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Published in final edited form as: Exp Hematol. 2024 Jan 17;131:104153. doi: 10.1016/j.exphem.2024.104153

Metabolic Regulation of Erythrocyte Development and Disorders

Junhua Lyu 1, Min Ni 2, Mitchell J Weiss 3, Jian Xu 1,*
PMCID: PMC10939827  NIHMSID: NIHMS1963713  PMID: 38237718

Abstract

The formation of new red blood cells (erythropoiesis) has served as a paradigm for understanding cellular differentiation and developmental control of gene expression. The metabolic regulation of this complex, coordinated process remains poorly understood. Each step of erythropoiesis, including lineage specification of hematopoietic stem cells, proliferation, differentiation, and terminal maturation into highly specialized oxygen-carrying cells, has unique metabolic requirements. Developing erythrocytes in mammals are also characterized by unique metabolic events such as loss of mitochondria with switch to glycolysis, ejection of nucleus and organelles, high level heme and hemoglobin synthesis, and antioxidant requirement to protect hemoglobin molecules. Genetic defects in metabolic enzymes including pyruvate kinase and glucose-6-phosphate dehydrogenase cause common erythrocyte disorders, whereas other inherited disorders such as sickle cell disease and β-thalassemia display metabolic abnormalities associated with disease pathophysiology. Here we describe recent discoveries on the metabolic control of red blood cell formation and function, highlight emerging concepts in understanding the erythroid metabolome, and discuss potential therapeutic benefits of targeting metabolism for red cell disorders.

UNIQUE METABOLIC NEEDS OF ERYTHROPOIESIS

Erythropoiesis presents some unique metabolic requirements as hematopoietic stem cells (HSC) undergo successive steps of lineage differentiation, proliferation, and terminal maturation into highly specialized oxygen transporting cells filled with hemoglobin [1, 2]. Approximately 25 trillion circulating red blood cells (RBC) account for ~84% of all cells in the adult human body. Over two million RBCs are produced every second to replace aged or damaged ones, representing ~40% of the total body mass turnover [3]. Defects in RBC production or function cause various forms of anemia, a major source of global morbidity and mortality affecting nearly one-third of the world’s population [4, 5]. Despite the need for generating significant biomass during erythrocyte differentiation, mature RBCs in mammals contain no nucleus or organelles including mitochondria, endoplasmic reticulum (ER), Golgi and lysosomes. In addition, RBCs rely on metabolic processes to be easily and reversibly deformable in order to squeeze through blood vessels in microcirculation.

Due in part to the easily accessible circulating RBCs in various organisms, erythrocyte has long served as a model for studying processes that regulate energy metabolism, membrane function, molecular transport, and iron and heme homeostasis [3, 6, 7]. In recent years, our knowledge of erythrocyte metabolism has increased with the applications of high throughput ‘omic’ technologies. Here we review recent advances in our understanding of metabolic processes essential for erythrocyte differentiation and function, and how dysregulation underlies pathological erythropoiesis.

METABOLIC REGULATION OF ERYTHROID LINEAGE SPECIFICATION AND DIFFERENTIATION

An exquisite control of HSC differentiation into the erythroid lineage is essential for continuous production of RBCs. The stepwise process of erythroid lineage commitment, differentiation, and maturation is characterized by progressive morphological and functional changes that accompany each cell division [2, 8] (Figure 1). Early erythroid progenitors are highly proliferative, while cell growth markedly decreases during later maturation. As hemoglobin content increases, erythroblasts enucleate to become reticulocytes, exit the bone marrow, extrude or degrade the remaining organelles including mitochondria, and complete the maturation process in the bloodstream. By analyzing the transcriptomic, proteomic and metabolomic changes between hematopoietic stem/progenitor cells (HSPCs) and differentiating erythroid cells in mice and humans, recent studies shed new insights into mitochondria-dependent and -independent pathways as critical regulators of erythropoiesis.

Figure 1. Dynamic regulation of erythroid cell metabolism.

Figure 1.

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Erythropoiesis is characterized by progressive morphological and functional remodeling and has served as a paradigm for studying transcriptional, epigenetic, and post-transcriptional mechanisms in tissue development and regeneration [93–96]. However, it remains elusive how developing erythrocytes coordinate the metabolic programs with other cellular processes to promote lineage specification, differentiation, and terminal maturation. Recent findings from multi-omic approaches start to uncover unique features of erythroid cell metabolism with translational implications of developing metabolism-targeted therapies for common red cell disorders.

