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Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency.

Rodrigues AS, Correia M, Gomes A, Pereira SL, Perestrelo T, Sousa MI, Ramalho-Santos J - PLoS ONE (2015)

Bottom Line: Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells.Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored.Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic profile, by using some of the signaling pathways found in the latter cells.

View Article: PubMed Central - PubMed

Affiliation: PhD Programme in Experimental Biology and Biomedicine, CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT

Introduction: The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency.

Methods/results: In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation.

Conclusions: DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic profile, by using some of the signaling pathways found in the latter cells.

No MeSH data available.


Related in: MedlinePlus

Assessing mitochondrial function.For each flow cytometry experiment results were analyzed in terms of Geometric Mean of Fluorescence for 20000 cells for each condition and are represented as percentage relative to the control. (A)- Quantification of mitochondrial membrane potential (MMP) potential using TMRM by flow cytometry. A significant increase in MMP was observed for CTR w/o LIF as well as for DCA 5 mM. Results are means ± SEM of 4 independent experiments. (B)- Oxygen consumption rate (OCR) was determined using the Seahorse XF24 analyzer. The three mitochondrial inhibitors were sequentially injected (after measurement points, 2, 4, and 6, as indicated) and the final concentrations of each were: oligomycin (1μM); FCCP (300mM); rotenone and antimicyn A (1μM). CTR w/o LIF presented a significant decrease in OCR in the presence of oligomycin. (C)- Determination of the respiration used to drive ATP production under basal conditions following the mathematical calculation: ATP production = Basal–Protocol Leak. This formula determines that CTR w/o LIF cells had higher mitochondrial respiration coupled to ATP production (D)- Measurement of extracellular acidification rate (ECAR) using the Seahorse XF24 analyzer. Cells were incubated in a medium without glucose and ECAR was accessed during consumption of Glucose (added at the third time point at a final concentration of 5mM) and in the presence of oligomycin (1μM added at the sixth point) and 2DG (10mM at the ninth point). At basal conditions, CTR w/o LIF had a lower glycolytic rate when compared to the control. The CTR condition displayed a significant higher glycolytic capacity. (E)- Determination of glucose breakdown to pyruvate using the mathematical calculation: first measurement after glucose injection minus the measurement prior to glucose injection. CTR w/o LIF cells had a significantly lower glycolytic capacity. F)- Results for the MTT assay are presented as percentage of formazan crystals relative to the control. Because the goal was to evaluate cell oxidative status, MTT results were normalized for total cell number. CTR w/o LIF and DCA 5 mM showed a significant increase in the oxidative state. A total of 10 independent experiments were performed. * < 0.05; ** p< 0.01.
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pone.0131663.g003: Assessing mitochondrial function.For each flow cytometry experiment results were analyzed in terms of Geometric Mean of Fluorescence for 20000 cells for each condition and are represented as percentage relative to the control. (A)- Quantification of mitochondrial membrane potential (MMP) potential using TMRM by flow cytometry. A significant increase in MMP was observed for CTR w/o LIF as well as for DCA 5 mM. Results are means ± SEM of 4 independent experiments. (B)- Oxygen consumption rate (OCR) was determined using the Seahorse XF24 analyzer. The three mitochondrial inhibitors were sequentially injected (after measurement points, 2, 4, and 6, as indicated) and the final concentrations of each were: oligomycin (1μM); FCCP (300mM); rotenone and antimicyn A (1μM). CTR w/o LIF presented a significant decrease in OCR in the presence of oligomycin. (C)- Determination of the respiration used to drive ATP production under basal conditions following the mathematical calculation: ATP production = Basal–Protocol Leak. This formula determines that CTR w/o LIF cells had higher mitochondrial respiration coupled to ATP production (D)- Measurement of extracellular acidification rate (ECAR) using the Seahorse XF24 analyzer. Cells were incubated in a medium without glucose and ECAR was accessed during consumption of Glucose (added at the third time point at a final concentration of 5mM) and in the presence of oligomycin (1μM added at the sixth point) and 2DG (10mM at the ninth point). At basal conditions, CTR w/o LIF had a lower glycolytic rate when compared to the control. The CTR condition displayed a significant higher glycolytic capacity. (E)- Determination of glucose breakdown to pyruvate using the mathematical calculation: first measurement after glucose injection minus the measurement prior to glucose injection. CTR w/o LIF cells had a significantly lower glycolytic capacity. F)- Results for the MTT assay are presented as percentage of formazan crystals relative to the control. Because the goal was to evaluate cell oxidative status, MTT results were normalized for total cell number. CTR w/o LIF and DCA 5 mM showed a significant increase in the oxidative state. A total of 10 independent experiments were performed. * < 0.05; ** p< 0.01.

