INTRODUCTION Beta-Thalassemia is a genetic disease due to mutations in the β-globin gene characterized by anemia, ineffective erythropoiesis, splenomegaly and iron-overload. Glucose tolerance abnormalities and diabetes are common complications in thalassemia patients, mostly as a consequence of iron-mediated pancreas and liver damage.

Transferrin receptor 2 (TFR2) is a player of the iron-erythropoiesis cross-talk. In the liver it contributes to the transcriptional activation of hepcidin, the master regulator of systemic iron metabolism, while in erythroid cells it negatively modulates erythropoietin signaling. Tfr2 selective inactivation in the bone marrow (BM) stimulates erythropoiesis in wild-type (WT) mice and ameliorates anemia and iron-overload in β-thalassemia models. Our transcriptomic data suggest that this effect is achieved increasing the metabolic activity of erythroid cells. Since similar alterations in polycythemia mice with massive erythropoiesis expansion were associated with a rearrangement of systemic energetic metabolism, hypoglycemia and adipose tissue atrophy, we hypothesized that BM Tfr2 deletion might prevent the metabolic alterations of β-thalassemia mice.

Methods Thalassemic mice with (Hbbth3/+) or without BM Tfr2 (Tfr2BMKO/Hbbth3/+) were generated through BM transplantation (BMT). As controls, Tfr2BMWT mice were obtained transplanting WT BM cells. Twenty weeks after BMT mice of each genotype were divided into 2 cohorts: one was fed a high-fat diet (HFD, 45% fat) for 14 weeks, and the other a control diet (CD) which has the same formulation, but standard fat content (10%). Complete blood count, glycemia and response to glucose (GTT) and insulin (ITT) tolerance test were evaluated at the beginning and at the end of the protocol.

Results Twenty weeks after BMT hematological parameters were compatible with the different genotypes, with Hbbth3/+ miceshowing a severe anemia, which was significantly improved by BM Tfr2 deletion. In non-fasting conditions, Tfr2BMWT,Hbbth3/+ and Tfr2BMKO/Hbbth3/+ mice displayed similar blood glucose levels. However, after 16 hours of starvation, Hbbth3/+ mice showed a trend toward higher blood glucose levels compared to Tfr2BMWT; the levels were about 30% reduced in Tfr2BMKO/Hbbth3/+ animals, suggesting a higher glucose uptake and/or consumption. This effect was insulin-independent and evident exclusively when circulating glucose was limiting. Indeed, mice of the 3 genotypes responded similarly to ITT and GTT tests.

Since the effect of Tfr2 inactivation became evident in stressed conditions and Hbbth3/+mice did not show metabolic abnormalities, we investigated the response of our models to an HFD. We confirmed that the diet did not affect the hematological parameters.

As expected, Tfr2BMWT and Hbbth3/+ mice fed the HFD gained weight rapidly as compared to CD-fed animals. Interestingly, HFD Tfr2BMKO/Hbbth3/+ mice showed no statistically significant body weight increase as compared to CD animals, in line with an increased energy expenditure.

In non-fasting conditions, blood glucose levels in animals of the 3 genotypes fed the CD were comparable and were similarly increased by HFD, likely because of the reduced glucose utilization which paralleled fat accumulation. On the contrary, after starvation, glycemia was reduced in both Tfr2BMWT and Tfr2BMKO/Hbbth3/+ mice, while in Hbbth3/+ animals blood glucose levels remained high. These findings highlighted for the first time metabolic abnormalities of β-thalassemia mice, which were corrected by BM Tfr2 deletion. HFD Tfr2BMWTand Hbbth3/+mice became more sensitive to exogenous glucose administration, maintaining higher glycemia than CD-fed controls in a GTT test. On the contrary, Tfr2BMKO/Hbbth3/+ mice fed both CD or HFD showed comparable GTT response, showing that BM Tfr2 deletion might prevent diabetes development in β-thalassemia mice.

CONCLUSIONS Overall these findings, together with transcriptomic data, suggest that Tfr2BMKO/Hbbth3/+ animals require high glucose utilization to sustain the augmented erythropoiesis, and that BM Tfr2 deletion corrects not only the erythroid and iron phenotype, but also prevents the development of metabolic alterations of β-thalassemia mice. This mechanism seems unrelated to the insulin-mediated glucose uptake, suggesting that other players could be involved in the regulation of erythroid cells metabolism.

No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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