HEME Degradation Enzyme Biliverdin IXb Reductase Is Required for Stem Cell Glutamine Metabolism

Bioenergetic requirements of pluripotent stem cells (PSC) vary with lineage fate, and cellular adaptations rely largely on substrate availability and mitochondrial function to balance TCA-derived anabolic and redox-regulated antioxidant functions. Heme (protoporphyrin IX complexed with iron) serves as an indispensable co-factor for all aerobic cells, and its cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles, the penultimate step regulated by two non-redundant biliverdin reductases (IXa, BLVRA and IXb, BLVRB) retaining isomeric specificity and NAD(P)H-dependent redox coupling linked to BR antioxidant function. Thus, heme biosynthesis (initiated by the condensation of glycine and succinyl CoA) can be considered a linear pathway that utilizes cataplerotic TCA-derived carbon (derived from glucose and glutamine) linked to the heme degradation pathway and bilirubin excretion. Given the overlapping glucose/glutamine requirements in both heme synthesis and PSC bioenergetics, we studied consequences of dysregulated heme metabolism by targeted BLVRB disruption in iPSCs derived from human umbilical cord CD34+ hematopoietic cells. Compared to control iPSCs, BLVRB-deficient iPSCs had no reciprocal BLVRA induction, no alterations in heme oxygenase expression, and no differences in isomeric BV accumulation, although phenotypically defective in antioxidant handling with exaggerated apoptotic cell death. To better delineate metabolic pathway perturbations causally implicated in BLVRB- deficient antioxidant function and cell viability, we generated gene/metabolite clusters and an extended network plot formulated on RNASeq data coupled with liquid chromatography/mass spectrometric-based targeted metabolomics profiling of 290 intracellular metabolites. Metabolic enrichment with topological pathway analysis identified a limited number of defective pathways involving purine/pyrimidine nucleotide synthesis, redox homeostasis (pentose phosphate pathway, PPP) and TCA cycle intermediates (a-ketoglutarate, fumarate, and malate). Mitochondrial staining using TMRE as a functional and quantifiable marker of the mitochondrial transmembrane potential (DYm) identified no defect across the genotypes, although BLVRB -deficient iPSCs demonstrated defective basal and maximal oxygen consumption rates (OCR) restricted to glutamine (but not glucose) utilization. Metabolic flux quantification using [U₆-¹³C] glucose revealed high-level and comparable (>97%) fractional labeling of ¹³C lactate across the genotypes, consistent with an active glycolytic metabolism that was comparable between control and BLVRB^-/- iPSCs. In contrast, isotopic tracing using [U-¹³C₅] glutamine demonstrated statistically-diminished glutamate-derived aKG accumulation (along with downstream TCA metabolites fumarate and malate isotopomers), results confirming rewired glutamine TCA entry, and reliance on glucose as a preferred substrate. Since the parallel PPP pathway utilizes glycolysis as the preferred substrate for generating both reducing equivalents (NADPH) and essential nucleotide component ribose-5-phosphate, we characterized three-dimensional embryoid body (EB) formation in the presence of the PPP pathway inhibitor 6-aminonicotinamide (6-AN), establishing statistically-decreased EB size (p<0.001) compared to control iPSCs. These data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism, and also suggest in principle that BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization, a key substrate for cancer and hematopoietic metabolism.