Mentions: Given that DCA has been shown to affect cell metabolism by inhibiting PDHK, with a concomitant cellular shift from glycolysis to oxidative phosphorylation, we next assessed mitochondrial function and metabolic status in ESCs cultured with the drug. Mitochondrial membrane potential (MMP) showed that with increasing concentrations of DCA mitochondria become more hyperpolarized even in the presence of LIF, which leads to a higher MMP for cells cultured in 5 mM DCA (p<0.05), similarly to cells grown without LIF (Fig 3A). To establish if DCA-induced changes could be related to higher mitochondrial activity we determined oxygen (O2) consumption rate (OCR) and glycolysis rate using the Seahorse XF24 extracellular flux analyzer. Taking into account that only exposure to 5mM DCA in the presence of LIF causes significant changes in morphology, proliferation rates, pluripotency status and MMP, we performed the analysis using only this concentration compared to the normal and negative control (cells without LIF). Mitochondrial OCR profiling (Fig 3B) showed that, although we have differences in MMP, these changes are not significant in terms of OCR for cells cultured with 5mM DCA. However, when considering our negative control a significant decrease in OCR in the presence of oligomycin was noted (P<0.05). This inhibitor has the ability to block ATP synthase at the F0 subunit, blocking proton conductance with loss of electron transfer and O2 consumption [8]. A detailed analysis of OCR profile allowed us to determine oxygen consumed for ATP production (Fig 3C), clearly demonstrating that differentiating cells have a higher percentage of OCR coupled to ATP production (P<0.05).


Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency.

Rodrigues AS, Correia M, Gomes A, Pereira SL, Perestrelo T, Sousa MI, Ramalho-Santos J - PLoS ONE (2015)

Assessing mitochondrial function.For each flow cytometry experiment results were analyzed in terms of Geometric Mean of Fluorescence for 20000 cells for each condition and are represented as percentage relative to the control. (A)- Quantification of mitochondrial membrane potential (MMP) potential using TMRM by flow cytometry. A significant increase in MMP was observed for CTR w/o LIF as well as for DCA 5 mM. Results are means ± SEM of 4 independent experiments. (B)- Oxygen consumption rate (OCR) was determined using the Seahorse XF24 analyzer. The three mitochondrial inhibitors were sequentially injected (after measurement points, 2, 4, and 6, as indicated) and the final concentrations of each were: oligomycin (1μM); FCCP (300mM); rotenone and antimicyn A (1μM). CTR w/o LIF presented a significant decrease in OCR in the presence of oligomycin. (C)- Determination of the respiration used to drive ATP production under basal conditions following the mathematical calculation: ATP production = Basal–Protocol Leak. This formula determines that CTR w/o LIF cells had higher mitochondrial respiration coupled to ATP production (D)- Measurement of extracellular acidification rate (ECAR) using the Seahorse XF24 analyzer. Cells were incubated in a medium without glucose and ECAR was accessed during consumption of Glucose (added at the third time point at a final concentration of 5mM) and in the presence of oligomycin (1μM added at the sixth point) and 2DG (10mM at the ninth point). At basal conditions, CTR w/o LIF had a lower glycolytic rate when compared to the control. The CTR condition displayed a significant higher glycolytic capacity. (E)- Determination of glucose breakdown to pyruvate using the mathematical calculation: first measurement after glucose injection minus the measurement prior to glucose injection. CTR w/o LIF cells had a significantly lower glycolytic capacity. F)- Results for the MTT assay are presented as percentage of formazan crystals relative to the control. Because the goal was to evaluate cell oxidative status, MTT results were normalized for total cell number. CTR w/o LIF and DCA 5 mM showed a significant increase in the oxidative state. A total of 10 independent experiments were performed. * < 0.05; ** p< 0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493017&req=5

