Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells

Abstract

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells.

Figure 1. Use of ²H glucose to label cytosolic NADPH

A) Atom-transition map depicting a model of deuterium transfer from [3-²H]glucose through glycolysis and the pentose phosphate pathway. Open large circles represent carbon and small red circles indicate deuterium label from [3-²H]glucose. Where measured, enrichment of M1 isotopomer (%) for glycolytic intermediates in parental H1299 cells is shown. B) Labeling of NADPH from [3-²H]glucose in parental H1299 cells over time. C) Saturated fatty acid labeling (myristate; C14:0, palmitate; C16:0 and stearate C18:0) from [3-²H]glucose in parental H1299 cells following incubation for 72 hours. D) Cholesterol labeling from [3-²H]glucose in parental H1299 cells cultured for 72 hours. E) Enrichment of lipogenic [²H]-NADPH by [3-²H]glucose estimated by a model for saturated fatty acid synthesis (ISA) in parental H1299 and A549 cells following incubation with tracer for 72 hours. Data plotted in a-d represent mean ± SD of at least three biological replicates. For e, data presented are mean ± 95% confidence interval of at least three biological replicates.

Figure 2. Use of ²H glucose to label NADH

A) Atom-transition map depicting a model of deuterium transfer from [4-²H]glucose through glycolysis and NAD+-dependent shuttle systems (malate dehydrogenase MDH, glycerol 3-phosphate dehydrogenase Gly3PDH, and lactate dehydrogenase LDH). Open large circles represent carbon and small green circles indicate deuterium label from [4-²H]glucose. B) Labeling of glycolytic intermediates from [4-²H]glucose in A549 (left panel) and H1299 (right panel) cells. Glyceraldehyde-3-phosphate (GAP) was below the limit of detection in H1299 cells, indicated by *. C) Labeling of NADH from [4-²H]glucose in parental H1299 cells over time. D) Palmitate labeling from [4-²H]glucose in A549 and H1299 cells following incubation with tracer for 72 hours. E) Time course labeling of NADPH from [4-²H]glucose in parental H1299. Data presented are mean ± SD of at least three biological replicates.

Figure 3. Generation and characterization of cell lines expressing inducible mutant IDH

A) Schematic demonstrating the transfer of deuterium (red dots) from NADPH to 2HG via the reaction catalyzed by mutant IDH enzymes. mtIDH1 is localized to the cytoplasm and mtIDH2 is localized to the mitochondria. By expressing compartment specific mutant enzymes and combining this with deuterated glucose tracer, it is possible to track 2HG production, and therefore the source of the NADPH used to make the 2HG in the cytosol or the mitochondria. B) Mutant IDH1-R132H is localized to the cytosol (mtIDH1-C) and mutant IDH2-R172K is localized to the mitochondria (mtIDH2-M) in H1299 and A549 cells. Cells were transduced with lentiviral constructs containing cDNA encoding C-terminal FLAG-tagged IDH1-R132H or IDH2-R172K under the control of a doxycycline-inducible promoter. Once stable cell lines were established, cells were treated with 0.1 µg/mL doxycycline for 24 hrs. Protein expression was analyzed by cellular fractionation and Western blotting using antibodies against FLAG, Cytochrome C (mitochondrial-specific marker) and Hsp70 (cytoplasmic-specific marker). C: Supernatant-100 fraction (cytoplasm); M: Mitochondria. White lines between blots in the horizontal direction indicate separate gels. C) Cell lines expressing inducible IDH mutants produce 2HG in a doxycycline-dependent manner. H1299 and A549 cells stably expressing inducible mtIDH1-C or mtIDH2-M constructs were treated with doxycycline (0.1 µg/mL) for 24 hrs. Amounts of 2HG (total ion counts: TIC) are shown relative to GFP control cells treated with vehicle. D) 2HG production, as measured by 2HG/αKG ratio, is much higher in cell lines harbouring endogenous mutations for IDH1 (R132C/+, HT1080) and IDH2 (R172S/+, SW1353) than cells expressing mtIDH1-C and mtIDH2-M. E) NADPH produced by the pentose phosphate pathway (6PGD) is cytosolic. Cells were cultured in [3-²H]-glucose (10mM) for 24 hrs before adding doxycycline (0.1µg/mL) for 24 hours to induce mutant IDH expression and amount of M1 label (%) from [3-²H]-glucose incorporated into 2HG and αKG was measured. F) NADH supports NADPH production in the mitochondria. Cells were incubated with 10mM [4-²H]-glucose for 24 hours and treated and analyzed as in E. Data represent mean ± SEM of at least three biological replicates.

