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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

Suppression of PDH activity reduces transfer of glucose carbon into citrate and lipids. a Schematic representing incorporation of 13C derived from glucose into metabolites of glycolysis and the TCA cycle. White and black circles are 12C and 13C, respectively. The solid line represents the first turn of the TCA cycle, and the dotted line represents the second turn. b Fractional abundance of citrate m + 2 and m + 4 during culture of H460 cells with [U-13C]glucose. c Fractional abundance of palmitate isotopologues after 24 h of culture in [U-13C]glucose. The inset shows the percentage of lipogenic acetyl-CoA derived from glucose carbon. d Labeling of lipid species from [U-14C]glucose. Data are the average and SD of biological triplicates, except for the inset in (c), where error bars represent 95 % CI. *P < 0.05; **P < 0.005. Abbreviations: CE cholesteryl esters, DAG diacylglycerol, FFA free fatty acids, Lac lactate, OAA oxaloacetate, PL phospholipids, Pyr pyruvate, Succ succinate, TAG triacylglycerol
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Fig2: Suppression of PDH activity reduces transfer of glucose carbon into citrate and lipids. a Schematic representing incorporation of 13C derived from glucose into metabolites of glycolysis and the TCA cycle. White and black circles are 12C and 13C, respectively. The solid line represents the first turn of the TCA cycle, and the dotted line represents the second turn. b Fractional abundance of citrate m + 2 and m + 4 during culture of H460 cells with [U-13C]glucose. c Fractional abundance of palmitate isotopologues after 24 h of culture in [U-13C]glucose. The inset shows the percentage of lipogenic acetyl-CoA derived from glucose carbon. d Labeling of lipid species from [U-14C]glucose. Data are the average and SD of biological triplicates, except for the inset in (c), where error bars represent 95 % CI. *P < 0.05; **P < 0.005. Abbreviations: CE cholesteryl esters, DAG diacylglycerol, FFA free fatty acids, Lac lactate, OAA oxaloacetate, PL phospholipids, Pyr pyruvate, Succ succinate, TAG triacylglycerol

Mentions: Cells were then cultured with [U-13C]glucose to determine the effects of PDHA1 silencing on intracellular glucose metabolism. In this labeling scheme, M + 2 and M + 4 citrate arise primarily from the contribution of uniformly-labeled acetyl-CoA to the TCA cycle in one or two turns (Fig. 2a). Kinetic labeling with [U-13C]glucose over 4 h revealed suppression of both the M + 2 and M + 4 isotopologues (Fig. 2b), as expected. Labeling for 24 h revealed no difference in 13C enrichment for lactate or alanine (Additional file 1: Figure S2a, j), but significant decreases in TCA cycle intermediates and non-essential amino acids arising from TCA cycle intermediates in PDHA1-silenced cells (Additional file 1: Figure S2b–g). Labeling in serine and glycine was modestly increased when PDHA1 was silenced (Additional file 1: Figure S2h, i).Fig. 2


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)

Suppression of PDH activity reduces transfer of glucose carbon into citrate and lipids. a Schematic representing incorporation of 13C derived from glucose into metabolites of glycolysis and the TCA cycle. White and black circles are 12C and 13C, respectively. The solid line represents the first turn of the TCA cycle, and the dotted line represents the second turn. b Fractional abundance of citrate m + 2 and m + 4 during culture of H460 cells with [U-13C]glucose. c Fractional abundance of palmitate isotopologues after 24 h of culture in [U-13C]glucose. The inset shows the percentage of lipogenic acetyl-CoA derived from glucose carbon. d Labeling of lipid species from [U-14C]glucose. Data are the average and SD of biological triplicates, except for the inset in (c), where error bars represent 95 % CI. *P < 0.05; **P < 0.005. Abbreviations: CE cholesteryl esters, DAG diacylglycerol, FFA free fatty acids, Lac lactate, OAA oxaloacetate, PL phospholipids, Pyr pyruvate, Succ succinate, TAG triacylglycerol
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Suppression of PDH activity reduces transfer of glucose carbon into citrate and lipids. a Schematic representing incorporation of 13C derived from glucose into metabolites of glycolysis and the TCA cycle. White and black circles are 12C and 13C, respectively. The solid line represents the first turn of the TCA cycle, and the dotted line represents the second turn. b Fractional abundance of citrate m + 2 and m + 4 during culture of H460 cells with [U-13C]glucose. c Fractional abundance of palmitate isotopologues after 24 h of culture in [U-13C]glucose. The inset shows the percentage of lipogenic acetyl-CoA derived from glucose carbon. d Labeling of lipid species from [U-14C]glucose. Data are the average and SD of biological triplicates, except for the inset in (c), where error bars represent 95 % CI. *P < 0.05; **P < 0.005. Abbreviations: CE cholesteryl esters, DAG diacylglycerol, FFA free fatty acids, Lac lactate, OAA oxaloacetate, PL phospholipids, Pyr pyruvate, Succ succinate, TAG triacylglycerol
Mentions: Cells were then cultured with [U-13C]glucose to determine the effects of PDHA1 silencing on intracellular glucose metabolism. In this labeling scheme, M + 2 and M + 4 citrate arise primarily from the contribution of uniformly-labeled acetyl-CoA to the TCA cycle in one or two turns (Fig. 2a). Kinetic labeling with [U-13C]glucose over 4 h revealed suppression of both the M + 2 and M + 4 isotopologues (Fig. 2b), as expected. Labeling for 24 h revealed no difference in 13C enrichment for lactate or alanine (Additional file 1: Figure S2a, j), but significant decreases in TCA cycle intermediates and non-essential amino acids arising from TCA cycle intermediates in PDHA1-silenced cells (Additional file 1: Figure S2b–g). Labeling in serine and glycine was modestly increased when PDHA1 was silenced (Additional file 1: Figure S2h, i).Fig. 2

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