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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

DCA effects on key proteins potentially involved in metabolic shifts.(A)- Protein levels for PDH, Phospho-PDH and PDHK1 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin levels and they are represented as percentage relative to the control; n = 4. A significant decrease in phosphorylated PDH (inactive form) was observed for CTR w/o LIF. (B)- Representative Western blot demonstrating the decrease in protein levels. (C)- qRT-PCR analysis for PDH, PDHK1 and PDHK2. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 4 independent experiments. A significant negative impact was observed for all genes in the experimental condition CTR w/o LIF. (D)- qRT-PCR analysis for PKM1/2 and GAPDH. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 3 independent experiments. Once more significant differences were observed for CTR w/o LIF. (E)- Protein levels for Hexokinase I and II, GAPDH and PKM1/2 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. PKM1/2 showed a significant decrease for all experimental conditions while for hexokinase II only CTR w/o LIF was significantly affected. (F)- Representative Western blot. (G)- Protein levels for Hif-1α and p53 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. Significant negative differences were observed for all experimental conditions. (H)- Representative blot. (I)- qRT-PCR analysis of Hif-1α, Hif-2α and p53. Results are presented as fold changes for 3 independent experiments, normalized to the reference house keeping gene βeta-Actin. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.
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pone.0131663.g005: DCA effects on key proteins potentially involved in metabolic shifts.(A)- Protein levels for PDH, Phospho-PDH and PDHK1 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin levels and they are represented as percentage relative to the control; n = 4. A significant decrease in phosphorylated PDH (inactive form) was observed for CTR w/o LIF. (B)- Representative Western blot demonstrating the decrease in protein levels. (C)- qRT-PCR analysis for PDH, PDHK1 and PDHK2. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 4 independent experiments. A significant negative impact was observed for all genes in the experimental condition CTR w/o LIF. (D)- qRT-PCR analysis for PKM1/2 and GAPDH. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 3 independent experiments. Once more significant differences were observed for CTR w/o LIF. (E)- Protein levels for Hexokinase I and II, GAPDH and PKM1/2 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. PKM1/2 showed a significant decrease for all experimental conditions while for hexokinase II only CTR w/o LIF was significantly affected. (F)- Representative Western blot. (G)- Protein levels for Hif-1α and p53 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. Significant negative differences were observed for all experimental conditions. (H)- Representative blot. (I)- qRT-PCR analysis of Hif-1α, Hif-2α and p53. Results are presented as fold changes for 3 independent experiments, normalized to the reference house keeping gene βeta-Actin. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.

Mentions: In a previous paper [8] we noted that pluripotency seemed to be accompanied by high levels of the phosphorylated form of PDH, so we wondered if DCA, which inhibits PDHK, was altering the PDH cycle, enhancing differentiation by activating PDH and shifting cells towards oxidative phosphorylation. Both the phosphorylated and non-phosphorylated forms of PDH, plus PDHK1, where analyzed by Western Blot and we observed a decrease in phosphorylated PDH (inactive form) and total PDH protein level, following DCA treatment and in the differentiation control (Fig 5A). Moreover mRNA levels mirrored protein results, with a significant negative impact for cells cultured in the absence of LIF (Fig 5C) for PDH (P<0.05); PDHK1 (P<0.01) and PDHK2 (P<0.01). This set of results points towards a more active PDH for cells grown without LIF and in the presence of DCA. Given that DCA is affecting pluripotency and the results suggests a shift in PDH regulation we assessed the protein levels for other key players in metabolic pathways.


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 key proteins potentially involved in metabolic shifts.(A)- Protein levels for PDH, Phospho-PDH and PDHK1 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin levels and they are represented as percentage relative to the control; n = 4. A significant decrease in phosphorylated PDH (inactive form) was observed for CTR w/o LIF. (B)- Representative Western blot demonstrating the decrease in protein levels. (C)- qRT-PCR analysis for PDH, PDHK1 and PDHK2. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 4 independent experiments. A significant negative impact was observed for all genes in the experimental condition CTR w/o LIF. (D)- qRT-PCR analysis for PKM1/2 and GAPDH. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 3 independent experiments. Once more significant differences were observed for CTR w/o LIF. (E)- Protein levels for Hexokinase I and II, GAPDH and PKM1/2 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. PKM1/2 showed a significant decrease for all experimental conditions while for hexokinase II only CTR w/o LIF was significantly affected. (F)- Representative Western blot. (G)- Protein levels for Hif-1α and p53 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. Significant negative differences were observed for all experimental conditions. (H)- Representative blot. (I)- qRT-PCR analysis of Hif-1α, Hif-2α and p53. Results are presented as fold changes for 3 independent experiments, normalized to the reference house keeping gene βeta-Actin. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131663.g005: DCA effects on key proteins potentially involved in metabolic shifts.(A)- Protein levels for PDH, Phospho-PDH and PDHK1 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin levels and they are represented as percentage relative to the control; n = 4. A significant decrease in phosphorylated PDH (inactive form) was observed for CTR w/o LIF. (B)- Representative Western blot demonstrating the decrease in protein levels. (C)- qRT-PCR analysis for PDH, PDHK1 and PDHK2. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 4 independent experiments. A significant negative impact was observed for all genes in the experimental condition CTR w/o LIF. (D)- qRT-PCR analysis for PKM1/2 and GAPDH. Results are presented as fold changes and normalized to the reference house keeping gene βeta-Actin. Results represent 3 independent experiments. Once more significant differences were observed for CTR w/o LIF. (E)- Protein levels for Hexokinase I and II, GAPDH and PKM1/2 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. PKM1/2 showed a significant decrease for all experimental conditions while for hexokinase II only CTR w/o LIF was significantly affected. (F)- Representative Western blot. (G)- Protein levels for Hif-1α and p53 were determined by western Blot and quantified by densiometric evaluation. Results were normalized to βeta-Actin protein levels and they are represented as percentage relative to the control; n = 4. Significant negative differences were observed for all experimental conditions. (H)- Representative blot. (I)- qRT-PCR analysis of Hif-1α, Hif-2α and p53. Results are presented as fold changes for 3 independent experiments, normalized to the reference house keeping gene βeta-Actin. * and # < 0.05; ** and ## p< 0.01; *** and ### p< 0.001 relative to the respective controls.
Mentions: In a previous paper [8] we noted that pluripotency seemed to be accompanied by high levels of the phosphorylated form of PDH, so we wondered if DCA, which inhibits PDHK, was altering the PDH cycle, enhancing differentiation by activating PDH and shifting cells towards oxidative phosphorylation. Both the phosphorylated and non-phosphorylated forms of PDH, plus PDHK1, where analyzed by Western Blot and we observed a decrease in phosphorylated PDH (inactive form) and total PDH protein level, following DCA treatment and in the differentiation control (Fig 5A). Moreover mRNA levels mirrored protein results, with a significant negative impact for cells cultured in the absence of LIF (Fig 5C) for PDH (P<0.05); PDHK1 (P<0.01) and PDHK2 (P<0.01). This set of results points towards a more active PDH for cells grown without LIF and in the presence of DCA. Given that DCA is affecting pluripotency and the results suggests a shift in PDH regulation we assessed the protein levels for other key players in metabolic pathways.

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