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. Author manuscript; available in PMC: 2022 Mar 7.
Published in final edited form as: Curr Opin Hematol. 2020 May;27(3):155–162. doi: 10.1097/MOH.0000000000000574

Erythrocyte adaptive metabolic reprogramming under physiological and pathological hypoxia

Angelo D’Alessandro 1, Yang Xia 2,3
PMCID: PMC8900923  NIHMSID: NIHMS1602419  PMID: 32141895

Abstract

Purpose of review-

The erythrocyte is the most abundant cell type in our body, acting as both a carrier/deliverer and sensor of oxygen (O2). Erythrocyte O2 delivery capacity is finely regulated by sophisticated metabolic control. In recent years, unbiased and robust human metabolomics screening and mouse genetic studies have advanced erythroid research revealing the differential role of erythrocyte hypoxic metabolic reprogramming in normal individuals at high altitudes and patients facing hypoxia, such as sickle cell disease (SCD) and chronic kidney disease (CKD). Here we summarize recent progress and highlight potential therapeutic possibilities.

Recent Findings-

Initial studies showed that elevated soluble CD73 (sCD73, converts AMP to adenosine) results in increased circulating adenosine that activates the A2B adenosine receptor (ADORA2B). Signaling through this axis is co-operatively strengthened by erythrocyte-specific synthesis of sphingosine-1-phosphate (S1P). Ultimately, these mechanisms promote the generation of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte specific allosteric modulator that decreases hemoglobin-O2 binding affinity and thus induces deoxygenated sickle Hb (deoxyHbS), deoxyHbS polymerization, sickling, chronic inflammation and tissue damage in SCD. Similar to SCD, plasma adenosine and erythrocyte S1P are elevated in humans ascending to high altitude. At high altitude these two metabolites are beneficial to induce erythrocyte metabolic reprogramming and the synthesis of 2,3-BPG and thus increase O2 delivery to counteract hypoxic tissue damage. Follow up studies showed that erythrocyte equilibrative nucleoside transporter 1 (eENT1) is a key purinergic cellular component controlling plasma adenosine in humans at high altitude and mice under hypoxia and underlies the quicker and higher elevation of plasma adenosine upon re-ascent due to prior hypoxia-induced degradation of eENT1. More recent studies demonstrated the beneficial role of erythrocyte ADORA2B-mediated 2,3-BPG production in CKD.

Summary-

aken together, these findings revealed the differential role of erythrocyte hypoxic metabolic reprogramming in normal humans at high altitude and patients with CKD vs SCD patients and immediately suggest differential and precision therapies to counteract hypoxia among these groups.

Keywords: erythrocyte; hypoxia; metabolic reprogramming; adenosine; sphingosine-1-phosphate; 2,3-bisphosphoglycerate; sickle cell disease; high altitude and chronic kidney disease

