Abstract
Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance.
Highlights
MethodsMice Generation of Prkab1tm1b(KOMP)Wtsi (hereafter referred to as Prkab1tm1b) mice was performed using ES cell clone EPD0033_3_C09
Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia
Reductions in erythrocyte number (Fig. 1C) and mean corpuscular hemoglobin concentration (Fig. 1D) were observed only in a sex-specific manner; erythrocytes in Prkab1tm1b/tm1b mice were significantly smaller (Fig. 1E), with an increased red blood cell distribution width (Fig. 1F) in both sexes. These altered erythrocyte indices indicate a microcytic anemia with anisocytosis, similar to that reported in mice deficient in Prkaa1 or Prkag1 [4,5,6,7]
Summary
Mice Generation of Prkab1tm1b(KOMP)Wtsi (hereafter referred to as Prkab1tm1b) mice was performed using ES cell clone EPD0033_3_C09. Histologic analysis Spleen, liver, and leg bones were fixed in formalin and embedded in paraffin, and sections were stained with hematoxylin and eosin or Perls’ Prussian blue according to standard methods These were assessed in a blinded manner for any pathologic abnormalities. Erythropoiesis analysis Staining of single-cell suspensions of spleen, bone marrow, and whole blood with CD71, Ter119, CD45, Syto 16, and Sytox blue was performed as previously described [12] and analyzed on a BD LSRII instrument (full details in Supplementary Methods). Erythrocytes were counted and adjusted to 2 Â 106 RBC/mL, and the two genotypes were pooled and injected via the tail vein into recipient mice (10 weeks old) to transfuse 2 Â 108 RBCs/genotype (full details in Supplementary Methods) Osmotic resistance assay This was performed essentially as described [4] with hematocrit adjusted to 0.8% with 0.9% saline solution. Statistical analysis All data was analyzed in Prism Version 6 (Graph Pad) and analyzed with an unpaired two-tailed Student t test, Mann–Whitney test or two-way analysis of variance as indicated in the figure legends
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