Mitochondria biogenesis and metabolism

Adult HSCs are maintained in a hypoxic bone marrow niche which promotes anaerobic glycolysis, the process of metabolizing glucose to lactate when oxygen (O2) is insufficient [9–11]. Anaerobic glycolysis also serves to preserve genomic integrity by maintaining low levels of reactive oxygen species (ROS), by-products of mitochondrial respiration that can induce DNA damage [12]. While quiescent long-term hematopoietic stem cells (LT-HSC) contain largely inactive or ‘nascent’ mitochondria, consistent with low ROS levels in these cells, the differentiation of HSCs is associated with increased mitochondria activity, activation of the mammalian target of rapamycin (mTOR) signaling, and elevated ROS generation [13–16] (Figure 1).

Mitochondria are required for many fundamental processes including oxidative phosphorylation (OXPHOS), ROS regulation, apoptosis, signaling, and heme biosynthesis [12]. Although erythroblasts progressively eliminate mitochondria during terminal maturation, mitochondrial biogenesis, respiration, and metabolism are enhanced in lineage-committed erythroid progenitors relative to HSPCs, at least in part through mTOR Complex 1 (mTORC1)-induced translation of mitochondria-associated mRNAs [17] (Figure 1). Genetic or pharmacological perturbation of mitochondria impairs erythroid cell differentiation, causing dysregulated gene expression, epigenetic modification, and intracellular metabolism [17]. Specifically, mitochondrial deficiency induced by loss of a major mitochondrial transcriptional regulator TFAM in erythroid cells leads to increased production of ketone bodies including β-hydroxybutyrate (βOHB), an inhibitor of class I and II histone deacetylases (HDACs). Inhibition of HDACs in mitochondria-deficient erythroblasts causes increased histone acetylation and persistent expression of HSPC-associated genes to impair erythroid differentiation [17]. These findings are consistent with the essential role of the mitochondrial respiratory chain for HSC function through regulating key metabolites associated with epigenetic regulation [18].

Gene transcription, epigenetics and metabolism are integral processes required for cell function. Cumulating evidence starts to unravel how their crosstalk regulates cell identity during development and disease [19–21]. For example, the metabolic state required for early erythroid lineage differentiation is controlled by a set of transcriptional regulators including TIF1γ and its downstream effector coenzyme Q (CoQ), a member of the mitochondrial respiratory chain [22]. TIF1γ is indispensable for erythropoiesis from zebrafish to humans by regulating the balance between nucleotide synthesis, mitochondrial respiration, and epigenetic modification, highlighting a critical role for cell-specific transcriptional control of mitochondrial metabolism in driving erythroid lineage commitment [22]. Similar metabolic and proteomic rewiring occurs in murine embryonic erythroblasts as they undergo terminal maturation during the circulation [23].

Although mitochondria and other organelles are progressively eliminated by mitophagy or autophagy in mature erythrocytes, their localization and activity are required for key processes during terminal maturation such as chromatin condensation and the expulsion of the nucleus (enucleation) [24, 25]. Interestingly, while healthy RBCs are devoid of mitochondria, the retention of functional mitochondria in mature RBCs is observed in the context of pathological conditions such as sickle cell disease (SCD) and systemic lupus erythematosus (SLE) [26–28]. It remains unclear whether and how these organelles and associated metabolic pathways contribute to erythrocyte defects.

Together, recent findings provide insights into understanding the mechanistic underpinnings for the general notion that erythrocytes are sensitive to deregulated gene transcription, protein translation or mitochondrial function. Understanding the crosstalk between gene regulatory processes and mitochondrial function will have important implications for developing new strategies to manage common RBC disorders and mitochondria-based syndromes [17].

