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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

IDH1 loss suppresses contribution of glutamine carbon to lipogenesis under PDH suppression. a Western blot for PDH E1α and IDH1. Cyclophilin B was used as a loading control. b Percentage labeling in mass isotopologues of palmitate after 24 h of culture with [U-13C]glutamine. Inset displays labeling in lipogenic acetyl-CoA derived from glutamine carbon. c Cell count after 4 days of culture in normal serum. Data are the average of biological triplicates with error bars representing SD.
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Fig6: IDH1 loss suppresses contribution of glutamine carbon to lipogenesis under PDH suppression. a Western blot for PDH E1α and IDH1. Cyclophilin B was used as a loading control. b Percentage labeling in mass isotopologues of palmitate after 24 h of culture with [U-13C]glutamine. Inset displays labeling in lipogenic acetyl-CoA derived from glutamine carbon. c Cell count after 4 days of culture in normal serum. Data are the average of biological triplicates with error bars representing SD.

Mentions: The simplest interpretation of the model in Fig. 5c is that distinct α-ketoglutarate and citrate pools arise because of subcellular compartmentalization in the mitochondria and cytosol. Considering the similarities between our system of PDHA1 silencing and PDH suppression during hypoxia and that hypoxic cells have been reported to undergo glutamine-dependent fatty-acid synthesis through cytosolic reductive carboxylation [43], we hypothesized that enhanced reductive carboxylation occurred in the cytosol. We therefore tested whether the cytosolic isoform of IDH (IDH1) was required for the enhanced glutamine-dependent palmitate labeling during PDHA1 silencing. CRISPR/Cas9 engineering was used to generate H460 cells containing biallelic IDH1 mutation, then doxycycline-inducible PDHA1 hairpins were introduced into these clones to regulate the levels of PDH E1α (Fig. 6a). Cell lines containing all combinations of IDH1 and PDHA1 expression were cultured with [U-13C]glutamine. Absence of IDH1 eliminated the increased glutamine-dependent palmitate labeling during PDHA1 silencing (Fig. 6b).Fig. 6


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)

IDH1 loss suppresses contribution of glutamine carbon to lipogenesis under PDH suppression. a Western blot for PDH E1α and IDH1. Cyclophilin B was used as a loading control. b Percentage labeling in mass isotopologues of palmitate after 24 h of culture with [U-13C]glutamine. Inset displays labeling in lipogenic acetyl-CoA derived from glutamine carbon. c Cell count after 4 days of culture in normal serum. Data are the average of biological triplicates with error bars representing SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: IDH1 loss suppresses contribution of glutamine carbon to lipogenesis under PDH suppression. a Western blot for PDH E1α and IDH1. Cyclophilin B was used as a loading control. b Percentage labeling in mass isotopologues of palmitate after 24 h of culture with [U-13C]glutamine. Inset displays labeling in lipogenic acetyl-CoA derived from glutamine carbon. c Cell count after 4 days of culture in normal serum. Data are the average of biological triplicates with error bars representing SD.
Mentions: The simplest interpretation of the model in Fig. 5c is that distinct α-ketoglutarate and citrate pools arise because of subcellular compartmentalization in the mitochondria and cytosol. Considering the similarities between our system of PDHA1 silencing and PDH suppression during hypoxia and that hypoxic cells have been reported to undergo glutamine-dependent fatty-acid synthesis through cytosolic reductive carboxylation [43], we hypothesized that enhanced reductive carboxylation occurred in the cytosol. We therefore tested whether the cytosolic isoform of IDH (IDH1) was required for the enhanced glutamine-dependent palmitate labeling during PDHA1 silencing. CRISPR/Cas9 engineering was used to generate H460 cells containing biallelic IDH1 mutation, then doxycycline-inducible PDHA1 hairpins were introduced into these clones to regulate the levels of PDH E1α (Fig. 6a). Cell lines containing all combinations of IDH1 and PDHA1 expression were cultured with [U-13C]glutamine. Absence of IDH1 eliminated the increased glutamine-dependent palmitate labeling during PDHA1 silencing (Fig. 6b).Fig. 6

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