Introduction –

Mature red blood cells (RBCs) lack nuclei and mitochondria and are the simplest and most abundant cell type in our body. They act as both a carrier/deliverer and sensor of oxygen (O2), which is vital to maintain normal function and survival of every single cell within our body. Notably, by the use of multiple innovative approaches during the early-mid 20th century, discoveries in RBC research have marked multiple milestones in the history of medical science and gave rise to modern molecular medicine. For example, in 1906 using state-of-the art light microscopy, James Herrick discovered elongated and sickle-shaped RBCs in an anemic patient[1]. This was the first example of a disease characterized at the cellular level and gave rise to the name sickle cell disease (SCD). In 1949, using an innovative gel electrophoresis approach, Linus Pauling showed that hemoglobin (Hb) is abnormal in RBCs of SCD patients[2]. In this way SCD was the first disease identified at the molecular level and this finding was the beginning of molecular medicine. In 1957, using a highly innovative protein sequencing strategy, Vernon Ingram identified the specific amino acid substitution in HbS[3, 4]. This was the first example of a genetic disease whose specific mutation was identified. In 1959, using X-ray crystallography, Max Perutz was able to unravel the structure of Hb[5, 6]. However, since the discovery in 1953 of the double helix, the twisted-ladder structure of DNA, by James Watson and Francis Crick[7–9], interest in the study of RBCs substantially decreased due to the lack of nuclei and DNA in mature RBCs. For more than 50 years progress in RBC research has been extremely limited, despite significant advances in the understanding of sensing of hypoxia (e.g., the discovery of Hypoxia Inducible Factor which led to the assignment of the 2019 Nobel Prize)[10–12]. However, the recent development of metabolomics, the newest member of the “omics” family, has provided a powerful new research strategy to accurately measure functional phenotypes that are the net result of genomic, transcriptomic and proteomic changes and respond faster to external stimuli than any other “OME”. Moreover, metabolomic profiling is especially appropriate for mature RBCs, where gene expression profiling is not an option due to the lack of a nucleus and de novo protein synthesis is hampered by the lack of ribosomes. This review summarizes our current progress in understanding the signaling pathways regulating hypoxic metabolic reprograming in RBCs, the pathological and physiological consequences of these metabolic changes, and the therapeutic potential of regulating RBC metabolism in healthy individuals at high altitude, those with inherited hemolytic diseases such as sickle cell disease (SCD) and individuals suffering from chronic kidney disease (CKD).

Erythrocyte glucose metabolism switch to glycolysis over pentose phosphate pathway under hypoxia is regulated by promoting deoxygenated hemoglobin trafficking to plasma membrane and in turn triggering the release of membrane bound glycolytic enzymes to the cytosol.

All living cells require energy for survival, proliferation and differentiation and to adapt to physiological and pathological stress conditions by metabolizing three major nutrients: glucose, amino acids and fatty acids. A change in nutrient metabolism for catabolic or anabolic purposes is termed “metabolic reprogramming”. Erythrocytes carry/deliver O2 to other cell types within our body but not use O2 by themselves. Thus, erythrocytes constantly face high level of reactive oxygen species (ROS). To maintain the O2 delivery capacity and counteract the challenge of high ROS within RBCs, the metabolism of glucose (their only nutrient) between glycolysis for energy need and the pentose phosphate pathway (PPP) coupling with the glutathione (GSH) cycle for anti-ROS is finely regulated under normoxia and hypoxia.

At normoxia, RBCs only release one or two O2 molecule carried by HbA tetramers. Thus, under normoxia approximately 75% of glucose is metabolized toward PPP to produce sufficient NADPH to couple efficiently with GSH cycle for anti-ROS activity. Under hypoxia, glucose metabolism is switched specially toward the erythroid-specific Rapoport-Luebering Shunt in glycolysis vs PPP in RBCs to produce more ATP and 2,3-bisphosphate (2,3-BPG) to induce O2 release capacity to counteract tissue hypoxia and damage. Some early elegant studies focused around the regulation of metabolic fluxes through glycolysis and the PPP in normal individuals under hypoxia, which ultimately resulted in the generation of a model involving the N-terminus of Band 3[13]. Band 3, also known as anion exchanger 1 (AE1) is the most abundant membrane protein in RBCs. AE1 N-term is a very acidic, intrinsically disordered domain that serves as a docking site for deoxygenated hemoglobin (deoxy-HbA) and glycolytic enzymes (e.g, phosphofructokinase, aldolase and glyceraldehyde 3-phosphate dehydrogenase). Under low oxygen tensions (hypoxia), deoxy-HbA outcompetes glycolytic enzymes that are binding at the N-term of AE1 in the membrane. In doing so, the released glycolytic enzymes promote the synthesis of high energy phosphate compounds including ATP and 2,3-bisphosphoglycerate (2,3-BPG), which further promote the release of O2 from hemoglobin by the mechanism of allosteric modulation (Fig.1). Dr. Doctor’s group reported a seminal study supporting an intriguing working model that the formation of deoxy-HbS polymers in SCD bind to AE1, displace glycolytic enzymes to enhance glycolysis vs PPP and thereby rendering sickle RBCs vulnerable to oxidative stress [14]. However, how deoxy-HbA or deoxy-HbS polymers transfer to the membrane and bind to Band 3 to trigger the release of glycolytic enzymes from the membrane to the cytosol and in turn induce 2,3-BPG production largely remained unknown until recent studies reviewed below.