Amino acid metabolism

Other than glucose as the natural substrate for erythrocyte metabolism, growing evidence supports critical roles for other nutrient sources in regulating erythroid lineage commitment and differentiation. Blocking glutamine uptake or utilization inhibits erythroid lineage differentiation from human HSPCs and increases the myelomonocytic lineage output [29]. Interestingly, glutamine-dependent nucleotide biosynthesis, but not ATP production, is required for erythroid specification of HSPCs. Together with the shunting of glucose through the pentose phosphate pathway (PPP) to generate ribose sugars for nucleotide biosynthesis, glutamine metabolism contributes to the increased nucleotide synthesis required for the markedly enhanced cell proliferation during erythroid differentiation [29]. Arginine metabolism is also critical for erythroid specification of human HSPCs through hypusination and activation of the eukaryotic translation initiation factor 5A (eIF5A) [30]. eIF5A-mediated translation of selected proteins, in particular mitochondrial ribosomal proteins, is required for normal erythropoiesis, whereas pathological erythropoiesis is associated with diminished eIF5A hypusination [30]. These findings highlight a functional crosstalk between amino acid metabolism, protein translation and mitochondrial function essential for erythroid differentiation.

Catabolism of several amino acids, including glycine, glutamine, and branched-chain amino acids (BCAAs), is critical for heme biosynthesis as substrates [31, 32]. One-carbon metabolism including the methionine and folate cycles are also central for erythropoiesis by providing one-carbon units (methyl groups) for the synthesis of DNA, polyamines, phospholipids, as well as glycine required for heme biosynthesis. During erythropoiesis, amino acids not only serve as the building blocks for protein synthesis, but also activate the mTOR signaling cascade to regulate multiple intracellular processes [33]. Blocking the uptake of the BCAA leucine in zebrafish and murine erythroid progenitors inhibits mTORC1 and impacts erythropoiesis and hemoglobin production [34]. EPO-JAK-AKT signaling [35] and intracellular iron [36] also enhance mTORC1 signaling during erythroid differentiation. mTORC1 promotes mitochondrial function through increased translation of mitochondria-associated mRNAs [17], while also inhibiting mitophagy that is essential for mitochondria clearance during terminal erythroid maturation [37]. Hence, future studies of the intricate and dynamic interactions between nutrient utilization, intracellular metabolism, mTOR signaling, and autophagy will be necessary to further elucidate the roles of nutrient signaling for erythroid cell function.

Iron and heme metabolism

Erythropoiesis requires the tightly regulated heme metabolism (see review by Chung et al. [31]). Erythropoiesis is also the largest consumer of iron in the human body. Both heme and iron are integral components of hemoglobin molecules essential for erythrocyte function. Iron deficiency is the most common cause of anemia characterized by smaller (microcytic) and hypoferremic red cells, whereas iron overload is also common and equally detrimental [38]. Iron metabolism is essential for many fundamental processes including mitochondrial function, DNA synthesis and repair, and enzymatic reactions required for cell proliferation and survival. As such, erythroid cells require highly regulated processes to coordinate the production of heme and hemoglobin with iron availability [39, 40]. Central aspects of this coordination are controlled posttranscriptionally by the orthologous RNA-binding proteins IRP1 and IRP2, which orchestrate the fate of mRNAs encoding key iron and heme metabolism proteins by binding to the cis-regulatory RNA structures called iron-responsive elements (IREs) [41]. IRPs inhibits translation initiation when bound to IREs located in the 5’UTR of the mRNAs encoding FTH1, FPN, FTL, ALAS2 or mitochondrial aconitase (ACO2), whereas IRP binding to the IREs within 3’UTR stabilizes TFR1 mRNAs [38]. IRP binding to IREs is regulated primarily by iron availability, although the function of most IRP/IRE-controlled mRNAs and the underlying mechanisms during in vivo erythropoiesis remain to be determined. Other mechanisms involving the transcriptional (e.g. BACH1/NRF2 or HIF2), posttranscriptional (e.g. miR-485–3p), and posttranslational (e.g. hepcidin) processes also act to control iron transport, utilization and storage in erythroid cells [38, 40, 42]. Hence, a key challenge for future studies is to elucidate how these regulatory mechanisms intersect to control iron and heme metabolism to fulfill the unique requirements during erythropoiesis.

