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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

Metabolism-related gene expression in mESC following DCA treatment.(A) Fold changes were calculated for the various genes using the -∆∆Ct method relative to the CTR condition. The values represent means ± SEM of four independent experiments. (B) Heat map of average gene expression represented as log10 of Ct values. An increase in gene expression is depicted in red, whereas a decrease in gene expression is represented by the green color. No differences in expression are depicted in black. Clustering was performed using the CFX manager software by Bio-Rad. * < 0.05; ** p< 0.01; ***p< 0.001.
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pone.0131663.g004: Metabolism-related gene expression in mESC following DCA treatment.(A) Fold changes were calculated for the various genes using the -∆∆Ct method relative to the CTR condition. The values represent means ± SEM of four independent experiments. (B) Heat map of average gene expression represented as log10 of Ct values. An increase in gene expression is depicted in red, whereas a decrease in gene expression is represented by the green color. No differences in expression are depicted in black. Clustering was performed using the CFX manager software by Bio-Rad. * < 0.05; ** p< 0.01; ***p< 0.001.

Mentions: Given that both metabolic changes and a shift towards differentiation were detected when culturing ESCs in the presence of DCA we next tried to identify possible candidate genes that could be involved in this process by designing a custom array. Although focusing on metabolic enzymes, we also decided to include other known players in metabolic shifts such as Hif-1α, c-Myc, p53 and stat3 (Fig 4A). Overall, differentiation decreases gene expression for almost all genes monitored. Interestingly, it is possible to distinguish between differentiation (absence of LIF) and DCA effects in the presence of LIF. DCA seems to try to counteract normal differentiation effects for some genes, notably Enolase 1 (Eno1), Glucuronidase beta (Gusb), c-Myc, Pdhk1, Stat3 and p53. On the other hand, when considering Aldolase a (Aldo a) (P<0.001); Hif-1α; HKII (P<0.001); Ldha (P<0.05 and P<0.001); Pdha1 (P<0.01 and P<0.001) and PKM (P<0.05) DCA seems to have a similar negative impact on these genes when compared to naturally differentiating cells cultured without LIF. Fig 4B supports the idea that DCA is causing a shift towards differentiation given that the clustergram analysis cluster cells exposed to DCA closer to ESCs cultured without LIF.


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)

Metabolism-related gene expression in mESC following DCA treatment.(A) Fold changes were calculated for the various genes using the -∆∆Ct method relative to the CTR condition. The values represent means ± SEM of four independent experiments. (B) Heat map of average gene expression represented as log10 of Ct values. An increase in gene expression is depicted in red, whereas a decrease in gene expression is represented by the green color. No differences in expression are depicted in black. Clustering was performed using the CFX manager software by Bio-Rad. * < 0.05; ** p< 0.01; ***p< 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131663.g004: Metabolism-related gene expression in mESC following DCA treatment.(A) Fold changes were calculated for the various genes using the -∆∆Ct method relative to the CTR condition. The values represent means ± SEM of four independent experiments. (B) Heat map of average gene expression represented as log10 of Ct values. An increase in gene expression is depicted in red, whereas a decrease in gene expression is represented by the green color. No differences in expression are depicted in black. Clustering was performed using the CFX manager software by Bio-Rad. * < 0.05; ** p< 0.01; ***p< 0.001.
Mentions: Given that both metabolic changes and a shift towards differentiation were detected when culturing ESCs in the presence of DCA we next tried to identify possible candidate genes that could be involved in this process by designing a custom array. Although focusing on metabolic enzymes, we also decided to include other known players in metabolic shifts such as Hif-1α, c-Myc, p53 and stat3 (Fig 4A). Overall, differentiation decreases gene expression for almost all genes monitored. Interestingly, it is possible to distinguish between differentiation (absence of LIF) and DCA effects in the presence of LIF. DCA seems to try to counteract normal differentiation effects for some genes, notably Enolase 1 (Eno1), Glucuronidase beta (Gusb), c-Myc, Pdhk1, Stat3 and p53. On the other hand, when considering Aldolase a (Aldo a) (P<0.001); Hif-1α; HKII (P<0.001); Ldha (P<0.05 and P<0.001); Pdha1 (P<0.01 and P<0.001) and PKM (P<0.05) DCA seems to have a similar negative impact on these genes when compared to naturally differentiating cells cultured without LIF. Fig 4B supports the idea that DCA is causing a shift towards differentiation given that the clustergram analysis cluster cells exposed to DCA closer to ESCs cultured without LIF.

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