Fig. 1. Glucose metabolism is finely regulated by O2 tension in mature normal RBCs via BAND 3-deoxy Hb-Glycolytic Enzyme switch.

Fig. 1.

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The BAND3-deoxyHb-Glycolytic Enzyme switch has been identified as a mechanism by which RBCs modulate metabolism based upon O2 tension. The N-terminus of BAND3 (BAND3 1–360) binds to (thus inactivating) key glycolytic enzymes including rate limiting phosphofructokinase (PFK), aldolase and glyceraldehyde 3-phosphatebdehydrogenase (GAPDH), inhibiting glycolysis and favoring PPP.

(A) This occurs in the oxygenated state, and thus drives increased generation of NADPH when there is higher oxidative stress, as O2 is a substrate for ROS-generating Fenton and Haber-Weiss reactions.

(B) Conversely, when in a low oxygen state (hypoxia), deoxy-Hb binds to BAND 3 in a way that displaces these glycolytic enzymes, promoting glycolysis over the PPP, leading to increased 2,3-BPG production and O2 delivery to peripheral tissues..

Detrimental role of adenosine and S1P-mediated RBC hypoxic metabolic reprogramming in SCD by promoting glycolysis, 2,3-BPG production, O2 release and sickling.

Cutting-edge metabolomics and state-of the art isotopically labeled nutrient flux analysis are two powerful unsupervised and high throughput approaches to monitor and trace intracellular metabolism. In recent years, using these two innovative approaches, multiple pioneering studies have defined “sophisticated intracellular signaling networks underlying RBC hypoxic metabolic reprogramming in promoting O2 delivery” in patients with SCD, the most common autosomal recessive hemolytic disorder with high morbidity and mortality. Due to the sickle mutation deoxy-HbS forms polymers, leading to the characteristic sickle-shaped RBCs, intravascular hemolysis, vaso-occlusion and multiple organ damage in SCD[15, 16]

Using innovative non-biased high throughput metabolomic screens, recent studies revealed the pathogenic roles of multiple metabolites and pathways in SCD and identified multiple innovative therapeutic targets. For example, adenosine, 2,3-BPG and sphingosine-1-phosphate (S1P) levels are highly elevated in the RBCs and plasma of humans and mice with SCD. Consistently, independent studies report that glycolytic intermediates and 2,3-BPG are induced in the erythrocytes of SCD patients[17]. Further pharmacological, genetic, cellular and biochemical studies revealed that elevated adenosine signaling via the A2B adenosine receptor (ADORA2B) induces 2,3-BPG, an RBC specific allosteric modulator that decreases Hb-O2 binding affinity, and thus promotes HbS deoxygenation, polymerization, sickling, inflammation, chronic pain and tissue damage (Fig.1)[18–26]. Elevated adenosine results from increased soluble CD73 (sCD73), a key enzyme that generates circulating adenosine from AMP. Moreover, both human and mouse genetic studies revealed that increased erythroid and plasma S1P in SCD mouse and humans are mediated by erythrocyte sphingosine kinase 1 (SphK1) activation via ADORA2B-mediated PKA and ERK signaling cascade. Mouse genetics, in vitro cultured human sickle RBCs and x-ray diffraction analysis of hemoglobin crystals demonstrated that elevated S1P functions intracellularly by binding directly to 2,3-BPG bound deoxy-HbS, stabilizing it in the deoxygenated state, and inducing the release of membrane anchored glycolytic enzymes from Band 3. As such, S1P induces sickling by switching RBC glucose metabolism toward glycolysis vs the PPP and thus induces 2,3-BPG production and increased deoxy-HbS (Fig.2). Elevated circulating S1P also contributes to chronic inflammation via activation of specific cell surface receptors in SCD mice (Fig.2). Overall, elevated sCD73-mediated increased circulating adenosine signaling via ADORA2B collaboratively works with intracellular S1P to promote glucose metabolism toward glycolysis vs PPP, resulting in increased 2,3-BPG, deoxy-HbS, sickling, chronic inflammation and tissue damage in SCD. Thus, lowering adenosine or S1P production by inhibiting CD73 or SphK1, inhibiting ADORA2B or S1P receptor-1 activation or interfering with downstream signaling cascades such as PKA or ERK are likely to be effective therapies for SCD.