Other metabolic pathways

Recent multi-omic analysis of erythroid cell differentiation started to uncover new mechanisms in erythrocyte biology. The zinc exporter Slc30a1 and importer Slc39a8, both regulated by heme and the erythroid transcriptional regulator GATA1, undergo developmental switch during erythrocyte differentiation [43]. Decreased zinc importer and sustained exporter expression reduce intracellular zinc to restrict terminal erythroid maturation, whereas increased zinc accelerates differentiation, illustrating a GATA1/heme-controlled trace metal machinery in governing cellular differentiation [43, 44]. GATA1 also binds to sterol-regulatory element binding protein 2 (SREBP2) in G1ER erythroid cells. This interaction was proposed to fine-tune cholesterol metabolism and hemoglobin expression required for terminal maturation of murine erythroid progenitors [45]. Enhanced lipid metabolism, in particular the catabolism of phosphatidylcholine (PC) and phosphocholine, is also required for terminal erythropoiesis by regulating lipid composition of red cell membranes and energy metabolism in mouse and human erythroid cells [46].

While erythropoiesis serves as an evolving paradigm for understanding the metabolic processes controlling lineage specification, emerging evidence also supports a central role for cell metabolism in regulating erythroid differentiation and proliferation in response to physiological and pathological cues, a condition termed as stress erythropoiesis [47–49]. In addition, dysregulated metabolic processes contribute to erythroid development under pathological conditions such as ineffective erythropoiesis, whereas genetic or pharmacological manipulations of metabolism could improve phenotypes [40]. Therefore, future studies are required to systematically analyze the metabolic programs during in vivo erythropoiesis across physiological and pathological conditions and identify shared or condition-specific pathways that may be leveraged as metabolism-based therapies.

METABOLIC (DE)REGULATION OF MATURE ERYTHROCYTES IN PHYSIOLOGY AND PATHOPHYSIOLOGY

Glycolysis

Unlike differentiating erythroid precursors, mature RBCs exclusively rely on glycolysis or ‘Embden-Meyerhof’ pathway (EMP) to generate ATP and fuel key processes of erythrocytes (Figure 2). Two metabolic pathways branched from glycolysis are also critical for RBC function. The 2,3-bisphosphoglycerate (2,3-BPG) shunt (or Rapoport-Luebering shunt) generates 2,3-BPG, also known as 2,3-DPG (2,3-diphosphoglycerate), that functions as an allosteric modulator of hemoglobin to promote oxygen release in tissues [7]. The pentose phosphate pathway (PPP or hexose monophosphate shunt) generates the reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is critical for converting oxidized glutathione (GSSG) to reduced glutathione (GSH), the major antioxidant in RBCs [50]. Reduced glutathione functions to protect RBC enzymes, membrane proteins and lipids, and especially hemoglobin from oxidative damage. Hence, adequate levels of ATP, 2,3-BPG, NADPH and GSH for various metabolic functions of erythrocytes are maintained by the main glycolytic pathway with its 2,3-BPG and pentose shunts.

Figure 2. Dysfunctional metabolism is associated with common red cell disorders.

Figure 2.

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Erythrocytes rely on metabolic processes to fuel biological processes, maintain cellular shapes and flexibility, and keep essential components in reduced and active form. The main metabolic pathways active in normal erythrocytes are anaerobic glycolysis (‘Embden-Meyerhof’ pathway) for energy production and two branch pathways including Rapoport-Luebering shunt (or 2,3-bisphosphoglycerate shunt) and pentose phosphate pathway (or hexose monophosphate shunt). Genetic defects on metabolic enzymes cause common red cell disorders, whereas other inherited disorders such as sickle cell disease display unique metabolic abnormalities. Understanding erythroid cell metabolome holds promise to uncover new targetable pathways for the improved management of erythrocyte disorders.

Membrane metabolism

Although the macromolecules of red cell membrane are produced by erythroid precursors during differentiation, the erythrocyte membrane is metabolically active and required for various red cell functions. The red cell membrane is composed of a phospholipid bilayer containing cholesterol molecules and proteins. Notably, the outer membrane is enriched with phosphatidyl choline and sphingomyelin, whereas the inner membrane consists of predominantly phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine (PS). The maintenance of phospholipid asymmetry is mediated by the ATP-dependent flippase (aminophospholipid translocase) activity. Exposure of PS due to loss of phospholipid asymmetry is an important apoptotic marker on the red cell surface, which promotes the removal of damaged or aged erythrocytes from the circulation [51].

