized and reduced roGFP. Each bar represents the average ± 1 SEM of mean fluorescence intensities obtained from 3 independent plates of cells. * = P<0.001. Similar results were independently obtained in 2 repeat experiments as well as in 2 experiments performed following a longer period of MycER activation (7 days, not shown). The 4 bars on the right represent control experiments in which WT cells expressing roGFP-cyto were exposed under the conditions described in (B). These values define the maximal possible degree of oxidation or reduction capable of being achieved under the most extreme conditions.
Fig 6
Fig 6. 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].
Fig 7
Fig 7. Model depicting the relationship between Myc and AMPK (yellow boxes) demonstrating their influence over common metabolic functions, although sometimes in opposite ways and for different purposes.
Communication between Myc and AMPK may occur via at least 2 distinct and semi-autonomous routes with different initiating events and consequences. The first involves the activation of AMPK via Myc-mediated depletion of cellular ATP stores arising as a consequence of energy-consuming anabolic processes such as proliferation (-ATP, blue box) (Fig 1D and [23]). The second involves AMPK activation via ROS generated from increases in mitochondrial metabolism or cytoplasmic signaling pathways. It is notable that, in the first case, AMPK activation is dependent upon ATP depletion whereas, in the second case, AMPK activation occurs regardless of ATP status. AMPK activation via ROS can thus anticipate impending ATP depletion and prevent or limit this by down-regulating ATP dependent processes. In the face of a pre-existing ATP deficit, other functions of AMPK such as the promotion of proliferative arrest might tend to override the effects of Myc over-expression. In contrast, activation of AMPK by ROS might reinforce an already highly proliferative and ATP-replete state by promoting pro-anabolic functions such as glycolysis and Oxphos without necessarily compromising proliferation.
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