Tracing compartment- specific redox pathways using stable isotopes and mass spectrometry Christian Metallo IECM 2017 Department of Bioengineering Moores Cancer Center Michal and Schomburg Biochemical Pathways 2 nd Ed. 2012 UCSD Diabetes Research Center
The challenge for biologists, biochemists, and engineers: Translate biochemistry to metabolic fluxes 20 ��� ���� � ���� · ����/���� 36 4 � � � ��� ��� 8 3 � � � ��� 8 5 • Fluxes describe the ultimate function of metabolic enzymes • This is where metabolomics/analytical chemistry meets cell biology • Metabolite level measurements only get you so far A + B C D Use isotopic tracers Analyze data as a system MODELING!!! X Y
Studying metabolism for flux sake Mass spectrometry 13 C and 2 H metabolic tracing Cultured mammalian cells 5 (cancer, iPSCs, adipocytes) Animal models Humans 3 8 4 Systems-based flux analysis 8 Identify drug targets and 20 pathway mechanisms 36 mol/(cell·hr) 1. Targeting metabolism in cancer (Grassian et al. Canc Res 2014; Svensson et al. Nat Med 2016; Parker et al. Met Eng 2017) 2. Cellular compartmentalization and redox metabolism (Lewis et al. Mol Cell 2014; Vacanti et al. Mol Cell 2014) 3. Metabolic changes during iPSC growth/differentiation (Badur et al. Biotech J. 2015; Zhang et al. Cell Rep 2016) 4. Regulation of macrophage metabolism (Cordes et al. JBC 2016) 5. Understanding adipose tissue metabolism and physiology in the context of T2DM (Green et al. Nat Chem Bio 2016)
Our approach to study cell physiology and metabolism 35% 60% 30% % labeling from Gluc 50% % labeling from Gluc 25% 40% 20% 30% 20% Palmitate 15% 10% 10% De novo lipogenesis 0% M0 M1 M2 M3 M4 M5 M6 5% 0% MS Oac # of isotopes per molecule CO 2 Cit AcCoA AcCoA CO 2 Pyr Pyr Cit Oac Allows quantitation of: CO 2 CO 2 aKG Mal Glucose 1. Contribution of different substrates to AcCoA CO 2 pools and mitochondrial metabolism Fum SucCoA 2. Fatty acid synthesis/de novo lipogenesis rates cytoplasm mitochondria 3. Directionality of TCA metabolism 4. Intracellular fluxes with MFA modeling = Carbon atom = 13 C atom
Reprogramming of TCA metabolism under hypoxia AcCoA Gluc Glutamine reduction Oac Glucose oxidation 100% Lac Pyr Cit CO 2 80% to lipogenic AcCoA % contribution Pyr CO 2 PDH NADP + 60% AcCoA PDK1 NADPH Oac Cit CO 2 40% HIF/ARNT aKG aKG Mal 20% 0% Fum Suc Glu Normoxia Hypoxia CO 2 Hypoxia VHL loss Glutamine Hypoxic and pseudohypoxic cells exhibit increased reductive carboxylation flux • Compartmentalization of metabolic processes is critical for cell function (but complicates analysis) • Redox metabolism is perturbed by hypoxic stresses Metallo et al. Nature (2012), Mullen et al. Nature (2012), Scott et al. JBC (2011), Wise et al. PNAS (2011)
Redox metabolism is highly compartmentalized Glucose NAD + NADH H6P T3P 3PG PEP AcCoA NADP + NAD + Oac NADH NADPH 6PGD Ser Lac Pyr NADP + Cit CO 2 NADPH Gly Pyr CO 2 R5P NADP + AcCoA NADPH Oac Cit CO 2 Pyridine nucleotides [NAD(P)H] orthogonally connect metabolic pathways via electron transfer aKG aKG Mal NADPH: redox homeostasis/reductive biosynthesis Fum Suc Glu CO 2 NADH: cellular bioenergetics Neither is transported in/out of the matrix Glutamine
Eukaryotes are highly compartmentalized 13 C tracing and metabolomics cannot resolve compartment-specific metabolism How are NADPH and NADH regenerated in the cytosol and mitochondria? AcCoA Oac Pyr Cit CO 2 Pyr CO 2 NADP + AcCoA NADPH Oac Cit CO 2 aKG aKG Mal H H Fum Suc Glu Glucose CO 2 Glutamine
Tracing the oxidative PPP with [ 2 H]glucose w/ Matt Vander Heiden (MIT) Lewis et al. Molecular Cell 2014
Tracing the oxidative PPP with [ 2 H]glucose
Contribution of the oxidative PPP to NADPH pools
NADH shuttles and mitochondrial metabolism regenerate NAD + for glycolysis 25% Label from [4-2H]glucose 20% 15% 10% Lactate 5% Malate G3P 0% 0 24 48 72 Hours 2H label enters TCA cycle via malate- aspartate shuttle
