Hyperactive Erythropoiesis Induced By Hematopoietic Tfr2 Deletion Corrects Glucose Abnormalities in β-Thalassemic Mice
Authors: Di Modica Simona Maria , Tanzi Emanuele , Pagani Alessia , Silvestri Laura , Nai Antonella 
Published: December 2024

Introduction: β-thalassemia is a genetic disease characterized by anemia, ineffective erythropoiesis and iron-overload due to inadequate hemoglobin production, which leads to premature cell death. Alterations of glucose metabolism and diabetes are common complications of the disease, usually ascribed to organ dysfunction because of iron accumulation. However, β-thalassemia carriers, who do not present iron-overload, have a higher risk of developing diabetes compared to the general population, suggesting that factors other than iron may contribute to glycometabolic abnormalities.

Transferrin Receptor 2 (TFR2), sensor of body iron concentration, is a negative regulator of erythropoietin (EPO) signalling in erythroid cells and its inactivation in the hematopoietic compartment increases red blood cell (RBC) and hemoglobin (Hb) production in both wild-type mice and murine models of genetic and acquired anemias.

In physiologic conditions, EPO-driven induction of erythroid cells' differentiation causes a shift of their energetic metabolism toward oxidative phosphorylation (OXPHOS). Interestingly, in polycythemia vera mice, characterized by EPO signalling overactivation, the high metabolic and mitochondrial activity of erythroid cells leads to systemic hypoglycemia. Thus, β-thalassemic erythroblasts, which fail in differentiating and producing red blood cells, consuming less glucose, might contribute to diabetes development in β-thalassemia.

Aims: Here we aim at verifying whether hematopoietic Tfr2 deletion, enhancing EPO signalling, might stimulate erythropoiesis increasing the metabolic activity of erythroid cells, thus preventing glucose abnormalities in β-thalassemia.

Methods: Wild-type (WT) and β-thalassemic (Hbbth3/+) mice with or without hematopoietic Tfr2 (Tfr2BMKO) were generated through bone marrow transplantation (BMT). A cohort of mice was sacrificed 8 weeks after BMT, when hematopoiesis was fully reconstituted, for RNA-seq analysis on sorted erythroid precursors at different stages of differentiation. A second cohort was used for the evaluation of complete blood count, fasting and non-fasting glycemia, and serum insulin levels at 8-10 weeks after BMT.

Results: As previously shown, 10 weeks after BMT, Hbbth3/+ mice are anemic, while Tfr2-deficientmice have RBC count and Hb levels higher than Tfr2BMWT and Hbbth3/+ controls respectively, confirming that hematopoietic Tfr2 deletion enhances erythropoiesis both in wild-type and thalassemic settings.

In line with patients' data, Hbbth3/+ mice are hyperglycemic both in basal conditions and after 16 hours of starvation, in the absence of differences in serum insulin levels, while Tfr2BMKO mice show reduced non-fasting glycemia both in wild-type and in thalassemic contexts. Tfr2BMKO/Hbbth3/+ animals have lower blood glucose levels than Hbbth3/+ also in fasting conditions, suggesting that the enhanced erythropoiesis driven by hematopoietic Tfr2 deletion promotes glucose consumption also in stress conditions. Interestingly, blood glycemia negatively correlates with both RBC count and Hb levels, supporting the hypothesis that alterations in erythropoiesis might significantly impact on systemic glucose metabolism. This effect is independent from the iron status, as evident by hypoglycemia of Tfr2BMKO mice, characterized by expanded erythropoiesis, but iron levels comparable to Tfr2BMWT.

In line with these findings, RNA-seq analysis shows increased expression of genes involved in glucose and lipid transport, glycolysis and OXPHOS in Tfr2-deficient erythroblasts compared to both wild-type and Hbbth3/+ cells, mainly at the most differentiated stages. Of note, the metabolic hyperactivation of Tfr2-deficient cells correlates with an increased expression of members of the EPO-JAK2-STAT5 pathway and relative target genes.

Conclusion: These results indicate that glucose uptake and metabolism are crucial to support terminal erythroid differentiation and that Tfr2-deficient cells increase glucose consumption to sustain enhanced erythropoiesis, thus leading to systemic hypoglycemia and correcting metabolic abnormalities in β-thalassemia. Overall, these findings substantiate hematopoietic Tfr2 targeting as a potential strategy to globally manage β-thalassemia and highlight an unexplored role for erythropoiesis in the regulation of systemic glucose homeostasis.