Erythrocyte membrane shape and deformability are controlled by the cytoskeletons, powered by the hydrolysis of ATP, at the inner surface. The metabolic activity of erythrocyte membrane is also required for maintaining osmotic stability through various ATP-dependent or - independent transport channels. The red cell membrane contains a diverse group of transmembrane proteins that serve as transport proteins for various ions and nutrients, adhesion proteins involved in the interactions of red cells with other cells in the circulation, and signaling receptors [51]. Thus, the refined analysis of the composition, function and regulation of membrane proteins and lipids will continue to provide new insights into erythrocyte metabolism, with important implications for understanding inherited red cell disorders and transfusion medicine [6, 51].

Enzyme defects in RBC disorders

As mature RBCs rely on anaerobic glycolysis for energy production and other functions, genetic defects of enzymes in glycolytic pathways can cause RBC disorders including hemolytic anemia (Figure 2). Most genetic defects in glycolytic enzymes are caused by deficiency of pyruvate kinase (PK), which catalyzes the formation of pyruvate from phosphoenolpyruvate (PEP) with the simultaneous generation of ATP. PK-deficient RBCs cannot produce sufficient ATP to maintain normal membrane function, resulting in leakage of potassium and water from the cell and increased calcium concentrations [52]. These abnormalities cause RBCs to lose flexibility, become rigid, and are susceptible to splenic sequestration and premature hemolysis [53].

Glucose 6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway. Mutations of G6PD gene are found in >500 million people worldwide, causing significant loss of G6PD enzymatic activity [54]. G6PD catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconolactone by reducing nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. NADPH is required for producing reduced glutathione in the erythrocyte cytoplasm to protect hemoglobin against oxidative damage. In G6PD deficiency, the presence of oxidizing agents can lead to oxidation and decreased solubility of hemoglobin molecules, causing acute hemolytic anemia [54].

Although much less common than PK deficiency and G6PD deficiency, there are many other erythrocyte abnormalities, often less known and underdiagnosed, caused by defects in glycolytic enzymes. These hereditary RBC disorders or erythrocyte enzymopathies can involve alterations of hexokinase (HK), glucose-6-phosphate isomerase (GPI), phosphoglycerate kinase (PGK), phosphofructose kinase (PFK), aldolase, and triosephosphate isomerase (TPI), among others [55] (Figure 2).

Metabolic abnormalities in major hemoglobin disorders

Recent advances in metabolomic approaches and genetic mouse modeling start to uncover new pathways that contribute to erythrocyte metabolism in physiology and pathophysiology [56]. In particular, adenosine, 2,3-BPG and sphingosine 1-phosphate (S1P) levels are elevated in RBCs and plasma of humans and mice with SCD, a major hemoglobin disorder caused mainly by a single amino acid substitution (glutamate to valine) at the 6th position of the β-globin (HBB) gene to produce hemoglobin S (HbS). Adenosine is generated by increased soluble CD73 (sCD73), whereas S1P is produced by erythrocyte sphingosine kinase 1 (SphK1) through adenosine-mediated signaling cascades. Elevated adenosine signaling increased 2,3-BPG levels to promote HbS deoxygenation, polymerization, sickling, and tissue damage [56, 57]. S1P also functions as an important metabolite to regulate RBC response to hypoxia by promoting oxygen release, thus serving as an intracellular hypoxia-response sphingolipid [58, 59]. Changes in intracellular levels of other metabolites, such as intermediates of the glycolytic and PPP pathways, amino acids (glycine, serine, glutamine, glutamate, and arginine), and nitric oxide (NO) metabolism (spermine, spermidine and citrulline), are also observed in RBCs of SCD patients, although the biological functions of these metabolites in SCD pathophysiology remain largely unclear [57, 60] (Figure 2).

The metabolic abnormalities in α- and β-thalassemias, the inherited hemoglobin disorders caused by insufficient production of the α- or β-chain of hemoglobin molecules due to mutations in the hemoglobin genes [61], are still poorly understood. Targeted profiling of a selected group of 40 metabolites by gas chromatography-mass spectrometry (GC-MS) in the serum of β-thalassemia patients and healthy controls revealed alterations in nearly all the metabolites, including those associated with glycolysis, glycerophospholipid, and fatty acid metabolism [62]. The use of more quantitative, unbiased, and high throughput metabolomic methods to study systemic and intracellular metabolism holds promise to uncover new metabolic pathways with translational implications to ameliorate the pathophysiology of thalassemias.