Do kinetic isotope effects affect results? • Deuterium lowers rates in enzyme reactions ( in vitro ) • Is this relevant to tracing through metabolic networks? • Allow “H” and “D” to compete by diluting • Compare labeling Cytosolic NADPH pathways NADH metabolism
(L)2HG and (D)2HG have different origins and are labeled distinctly via 2H tracers (L)2HG (D)2HG MDH and LDH generate (L)2HG from NADH Oncogenic IDH1 generates 2HG from cytosolic NADPH 2HG is distinctly labeled by these tracers
Can we probe NADPH metabolism in mitochondria? Glucose Lipids NAD + NADH H6P T3P 3PG PEP AcCoA NADP + NAD + Oac NADH NADPH 6PGD Ser Lac Pyr NADP + Cit CO 2 NADPH Gly Pyr CO 2 2HG R5P AcCoA 2HG mIDH1 Oac Cit CO 2 IDH mutations identified in low-grade mIDH2 gliomas, AML, and other tumors aKG aKG Mal (Parsons et al. Science 2008 and others) Fum Suc Gain-of-function mutations in IDH1 and IDH2 Glu CO 2 Mutant enzymes reductively generate (D)2HG using aKG and NADPH mitochondria (Dang et al. Nature 2009) Glutamine cytosol
Using 2-HG production as a reporter of compartment-specific NADPH pools Minimal effect on central carbon metabolism Grassian et al. Cancer Research (2014)
Using 2-HG production as a reporter of compartment-specific NADPH pools 2H detection on 2HG provides readout of cytosolic vs. mitochondrial NADPH metabolism Cytosolic NADPH trace (via oxidative PPP) NADH trace (via glycolysis)
Can we use this reporter to annotate compartment-specific metabolic pathways? Folate-mediated one carbon metabolism Tibbetts and Appling, Ann. Rev. Nutr. 2010
Can we use this reporter to annotate compartment-specific metabolic pathways? Folate-mediated one carbon metabolism (L) ( ) ( )
Discerning compartment-specific serine metabolism using cofactor tracing % labeled 2HG (L) ( ) ( ) mtIDH1 ‐ C mtIDH2 ‐ M NADPH produced from serine only observed in mitochondria
Discerning compartment-specific serine metabolism using cofactor tracing and mIDH reporters (L) ( ) ( ) Serine, glycine, and folate-mediated one carbon metabolism generate mitochondrial reducing equivalents
Discerning compartment-specific serine metabolism using cofactor tracing NAD+ 2 H-NADH NNT Cytosolic reactions consume NADPH/produce serine NADPH from the oxidative PPP appears on serine
Resolving compartment-specific NADPH metabolism using 2H tracers and mutant IDH 25% 20% [4-2H]glucose Label from 15% 10% Lactate Malate 5% G3P 0% 0 24 48 72 Hours • 2H tracers allow for quantitation of NAD(P)H metabolism • Oncogenic IDH1 and IDH2 used as reporters for compartment-specific NADPH labeling Lewis et al. Mol Cell 2014
How is NAD(P)H metabolism reprogrammed under hypoxia? Hypoxia Oxidation of GAPDH under hypoxia leads to increased loss of isotope Increased exchange flux at TPI/aldolase
Hypoxia increases flux through the oxidative pentose phosphate pathway Hypoxia GAPDH oxidation leads to increased (15-40%) oxidative PPP contribution to NADPH pools
Hypoxic induction of reductive carboxylation is mediated by cytosolic oxPPP flux and IDH1 AcCoA Gluc Oac Lac Pyr Cit CO 2 Pyr CO 2 PDH NADP + AcCoA PDK1 NADPH Oac Cit CO 2 HIF/ARNT aKG aKG Mal Fum Suc Glu CO 2 Hypoxia Glutamine
Acknowledgements Metallo Lab UCSD MIT Salk Institute Martina Wallace Pedro Cabrales Matt Vander Heiden Reuben Shaw Thekla Cordes Rohit Loomba SBMRI UMass Worcester Le You Anne Murphy Jorge Moscat Mehmet Badur Ajit Divakaruni Dave Guertin Maria Diaz-Meco Courtney Green UC Berkeley Avi Kumar UCSD/VAMC SD UPenn Austin Lefebvre Dan Nomura Ted Ciaraldi Katy Wellen Noah Myers Bob Henry Hui Zhang Seth Parker Nate Vacanti Support NSF CAREER Award DOD Lung Cancer Research Program NIH/NCI Searle Scholars Program California Institute of Regenerative Medicine Hellman Faculty Fellowship Lowy Medical Research Foundation Camille and Henry Dreyfus Teacher-Scholar Award
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