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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

Effect of DCA on morphology, viability, cell number and proliferation.(A) Phase microscopy photographs of ESC colonies in all conditions with a magnification of 200x. Control ESCs (CTR) present tightly packed round colonies with well-defined borders. ESCs cultured in the absence of LIF (CTR w/o LIF) present colonies that are differentiating. ESCs incubated in the presence of LIF plus 3 mM or 5 mM DCA, show high differentiation levels, similar to the differentiation control. (B) Percentage of viable versus dead cells. Cells stained green were counted as live, while PI positive cells were counted as dead. 100 cells were counted per condition for a total of n = 8 independent experiments. Results are expressed as means and error bars represent SEM for 30 experiments. (C) Total number of cells counted using a Neubauer chamber prior to collection for ATP and flow cytometry experiments. Results are expressed as means and error bars represent SEM. (D)- Cells in all experimental conditions were stained for PCNA in the cells nucleus and protein levels were assessed by flow cytometry. 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. 4 independent experiments were performed. * < 0.05; ** p< 0.01.
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pone.0131663.g001: Effect of DCA on morphology, viability, cell number and proliferation.(A) Phase microscopy photographs of ESC colonies in all conditions with a magnification of 200x. Control ESCs (CTR) present tightly packed round colonies with well-defined borders. ESCs cultured in the absence of LIF (CTR w/o LIF) present colonies that are differentiating. ESCs incubated in the presence of LIF plus 3 mM or 5 mM DCA, show high differentiation levels, similar to the differentiation control. (B) Percentage of viable versus dead cells. Cells stained green were counted as live, while PI positive cells were counted as dead. 100 cells were counted per condition for a total of n = 8 independent experiments. Results are expressed as means and error bars represent SEM for 30 experiments. (C) Total number of cells counted using a Neubauer chamber prior to collection for ATP and flow cytometry experiments. Results are expressed as means and error bars represent SEM. (D)- Cells in all experimental conditions were stained for PCNA in the cells nucleus and protein levels were assessed by flow cytometry. 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. 4 independent experiments were performed. * < 0.05; ** p< 0.01.

Mentions: Although DCA has been used to show that a glycolytic turnover is necessary to induce pluripotency [6], there are no data regarding its effect on pluripotent stem cells. Fig 1A shows that control (pluripotent) cells are arranged in tightly packed round colonies with well-defined borders. On the other hand, cells growing without LIF (as a differentiation control) present spread out colonies that are not well defined, characteristic of differentiated cells. When exposed to 5mM DCA, and to a lesser extent to 3mM DCA, cells show differentiation levels, similar to the differentiation control, although they were cultured with the pluripotency factor LIF. Next, we assessed cellular viability, and a significant decrease in the percentage of live cells for cells grown without LIF and exposed to the higher concentration of DCA in the presence of LIF was observed (P<0.01 and P<0.05, respectively) (Fig 1B). In parallel with viability results, there is a significant decrease in total cell numbers for all experimental conditions when compared to pluripotent control cells (P<0.01) (Fig 1C). To determine if the differences in cell number could be also due to changes in proliferation, we stained cells for PCNA, which is highly expressed in rapidly proliferating cells [30] (Fig 1D). Results show a significant increase in PCNA levels in cells cultured without LIF (P<0.01) and cells incubated with 5mM DCA plus LIF (P<0.01), suggesting that they are both more proliferative. Overall our experimental conditions are affecting cellular viability and number, but cells maintain proliferation capability.


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)

Effect of DCA on morphology, viability, cell number and proliferation.(A) Phase microscopy photographs of ESC colonies in all conditions with a magnification of 200x. Control ESCs (CTR) present tightly packed round colonies with well-defined borders. ESCs cultured in the absence of LIF (CTR w/o LIF) present colonies that are differentiating. ESCs incubated in the presence of LIF plus 3 mM or 5 mM DCA, show high differentiation levels, similar to the differentiation control. (B) Percentage of viable versus dead cells. Cells stained green were counted as live, while PI positive cells were counted as dead. 100 cells were counted per condition for a total of n = 8 independent experiments. Results are expressed as means and error bars represent SEM for 30 experiments. (C) Total number of cells counted using a Neubauer chamber prior to collection for ATP and flow cytometry experiments. Results are expressed as means and error bars represent SEM. (D)- Cells in all experimental conditions were stained for PCNA in the cells nucleus and protein levels were assessed by flow cytometry. 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. 4 independent experiments were performed. * < 0.05; ** p< 0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493017&req=5

pone.0131663.g001: Effect of DCA on morphology, viability, cell number and proliferation.(A) Phase microscopy photographs of ESC colonies in all conditions with a magnification of 200x. Control ESCs (CTR) present tightly packed round colonies with well-defined borders. ESCs cultured in the absence of LIF (CTR w/o LIF) present colonies that are differentiating. ESCs incubated in the presence of LIF plus 3 mM or 5 mM DCA, show high differentiation levels, similar to the differentiation control. (B) Percentage of viable versus dead cells. Cells stained green were counted as live, while PI positive cells were counted as dead. 100 cells were counted per condition for a total of n = 8 independent experiments. Results are expressed as means and error bars represent SEM for 30 experiments. (C) Total number of cells counted using a Neubauer chamber prior to collection for ATP and flow cytometry experiments. Results are expressed as means and error bars represent SEM. (D)- Cells in all experimental conditions were stained for PCNA in the cells nucleus and protein levels were assessed by flow cytometry. 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. 4 independent experiments were performed. * < 0.05; ** p< 0.01.
Mentions: Although DCA has been used to show that a glycolytic turnover is necessary to induce pluripotency [6], there are no data regarding its effect on pluripotent stem cells. Fig 1A shows that control (pluripotent) cells are arranged in tightly packed round colonies with well-defined borders. On the other hand, cells growing without LIF (as a differentiation control) present spread out colonies that are not well defined, characteristic of differentiated cells. When exposed to 5mM DCA, and to a lesser extent to 3mM DCA, cells show differentiation levels, similar to the differentiation control, although they were cultured with the pluripotency factor LIF. Next, we assessed cellular viability, and a significant decrease in the percentage of live cells for cells grown without LIF and exposed to the higher concentration of DCA in the presence of LIF was observed (P<0.01 and P<0.05, respectively) (Fig 1B). In parallel with viability results, there is a significant decrease in total cell numbers for all experimental conditions when compared to pluripotent control cells (P<0.01) (Fig 1C). To determine if the differences in cell number could be also due to changes in proliferation, we stained cells for PCNA, which is highly expressed in rapidly proliferating cells [30] (Fig 1D). Results show a significant increase in PCNA levels in cells cultured without LIF (P<0.01) and cells incubated with 5mM DCA plus LIF (P<0.01), suggesting that they are both more proliferative. Overall our experimental conditions are affecting cellular viability and number, but cells maintain proliferation capability.

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