Fig. 2. Detrimental role of adenosine and S1P-mediated erythrocyte metabolic reprogramming in SCD.

Fig. 2.

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Elevated sCD73 activity leads to increased plasma adenosine in SCD. Increased plasma adenosine signaling via ADORA2B results in activation of AMPK and SphK1 in sickle RBCs, respectively. Increased AMPK directly phosphorylates and activates BPG mutase and thus induce 2,3-BPG production. Meanwhile, activated SphK1-mediated elevated S1P functions intracellularly to induce 2,3-BPG production via glycolysis by directly binding to 2,3-BPG-deoxygenated HbS complex, stabilizing this complex anchoring membrane and thus triggering glycolytic enzymes release from membrane the cytosol. Thus, plasma adenosine signaling via ADORA2B collaboratively working with SphK1-induced elevation of erythrocyte S1P is detrimental to promote erythrocyte hypoxia metabolic reprogramming, leading to increased 2,3-BPG production, deoxy-HbS, deoxy-HbS polymerization and thus sickling.

Beneficial role of adenosine and S1P-mediated RBC hypoxic metabolic reprogramming in normal humans at high altitude by increased glycolysis, 2,3-BPG production and O2 delivery to counteract tissue ischemic damage.

It has been known for more than fifty years that 2,3-BPG levels and O2 delivery capacity of RBCs are induced by hypoxia in normal individuals at high altitude[27, 28]. However, the molecular basis underlying hypoxia-induced 2,3-BPG levels and O2 delivery was unknown prior to recent human high altitude discoveries. These studies revealed that plasma adenosine and erythrocyte S1P are elevated in humans ascending to high altitude up to 16 days and in mice exposed to hypoxia. Functionally, these two metabolites are beneficial to counteract tissue hypoxia by promoting metabolic reprogramming switching RBC glucose metabolism toward glycolysis vs PPP and thus inducing 2,3-BPG production and O2 delivery. Dual mechanisms are revealed by both human and mouse studies. 1) S1P binds directly to 2,3-BPG bound deoxy-HbA, stabilizing the complex in the deoxygenated state. Deoxy-HbA binds to Band 3, causing the release of glycolytic enzymes to the cytosol, thereby promoting glycolysis. 2) AMPK is a downstream signaling molecule of ADORA2B that directly phosphorylates and activates BPG mutase (Fig.3)[29],[30]. Thus, enhancing ADORA2B-AMPK-mutase or SphK1/S1P signaling networks are likely beneficial for normal individuals at high altitude and mice exposed to hypoxia to counteract tissue hypoxia and damage.

Fig. 3. Beneficial role of adenosine and S1P-mediated metabolic reprogramming in normal RBCs under high altitude and purinergic memory upon re-ascent.

Fig. 3.