TRANSLATIONAL IMPLICATIONS OF TARGETING METABOLISM FOR ERYTHROID DISORDERS

An improved understanding of the metabolic processes regulating normal and disordered erythropoiesis has important implications for therapeutic interventions. In this section, we discuss the emerging metabolism-based therapeutic strategies by focusing on the major red cell disorders SCD and β-thalassemia.

Currently there are four drugs approved for treating SCD, which include hydroxyurea, voxelotor, L-glutamine, and crizanlizumab. Other treatment options include blood transfusion, hematopoietic cell transplantation, and the emerging gene therapies [63–65]. Hydroxyurea, which has been used to treat SCD for over two decades, increases the production of fetal hemoglobin (HbF) to prevent HbS polymerization, although the precise mechanisms for these effects remain elusive [66, 67]. Voxelotor (brand name: Oxbryta) increases hemoglobin levels and reduces hemolysis in SCD patients by increasing hemoglobin affinity for oxygen to prevent hypoxia-induced sickling [68]. L-glutamine (brand name: Endari) reduces SCD-associated complications including pain crises and hospitalizations [69]. Given that glutamine is a precursor for the synthesis of reduced forms of glutathione (GSH) and nicotinamide adenine dinucleotide (NAD), its clinical benefit is speculated to be due to the decreased susceptibility of sickle RBCs to oxidative damage; however, the precise metabolic pathways and mechanisms of action are still a matter of debate [70]. Crizanlizumab (brand name: Adakveo), a monoclonal antibody that blocks P-selectin-mediated adhesion of RBCs to the vascular endothelium, reduces the frequency of vaso-occlusive crisis (VOC) and other complications of SCD [71]. Of note, none of the current treatments for SCD is likely to be universally effective or accessible, and there remains an unmet need for improved therapeutic options.

Recent metabolic analysis of normal and disordered erythrocytes has provided new and potentially ‘actionable’ targets for developing new therapeutic strategies [72]. For example, elevated levels of 2,3-BPG, the product of the Rapoport-Luebering pathway branched from glycolysis, are associated with hypoxic conditions and the pathogenesis of SCD [58, 73, 74]. Strategies to decrease the production of 2,3-BPG, such as by targeting the synthase activity of bisphophoglycerate mutase (BPGM) in the Rapoport-Luebering pathway, may be beneficial for SCD. Pyruvate kinase (PK) catalyzes the last and rate-limiting step of the glycolytic Embden-Meyerhof pathway in erythrocytes and is responsible for producing 50% of ATP during glycolysis [53]. The PK activator mitapivat (AG-348 or Pyrukynd) is approved for treating inherited hemolytic anemia caused by PK deficiency [75, 76]. Importantly, PK activators may also be helpful to decrease 2,3-BPG levels involved in SCD pathogenesis [77]. Thus, the involvement of PK activity in the regulation of both ATP and 2,3-BPG makes it a promising target. PK activators mitapivat and FT-4202 are currently in clinical trials for treating SCD patients and show favorable safety profiles [78, 79]. Importantly, dose-dependent increases in ATP and decreases in 2,3-BPG, together with reductions in hemolytic markers, were observed in SCD patients treated with mitapivat [78, 79]. Although the underlying mechanisms for the improved metabolic profiles remain to be determined, these studies provide the proof-of-concept that PK activators have potential therapeutic benefits in patients with SCD.

β-thalassemia is characterized by ineffective erythropoiesis, exhibiting an increase in erythroblast proliferation that fails to differentiate and produce mature RBCs [80]. Common treatment options for β-thalassemia include blood transfusion, iron chelation, hematopoietic cell transplantation, and gene therapy approaches [65, 81]. Of note, several strategies targeting erythrocyte metabolism are being evaluated as potential therapies. Anemia patients affected by ineffective erythropoiesis including β-thalassemia suffer from iron overload due to chronic blood transfusion and/or increased iron absorption [40]. In β-thalassemia, the balance of α-globin and β-globin chains is lost, causing a relative excess of α-globin chains. The unbound α-globin can bind free heme to form insoluble aggregates called hemichromes that induce oxidative stress and damage RBC membranes [40]. For these reasons, genetic and pharmacologic approaches to restrict erythroid iron intake, which limits heme synthesis, are being evaluated as potential treatments for β-thalassemia [40].

