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Metabolic plasticity maintains proliferation in pyruvate dehydrogenase deficient cells.

Rajagopalan KN, Egnatchik RA, Calvaruso MA, Wasti AT, Padanad MS, Boroughs LK, Ko B, Hensley CT, Acar M, Hu Z, Jiang L, Pascual JM, Scaglioni PP, DeBerardinis RJ - Cancer Metab (2015)

Bottom Line: However, evidence supports the benefits of constraining maximal PDH activity under certain contexts, including hypoxia and oncogene-induced cell growth.PDH suppression also shifted the source of lipogenic acetyl-CoA from glucose to glutamine, and this compensatory pathway required a net reductive isocitrate dehydrogenase (IDH) flux to produce a source of glutamine-derived acetyl-CoA for fatty acids.We also identify the compensatory mechanisms that are activated under PDH deficiency, namely scavenging of extracellular lipids and lipogenic acetyl-CoA production from reductive glutamine metabolism through IDH1.

View Article: PubMed Central - PubMed

Affiliation: Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-8502 USA.

ABSTRACT

Background: Pyruvate dehydrogenase (PDH) occupies a central node of intermediary metabolism, converting pyruvate to acetyl-CoA, thus committing carbon derived from glucose to an aerobic fate rather than an anaerobic one. Rapidly proliferating tissues, including human tumors, use PDH to generate energy and macromolecular precursors. However, evidence supports the benefits of constraining maximal PDH activity under certain contexts, including hypoxia and oncogene-induced cell growth. Although PDH is one of the most widely studied enzyme complexes in mammals, its requirement for cell growth is unknown. In this study, we directly addressed whether PDH is required for mammalian cells to proliferate.

Results: We genetically suppressed expression of the PDHA1 gene encoding an essential subunit of the PDH complex and characterized the effects on intermediary metabolism and cell proliferation using a combination of stable isotope tracing and growth assays. Surprisingly, rapidly dividing cells tolerated loss of PDH activity without major effects on proliferative rates in complete medium. PDH suppression increased reliance on extracellular lipids, and in some cell lines, reducing lipid availability uncovered a modest growth defect that could be completely reversed by providing exogenous-free fatty acids. PDH suppression also shifted the source of lipogenic acetyl-CoA from glucose to glutamine, and this compensatory pathway required a net reductive isocitrate dehydrogenase (IDH) flux to produce a source of glutamine-derived acetyl-CoA for fatty acids. By deleting the cytosolic isoform of IDH (IDH1), the enhanced contribution of glutamine to the lipogenic acetyl-CoA pool during PDHA1 suppression was eliminated, and growth was modestly suppressed.

Conclusions: Although PDH suppression substantially alters central carbon metabolism, the data indicate that rapid cell proliferation occurs independently of PDH activity. Our findings reveal that this central enzyme is essentially dispensable for growth and proliferation of both primary cells and established cell lines. We also identify the compensatory mechanisms that are activated under PDH deficiency, namely scavenging of extracellular lipids and lipogenic acetyl-CoA production from reductive glutamine metabolism through IDH1.

No MeSH data available.


Related in: MedlinePlus

PDH suppression increases dependence on extracellular lipids for growth. a Uptake of BODIPY-labeled fatty acids imaged by fluorescence microscopy after 24 h of culture. b Quantification of BODIPY fluorescence intensity. c Doubling time calculated from 12-day cultures under normal (left) and delipidated (right) conditions. d Cell count after 4 days of culture in delipidated serum, with or without a 50 μM each of palmitate and oleate. Data are the average and SD of biological triplicates. *P < 0.05; **P < 0.005. Abbreviations: FFAs, free fatty acids
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Fig3: PDH suppression increases dependence on extracellular lipids for growth. a Uptake of BODIPY-labeled fatty acids imaged by fluorescence microscopy after 24 h of culture. b Quantification of BODIPY fluorescence intensity. c Doubling time calculated from 12-day cultures under normal (left) and delipidated (right) conditions. d Cell count after 4 days of culture in delipidated serum, with or without a 50 μM each of palmitate and oleate. Data are the average and SD of biological triplicates. *P < 0.05; **P < 0.005. Abbreviations: FFAs, free fatty acids

Mentions: Despite its effects on glucose-dependent TCA cycling and lipid synthesis, PDHA1 silencing did not significantly alter the rate of H460 cell proliferation (Additional file 1: Figure S4a). Doubling times calculated from the growth curve were, at most, only marginally increased with one of the hairpins (Fig. 3c, left). Surprisingly, even deletion of PDHA1 exon 8 only marginally decreased the growth rate of MEFs (Additional file 1: Figure S3g), although PDHA1-deleted cells were smaller than their wild-type controls (Additional file 1: Figure S3f).Fig. 3


Metabolic plasticity maintains proliferation in pyruvate dehydrogenase deficient cells.