Figure 4. Kinetic isotope effect minimally affects [3-²H]glucose and [4-²H]glucose metabolism

A) [3-²H]glucose was titrated with unlabeled glucose and added to H1299 cells expressing mtIDH1-C. Contribution from [3-²H]glucose to lipogenic NADPH (left panel, normalized to contribution at 100% [3-²H]glucose media enrichment) and 2HG (right panel) was measured. Dashed lined (left panel) represents 1:1 contribution of [3-²H]glucose to lipogenic NADPH to enrichment of [3-²H]glucose in media. B) [4-²H]glucose was titrated with unlabeled glucose and added to H1299 cells expressing mtIDH2-M. Labeling from [4-²H]glucose on lactate and malate (left panel); aspartate, citrate, and fumarate (middle panel); and 2HG (right panel) was measured. C) [3-²H]glucose was titrated with unlabeled glucose in HT1080 cells harbouring endogenous IDH1 mutations (R132C/+). Contribution from [3-²H]glucose to lipogenic NADPH (left panel, normalized to contribution at 100% [3-²H]glucose media enrichment) and M1 labeling on 2HG (right panel) was quantified at 0, 25, 50, 75, and 100 percent dilution with unlabeled glucose (left panel) in HT1080 cells cultured with [3-²H]glucose diluted with unlabeled glucose at 0, 25, 50, 75, and 100 percent enrichment. Data presented are shown as mean ± SD of three biological replicates for panels A (right panel), B, and C (right panel). Data represent mean ± 95% confidence interval for at least three biological replicates for panels A (left panel) and C (left panel)

Figure 5. Characterizing serine/glycine metabolism in the cytosol/mitochondria

A) Serine (left panel) and glycine (right panel) labeling in A549 mtIDH1-C and mtIDH2-M cells cultured with [U-¹³C₃]serine. Cells were incubated with [U-¹³C₃]serine for 24 hours prior to dox-induction (0.1 µg/mL) for an additional 48 hours. Middle panel demonstrates interconversion of serine and glycine by SHMT. Data plotted represent mean ± SD for three biological replicates. B) A schematic of folate-mediate one carbon metabolism in cytosolic and mitochondrial compartments catalyzed via SHMT and MTHFD. Deuterium transfer from [3,3-²H₂]serine is shown for pathways containing SHMT and MTHFD and is indicated by small red or blue circles for cytosolic and mitochondrial isozymes, respectively. The extra deuterium on [2,3,3-²H₃]serine is indicated by an orange (cytosolic) or a turquoise (mitochondrial) circle. Deuterium transfer from [2,2-²H₂]glycine is shown for the glycine cleavage system (GCS) pathway indicated by small green circles. C) 2HG labeling from [3,3-²H₂]serine, [2,3,3-²H₃]serine or [2,2-²H₂]glycine in A549 mtIDH1-C and mtIDH2-M cells. Cells were incubated with either tracer for 24 hours prior to dox-induction (0.1 µg/mL) for an additional 48 hours. No label was detected on 2HG in mtIDH1-C cells from either [3,3-²H₂]serine or [2,3,3-²H₃]serine, nor was label detected on 2HG from [2,2-²H₂]glycine in mtIDH1-C and mtIDH2-M cells (indicated by *). D) Fatty acid labeling from A549 mtIDH1-C and mtIDH2-M cells cultured with either [3,3-²H₂]serine, [2,3,3-²H₃]serine, or [2,2-²H₂]glycine. Cells were incubated with tracer for 24 hours prior to dox-induction (0.1 µg/mL) for an additional 48 hours. E) Serine and glycine labeling in A549 mtIDH1-C and mtIDH2-M cells cultured with [3-²H]glucose. Cells were incubated with tracer for 24 hours prior to dox-induction (0.1 µg/mL) for 48 hours. Data represent mean ± SEM of at least three biological replicates for panels C–E.