Additional strategies that aim to improve erythroid differentiation have shown improvement of phenotypes in preclinical and clinical studies. In particular, the recently approved drug luspatercept (ACE-536), which acts as a ligand trap for TGFβ-like molecules, increases the differentiation of late erythroblasts, improves iron metabolism, reduces hemichromes, and ameliorates anemia in a dose-dependent manner [82–84]. Although luspatercept improves several aspects of erythrocyte metabolism, the metabolic pathways relevant to disease pathophysiology and the underlying mechanisms are poorly understood.

Oral administration of the PK activator mitapivat ameliorated ineffective erythropoiesis in Hbbth3/+ β-thalassemia mice, resulting in increased ATP, reduced ROS, and improved mitochondrial clearance [85]. Given the effect of PK activator on improving energy metabolism, clinical studies are underway to evaluate its efficacy for treating α- or β-thalassemia [86, 87]. The high rate of hemoglobin response and good tolerability, along with increased ATP concentrations and decreased markers of hemolysis, support further development of PK activation as a potential treatment for thalassemias. Despite the observed therapeutic benefits of PK activation in SCD and thalassemias, the mechanism of action and the metabolic processes responsible for the hemoglobin response remain unclear. Lastly, the removal of excess α-globin in β-thalassemic erythroid precursors is controlled by ULK1-dependent autophagy, whereas inhibiting mTORC1 stimulates this pathway to alleviate β-thalassemia pathologies [88, 89]. In light of recent advances in targeting autophagy and/or mTOR signaling in human disease [33, 90], it is of interest to further evaluate these pathways as potential targets for treating β-thalassemia or other hematologic disorders associated with ineffective erythropoiesis.

SUMMARY AND PERSPECTIVES

The metabolic analysis of cell function has historically relied on in vitro cell culture models in the presence of supraphysiological concentrations of major nutrients including glucose and glutamine [91]. A comprehensive analysis of metabolite changes and pathway activities in highly enriched cell populations during in vivo erythropoiesis has been lacking. Therefore, applying the emerging metabolomic techniques to simultaneously measure the concentrations of metabolites and follow their activities in isotope tracing experiments [91, 92] will likely uncover new pathways and their biological functions in erythrocyte biology. In addition, the use of heterogenous cell populations for metabolism research does not allow for the analysis of the dynamic metabolic functions and crosstalk between phenotypically defined subpopulations. With the advent of more sensitive and higher throughput metabolomic techniques, future studies of highly purified cell populations or even single cells will be necessary to define metabolic interactions within and between distinct cell states.

Despite recent advances in understanding erythroid cell metabolism, there are important questions that remain to be addressed. Specifically, what are the nutrient sources for differentiating erythroid cells under physiological and/or pathological conditions? Can we understand and exploit metabolic crosstalk between erythroid cells and their microenvironments? How to integrate metabolomics with other omic data to identify new pathways? These analyses will address how gene regulatory processes cooperate with metabolic programs to control lineage differentiation, and how altered metabolism impacts gene expression under pathological conditions. Lastly, which metabolic properties predict severity of erythrocyte disorders, and can these properties be modulated to impact disease course? Addressing these questions holds promise for not only improving our knowledge of erythroid cell metabolome but also identifying new actionable targets for the treatment of common red cell disorders.

Highlights.

  • Erythrocytes have unique metabolic requirements for development and function

  • Dysfunctional metabolism contributes to common erythrocyte disorders

  • Understanding the metabolome may identify new therapies for red cell disorders

Acknowledgments

We apologize for being unable to cite and discuss numerous studies in this field due to space constraints. We thank Hieu Vu and the members of the Xu laboratory for discussions and comments. J.X. is a Scholar of The Leukemia & Lymphoma Society (LLS) and an American Society of Hematology (ASH) Scholar. This work was supported by the NIH grants R01DK111430, R01CA230631, and R01CA259581 to J.X, and R01HL165798 to M.J.W.

Footnotes

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Conflict of Interest Disclosure

The authors do not have any conflicts of interest to declare in relation to this work.

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