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Elevated sCD73 activity leads to increased plasma adenosine in normal individuals under high altitude and mice exposed to hypoxia. Increased plasma adenosine signaling via ADORA2B results in activation of AMPK and SphK1 in normal RBCs under hypoxia, respectively. Increased AMPK directly phosphorylates and activates BPG mutase and thus induce 2,3-BPG production in normal RBCs under hypoxia. Meanwhile, activated SphK1-mediated elevated S1P functions intracellularly to induce 2,3-BPG production via glycolysis by directly binding to 2,3-BPG-deoxygenated HbS complex, stabilizing this complex anchoring membrane and thus triggering glycolytic enzymes release from membrane the cytosol under hypoxia. Thus, plasma adenosine signaling via ADORA2B collaboratively working with SphK1-induced elevation of erythrocyte S1P is detrimental to promote erythrocyte hypoxia metabolic reprogramming, leading to increased 2,3-BPG production and O2 delivery to counteract tissue hypoxia in normal individuals. Adenosine signaling via ADORA2B leads to PKA phosphorylation, polyubiquitination and proteasomal degradation of equlibrative nucleotide transporter 1 (eENT1) in normal mature erythrocytes under high altitude hypooxia. Thus, quicker and higher elevation of soluble CD73 activity and proteasomal degradation of eENT1-mediated reduction of eENT1 work together to set up a hypoxic memory for quick adaptation upon re-ascent to high altitude by rapidly inducing production of plasma adenosine and decreasing elimination of extracellular adenosine by reduced eENT1-mediated RBC uptake as a transient hypoxic memory.

Reduced erythrocyte equilibrative nucleoside transporter 1 (eENT1) and quicker elevation of sCD73 activity as a result of previous hypoxia sets up a hypoxic memory allowing for a rapid increase in plasma adenosine upon re-ascent to high altitude –

It is well known that humans can remember high altitude hypoxic stress and adapt to hypoxia much better and quicker upon re-ascent. However, the molecular basis underlying this phenomenon remains unclear. Recent studies showed that quicker and higher elevation of soluble CD73 activity and proteasomal degradation of eENT1 work together to set up a hypoxic memory for quick adaptation upon re-ascent to high altitude by rapidly inducing production of plasma adenosine and decreasing elimination of extracellular adenosine by reduced eENT1-mediated RBC uptake (Fig.3). Mechanistic studies showed that initial hypoxia-induced elevation of adenosine signaling via ADORA2B leads to PKA activation, subsequent phosphorylation of eENT1 and eventually polyubiquitination and degradation of eENT1. As such, erythrocytes being exposed to previous hypoxia contain much lower eENT1 levels and display much lower ability to uptake circulating adenosine upon re-ascent until new RBCs replace the old RBCs that were previously exposed to hypoxia. Thus, the erythrocyte purinergic signaling-mediated memory for quicker acclimatization to hypoxia upon re-ascent is only transient as long as RBCs with reduced eENT1 content are present.

Beneficial role of erythrocyte hypoxic metabolic reprogramming in pathological hypoxia: chronic kidney disease (CKD) –

Hypoxia is a dangerous condition for both normal individuals facing high altitude hypoxia and patients with chronic kidney disease (CKD), cardiovascular disease (CVD), respiratory diseases and even aging[11, 31–34]. At present, substantial effort and research in these diseases have largely focused on ischemic damage and dysfunction of specific end organs (heart, lung, kidney). CKD is one of the most devastating and costly medical conditions associated with high morbidity and mortality, affecting ~ 1 in 10 adults (more than 20 million individuals in the United States) and 8–16% of the adult population worldwide[35–38]. No effective therapy is available except renal dialysis or organ transplantation when CKD has progressed to end stage renal disease. A large body of evidence indicates that CKD is driven by renal tissue hypoxia[39]. As with high altitude hypoxia in normal individuals, RBC 2,3-BPG levels are also elevated in many diseases associated with chronic hypoxia including CKD and CVD[40–44]. However, the function and underlying mechanism of increased RBC 2,3-BPG in CKD and CVD patients remains a mystery. A recent mouse genetic study showed that elevated adenosine signaling via ADORA2B-mediated RBC metabolic reprogramming is an early protective mechanism to ameliorate renal hypoxia and slow progression of CKD by promoting AMPK and BPG mutase activation and thus 2,3-BPG production and O2 delivery capacity[45]. Follow up human studies validated mouse studies revealing that erythrocyte AMPK and BPG mutase activity and 2,3-BPG levels are elevated in the patients with CKD and correlated to the disease severity[45]. Thus, targeting this innovative pathway will likely promote adenosine-mediated hypoxic metabolic reprogramming and O2 delivery to counteract renal hypoxia, tissue damage and progression to fibrosis in CKD.

