Limits...
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

DCA effects on Pluripotency.(A)- Quantification for the alkaline phosphatase assay: colonies stained red were counted as (positive) pluripotent colonies whereas colonies without staining were counted as negative. A total of 10 independent experiments were analyzed for all experimental conditions resulting in a significant negative impact for CTR w/o LIF and DCA 5 mM. (B)- Quantification of pluripotency markers Oct4 and Nanog expression levels using high-resolution ELISA. All experimental conditions present a significant decrease when compared to the control for both pluripotency factors. (C)- qRT-PCR analysis for the Oct4 and Nanog mRNA gene levels results are represented as fold changes normalized to the Control after normalization for endogenous βeta-actin. These results mirror the protein levels regarding CTR w/o LIF and DCA 5 mM. Four independent experiments were performed. All the results are expressed as means and error bars represents SEM. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493017&req=5

pone.0131663.g002: DCA effects on Pluripotency.(A)- Quantification for the alkaline phosphatase assay: colonies stained red were counted as (positive) pluripotent colonies whereas colonies without staining were counted as negative. A total of 10 independent experiments were analyzed for all experimental conditions resulting in a significant negative impact for CTR w/o LIF and DCA 5 mM. (B)- Quantification of pluripotency markers Oct4 and Nanog expression levels using high-resolution ELISA. All experimental conditions present a significant decrease when compared to the control for both pluripotency factors. (C)- qRT-PCR analysis for the Oct4 and Nanog mRNA gene levels results are represented as fold changes normalized to the Control after normalization for endogenous βeta-actin. These results mirror the protein levels regarding CTR w/o LIF and DCA 5 mM. Four independent experiments were performed. All the results are expressed as means and error bars represents SEM. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.

Mentions: We next investigated whether DCA affected pluripotency even when ESCs are maintained under pluripotency conditions (with LIF). The alkaline phosphatase (AP) assay (Fig 2A) revealed that cells without LIF have a significant lower number of positive colonies when compared to the control (P<0.01) and the same was true for cells cultured with LIF and 5 mM DCA (P<0.05). When protein levels for the pluripotency markers Oct4 and Nanog were quantified (Fig 2B), a significant decrease was observed for all conditions when compared to the control for both factors. Cells without LIF and those exposed to 5mM DCA had the lowest protein levels (P<0.001) and the same tendency was observed for mRNA levels (Fig 2C). Concomitantly, mRNA levels dropped significantly in absence of LIF (P<0.001) for both genes while in the presence of 5mM DCA plus LIF (Fig 2C) the decrease in Oct4 was more pronounced when compared to Nanog levels, and once more the results for 5 mM DCA paralleled the negative (differentiation) control.


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)

DCA effects on Pluripotency.(A)- Quantification for the alkaline phosphatase assay: colonies stained red were counted as (positive) pluripotent colonies whereas colonies without staining were counted as negative. A total of 10 independent experiments were analyzed for all experimental conditions resulting in a significant negative impact for CTR w/o LIF and DCA 5 mM. (B)- Quantification of pluripotency markers Oct4 and Nanog expression levels using high-resolution ELISA. All experimental conditions present a significant decrease when compared to the control for both pluripotency factors. (C)- qRT-PCR analysis for the Oct4 and Nanog mRNA gene levels results are represented as fold changes normalized to the Control after normalization for endogenous βeta-actin. These results mirror the protein levels regarding CTR w/o LIF and DCA 5 mM. Four independent experiments were performed. All the results are expressed as means and error bars represents SEM. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493017&req=5

pone.0131663.g002: DCA effects on Pluripotency.(A)- Quantification for the alkaline phosphatase assay: colonies stained red were counted as (positive) pluripotent colonies whereas colonies without staining were counted as negative. A total of 10 independent experiments were analyzed for all experimental conditions resulting in a significant negative impact for CTR w/o LIF and DCA 5 mM. (B)- Quantification of pluripotency markers Oct4 and Nanog expression levels using high-resolution ELISA. All experimental conditions present a significant decrease when compared to the control for both pluripotency factors. (C)- qRT-PCR analysis for the Oct4 and Nanog mRNA gene levels results are represented as fold changes normalized to the Control after normalization for endogenous βeta-actin. These results mirror the protein levels regarding CTR w/o LIF and DCA 5 mM. Four independent experiments were performed. All the results are expressed as means and error bars represents SEM. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.
Mentions: We next investigated whether DCA affected pluripotency even when ESCs are maintained under pluripotency conditions (with LIF). The alkaline phosphatase (AP) assay (Fig 2A) revealed that cells without LIF have a significant lower number of positive colonies when compared to the control (P<0.01) and the same was true for cells cultured with LIF and 5 mM DCA (P<0.05). When protein levels for the pluripotency markers Oct4 and Nanog were quantified (Fig 2B), a significant decrease was observed for all conditions when compared to the control for both factors. Cells without LIF and those exposed to 5mM DCA had the lowest protein levels (P<0.001) and the same tendency was observed for mRNA levels (Fig 2C). Concomitantly, mRNA levels dropped significantly in absence of LIF (P<0.001) for both genes while in the presence of 5mM DCA plus LIF (Fig 2C) the decrease in Oct4 was more pronounced when compared to Nanog levels, and once more the results for 5 mM DCA paralleled the negative (differentiation) control.

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