Rajagopalan KN, Egnatchik RA, Calvaruso MA, Wasti AT, Padanad MS, Boroughs LK, Ko B, Hensley CT, Acar M, Hu Z, Jiang L, Pascual JM, Scaglioni PP, DeBerardinis RJ - Cancer Metab (2015)

PDH suppression increases dependence on extracellular lipids for growth. a Uptake of BODIPY-labeled fatty acids imaged by fluorescence microscopy after 24 h of culture. b Quantification of BODIPY fluorescence intensity. c Doubling time calculated from 12-day cultures under normal (left) and delipidated (right) conditions. d Cell count after 4 days of culture in delipidated serum, with or without a 50 μM each of palmitate and oleate. Data are the average and SD of biological triplicates. *P < 0.05; **P < 0.005. Abbreviations: FFAs, free fatty acids
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487196&req=5

Fig3: PDH suppression increases dependence on extracellular lipids for growth. a Uptake of BODIPY-labeled fatty acids imaged by fluorescence microscopy after 24 h of culture. b Quantification of BODIPY fluorescence intensity. c Doubling time calculated from 12-day cultures under normal (left) and delipidated (right) conditions. d Cell count after 4 days of culture in delipidated serum, with or without a 50 μM each of palmitate and oleate. Data are the average and SD of biological triplicates. *P < 0.05; **P < 0.005. Abbreviations: FFAs, free fatty acids
Mentions: Despite its effects on glucose-dependent TCA cycling and lipid synthesis, PDHA1 silencing did not significantly alter the rate of H460 cell proliferation (Additional file 1: Figure S4a). Doubling times calculated from the growth curve were, at most, only marginally increased with one of the hairpins (Fig. 3c, left). Surprisingly, even deletion of PDHA1 exon 8 only marginally decreased the growth rate of MEFs (Additional file 1: Figure S3g), although PDHA1-deleted cells were smaller than their wild-type controls (Additional file 1: Figure S3f).Fig. 3

Bottom Line: However, evidence supports the benefits of constraining maximal PDH activity under certain contexts, including hypoxia and oncogene-induced cell growth.PDH suppression also shifted the source of lipogenic acetyl-CoA from glucose to glutamine, and this compensatory pathway required a net reductive isocitrate dehydrogenase (IDH) flux to produce a source of glutamine-derived acetyl-CoA for fatty acids.We also identify the compensatory mechanisms that are activated under PDH deficiency, namely scavenging of extracellular lipids and lipogenic acetyl-CoA production from reductive glutamine metabolism through IDH1.

View Article: PubMed Central - PubMed

Affiliation: Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-8502 USA.

ABSTRACT

Background: Pyruvate dehydrogenase (PDH) occupies a central node of intermediary metabolism, converting pyruvate to acetyl-CoA, thus committing carbon derived from glucose to an aerobic fate rather than an anaerobic one. Rapidly proliferating tissues, including human tumors, use PDH to generate energy and macromolecular precursors. However, evidence supports the benefits of constraining maximal PDH activity under certain contexts, including hypoxia and oncogene-induced cell growth. Although PDH is one of the most widely studied enzyme complexes in mammals, its requirement for cell growth is unknown. In this study, we directly addressed whether PDH is required for mammalian cells to proliferate.

Results: We genetically suppressed expression of the PDHA1 gene encoding an essential subunit of the PDH complex and characterized the effects on intermediary metabolism and cell proliferation using a combination of stable isotope tracing and growth assays. Surprisingly, rapidly dividing cells tolerated loss of PDH activity without major effects on proliferative rates in complete medium. PDH suppression increased reliance on extracellular lipids, and in some cell lines, reducing lipid availability uncovered a modest growth defect that could be completely reversed by providing exogenous-free fatty acids. PDH suppression also shifted the source of lipogenic acetyl-CoA from glucose to glutamine, and this compensatory pathway required a net reductive isocitrate dehydrogenase (IDH) flux to produce a source of glutamine-derived acetyl-CoA for fatty acids. By deleting the cytosolic isoform of IDH (IDH1), the enhanced contribution of glutamine to the lipogenic acetyl-CoA pool during PDHA1 suppression was eliminated, and growth was modestly suppressed.

Conclusions: Although PDH suppression substantially alters central carbon metabolism, the data indicate that rapid cell proliferation occurs independently of PDH activity. Our findings reveal that this central enzyme is essentially dispensable for growth and proliferation of both primary cells and established cell lines. We also identify the compensatory mechanisms that are activated under PDH deficiency, namely scavenging of extracellular lipids and lipogenic acetyl-CoA production from reductive glutamine metabolism through IDH1.

No MeSH data available.


Related in: MedlinePlus