CONCLUSION.

RBCs, as the only cells delivering O2 to peripheral tissues to maintain their normal function, are extremely sensitive to hypoxia[46]. However, studies of RBCs’ functional response to hypoxia are extremely limited. Using cutting-edge metabolomics and state-of the art isotopically labeled nutrient flux analysis, two powerful unbiased and high throughput approaches, recent pioneering research defined “sophisticated intracellular signaling networks underlying RBC hypoxic metabolic reprogramming in promoting O2 delivery” in SCD patients[16, 18–26], normal individuals adapting to high altitude[29, 30, 47, 48] and in patients with CKD. Thus, these discoveries support a novel concept that “erythrocyte hypoxia metabolic reprogramming is a compensatory response counteracting tissue hypoxia in normal humans under high altitude and kidney damage and its progression in CKD patients. However, these adaptive erythrocyte hypoxia metabolic reprogramming-mediated decreased Hb-O2 binding affinity is detrimental for those with SCD due to the polymerization of deoxyHbS and sickling. Significantly, the discovery of the differential consequences of RBC hypoxic metabolic reprogramming in SCD versus healthy individuals facing high altitude and patients with CKD highlights the need for new and differential therapeutic strategies for SCD and normal individuals and CKD patients facing hypoxia. It is our hope that the significant new insight into erythrocyte adaptive metabolic reprogramming-mediated O2 delivery counteracting tissue hypoxia and the progression of CKD, but promoting sickling and disease progression in SCD, pave the way for innovative and precision therapeutics for both physiological conditions and pathological hypoxia related diseases.

Key points:

  1. Elevated plasma adenosine signaling via ADORA2B collaboratively working with SphK1-induced elevation of erythrocyte S1P to promote erythrocyte hypoxia metabolic reprogramming is detrimental to sickle cell disease by promoting 2,3-BPG production, increased deoxy-HbS, deoxy-HbS polymerization and thus sickling.

  2. Elevated plasma adenosine via ADORA2B signaling network coupled with increased SphK1 activation-mediated elevation of erythrocyte S1P to promote erythrocyte hypoxia metabolic reprogramming is beneficial to counteract tissue hypoxia in normal humans at high altitude by promoting 2,3-BPG production and O2 release. Note; deoxy-HbA does not polymerize.

  3. Elevated adenosine via ADORA2B-AMPK signaling cascade mediated erythrocyte 2,3-BPG induction is beneficial to counteract renal hypoxia, damage and its progression to CKD.

  4. Enhancing adenosine-ADORA2B-SphK1-AMPK-BPG mutase signaling cascade is likely to counteract tissue hypoxia in normal individuals under high altitude or patients facing chronic hypoxia but detrimental for patients with SCD due to its point mutation of Hbβ.

Acknowledgements:

Financial support and sponsorship

This work is supported by National Heart, Lung and Blood Institute R01HL137990, R01HL136969 and McGoven fund of UTHealth-Medical School (YX) and National Heart, Lung and Blood Institute R01HL146442 and R01HL148151, National Institute of General Medical Sciences RM1GM131968, and Boettcher Webb-Waring Early Career Award 2017 (AD).

Footnotes

Conflicts of interest: Though unrelated to the contents of this manuscript, AD is the founder and CSO of Omix Technologies Inc and a Scientific Advisory Board member for Hemanext Inc.

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