pone.0131663.g003: Assessing mitochondrial function.For each flow cytometry experiment results were analyzed in terms of Geometric Mean of Fluorescence for 20000 cells for each condition and are represented as percentage relative to the control. (A)- Quantification of mitochondrial membrane potential (MMP) potential using TMRM by flow cytometry. A significant increase in MMP was observed for CTR w/o LIF as well as for DCA 5 mM. Results are means ± SEM of 4 independent experiments. (B)- Oxygen consumption rate (OCR) was determined using the Seahorse XF24 analyzer. The three mitochondrial inhibitors were sequentially injected (after measurement points, 2, 4, and 6, as indicated) and the final concentrations of each were: oligomycin (1μM); FCCP (300mM); rotenone and antimicyn A (1μM). CTR w/o LIF presented a significant decrease in OCR in the presence of oligomycin. (C)- Determination of the respiration used to drive ATP production under basal conditions following the mathematical calculation: ATP production = Basal–Protocol Leak. This formula determines that CTR w/o LIF cells had higher mitochondrial respiration coupled to ATP production (D)- Measurement of extracellular acidification rate (ECAR) using the Seahorse XF24 analyzer. Cells were incubated in a medium without glucose and ECAR was accessed during consumption of Glucose (added at the third time point at a final concentration of 5mM) and in the presence of oligomycin (1μM added at the sixth point) and 2DG (10mM at the ninth point). At basal conditions, CTR w/o LIF had a lower glycolytic rate when compared to the control. The CTR condition displayed a significant higher glycolytic capacity. (E)- Determination of glucose breakdown to pyruvate using the mathematical calculation: first measurement after glucose injection minus the measurement prior to glucose injection. CTR w/o LIF cells had a significantly lower glycolytic capacity. F)- Results for the MTT assay are presented as percentage of formazan crystals relative to the control. Because the goal was to evaluate cell oxidative status, MTT results were normalized for total cell number. CTR w/o LIF and DCA 5 mM showed a significant increase in the oxidative state. A total of 10 independent experiments were performed. * < 0.05; ** p< 0.01.
Mentions: Given that DCA has been shown to affect cell metabolism by inhibiting PDHK, with a concomitant cellular shift from glycolysis to oxidative phosphorylation, we next assessed mitochondrial function and metabolic status in ESCs cultured with the drug. Mitochondrial membrane potential (MMP) showed that with increasing concentrations of DCA mitochondria become more hyperpolarized even in the presence of LIF, which leads to a higher MMP for cells cultured in 5 mM DCA (p<0.05), similarly to cells grown without LIF (Fig 3A). To establish if DCA-induced changes could be related to higher mitochondrial activity we determined oxygen (O2) consumption rate (OCR) and glycolysis rate using the Seahorse XF24 extracellular flux analyzer. Taking into account that only exposure to 5mM DCA in the presence of LIF causes significant changes in morphology, proliferation rates, pluripotency status and MMP, we performed the analysis using only this concentration compared to the normal and negative control (cells without LIF). Mitochondrial OCR profiling (Fig 3B) showed that, although we have differences in MMP, these changes are not significant in terms of OCR for cells cultured with 5mM DCA. However, when considering our negative control a significant decrease in OCR in the presence of oligomycin was noted (P<0.05). This inhibitor has the ability to block ATP synthase at the F0 subunit, blocking proton conductance with loss of electron transfer and O2 consumption [8]. A detailed analysis of OCR profile allowed us to determine oxygen consumed for ATP production (Fig 3C), clearly demonstrating that differentiating cells have a higher percentage of OCR coupled to ATP production (P<0.05).

Bottom Line: Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells.Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored.Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic profile, by using some of the signaling pathways found in the latter cells.

View Article: PubMed Central - PubMed

Affiliation: PhD Programme in Experimental Biology and Biomedicine, CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT

Introduction: The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency.

Methods/results: In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation.

Conclusions: DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic profile, by using some of the signaling pathways found in the latter cells.

No MeSH data available.


Related in: MedlinePlus