Ex Vivo Glucose Tracing in Red Blood Cells from Patients with Hereditary Xerocytosis Reveals Enhanced Glycolytic Flux

Background: Hereditary xerocytosis (HX) is a rare cause of hemolytic anemia characterized by abnormalities of red blood cell (RBC) hydration. The majority of cases are caused by gain-of-function (GoF) variants in PIEZO1, which encodes a mechano-sensitive calcium channel in the RBC membrane. Such variants delay inactivation of the channel, leading to increased calcium influx. This in turn activates the Gardos channel, resulting in increased potassium efflux with concomitant loss of water. The consequent dehydration of the RBC reduces cellular deformability and induces premature splenic clearance. RBCs have no mitochondria and rely solely on glycolysis for the generation of adenosine triphosphate (ATP). ATP is essential for the RBC to maintain its ion balance, anti-oxidative defense mechanisms and deformability. Previously, we demonstrated decreased activity of the key glycolytic enzyme pyruvate kinase (PK) in RBCs from HX patients. The metabolic effects of this decreased activity are largely unknown.

Aim: To explore the effects of decreased PK activity on glucose metabolism of HX RBCs using ex vivo glucose tracing.

Methods: Four healthy controls (HCs) and four patients with HX caused by a GoF variant in PIEZO1 were studied. PIEZO1 variants were: c.1792G>A p.(Val598Met); c.7367G>A p.(Arg2456His) (2 relatives); and c.7483-7488dupCTGGAG p.(Leu2495_Glu2496dup). RBCs were purified and incubated at 37 ⁰C in RPMI medium for 30 minutes. Then, medium was changed to glucose-free RPMI supplemented with ¹³C₆-carbon labeled glucose and RBCs were incubated up to 360 minutes. RBCs and medium samples were collected prior to medium change and at 0, 30, 60, 120, 240, and 360 minutes. Metabolites were extracted from pelleted RBCs and medium using methanol and measured using ultra high pressure liquid chromatography coupled to high resolution mass spectrometry. Peaks were integrated using TraceFindr software and unpaired t-tests with Welch’s correction were performed in GraphPad Prism.

Results: After changing the medium to glucose-free medium supplemented with ¹³C₆-carbon labeled glucose, negligible amounts of unlabeled glucose remained. Hence, only ¹³C₆-carbon labeled glucose was taken up and metabolized by the RBCs. Over time, we observed a decrease in the level of unlabeled and an increase in the level of ¹³C₆-carbon labeled glycolytic metabolites. In HCs, unlabeled glycolytic metabolites were almost completely replaced by ¹³C₆-carbon labeled metabolites after 360 minutes. This turnover was already observed after 240 minutes in HX RBCs. HX RBCs also demonstrated increased production of ¹³C₆-carbon labeled pyruvate (HC 11.6 µM vs HX 25.6 µM, p=0.04), intracellular lactate (HC 98.6 µM vs HX 302.0 µM, p=0.002) and secreted lactate (HC 279.3 µM vs HX 910.8 µM, p<0.0001), the end products of glycolysis. These results indicate enhanced glycolytic flux. Furthermore, we observed increased production of ¹³C₆-carbon labeled glucose-6-phosphate in HX RBCs compared to HCs (HC 5.6 µM vs HX 8.4 µM, not significant), which could indicate a slightly elevated glucose uptake.

Conclusion: We used glucose tracing to study glycolytic flux in RBCs from healthy donors and patients with HX due to a PIEZO1 GoF defect. We demonstrate that RBCs from HX patients show a faster turnover of unlabeled glycolytic metabolites, as well as increased production of ¹³C₆-carbon labeled end products of glycolysis. These results strongly suggest that glycolytic flux is enhanced in HX patients. Glycolysis might be increased in HX patients to produce more ATP to counteract the disturbed ion balance. Interestingly, this enhanced glycolytic flux occurs in presence of the previously reported decrease in PK activity. Further studies are warranted to establish if and how increased mechanosensitivity and intracellular calcium levels are associated with enhanced glycolysis, and if the increased reticulocyte count typically seen in HX patients plays a role. Notably, small molecule activators of PK are currently being tested in various hereditary anemias and could potentially also have a beneficial effect for HX RBCs by enhancing glycolytic flux, and thereby ATP production, even further. Finally, the results of this study suggest that glucose tracing is a useful tool to better understand HX (patho)physiology and it has the potential to study the effect of PK activation therapy in patients with HX and other hereditary red blood cell disorders.