AMPKalpha1 deletion shortens erythrocyte life span in mice: role of oxidative stress

Abstract

AMP-activated protein kinase (AMPK) is an energy sensor essential for maintaining cellular energy homeostasis. Here, we report that AMPKalpha1 is the predominant isoform of AMPK in murine erythrocytes and mice globally deficient in AMPKalpha1 (AMPKalpha1(-/-)), but not in those lacking AMPKalpha2, and the mice had markedly enlarged spleens with dramatically increased proportions of Ter119-positive erythroid cells. Blood tests revealed significantly decreased erythrocyte and hemoglobin levels with increased reticulocyte counts and elevated plasma erythropoietin concentrations in AMPKalpha1(-/-) mice. The life span of erythrocytes from AMPKalpha1(-/-) mice was less than that in wild-type littermates, and the levels of reactive oxygen species and oxidized proteins were significantly increased in AMPKalpha1(-/-) erythrocytes. In keeping with the elevated oxidative stress, treatment of AMPKalpha1(-/-) mice with the antioxidant, tempol, resulted in decreased reticulocyte counts and improved erythrocyte survival. Furthermore, the expression of Foxo3 and reactive oxygen species scavenging enzymes was significantly decreased in erythroblasts from AMPKalpha1(-/-) mice. Collectively, these results establish an essential role for AMPKalpha1 in regulating oxidative stress and life span in erythrocytes.

FIGURE 1.

AMPKα1 is the predominant isoform of AMPK expressed in mouse erythrocytes, and its deletion causes splenomegaly. A, AMPKα1 and total AMPKα protein expression in erythrocytes from wild-type, AMPKα1^−/−, and AMPKα2^−/− mice were detected by immunoblotting. Δ indicates nonspecific bands. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, representative picture of spleens from wild-type and AMPKα1^−/− mice. C, increased spleen index in AMPKα1^−/− mice compared with wild-type mice. Data were expressed as means ± S.D. (**, p < 0.01 versus WT, n = 12 in each group). D, hematoxylin and eosin-stained sections of spleen, showing clearly defined white pulp and red pulp in wild-type spleen, and evidence for extramedullary erythropoiesis in AMPKα1^−/− spleen. Thick arrow indicates white pulp and thin arrow indicates red pulp. E, Prussian blue-stained sections of spleen showing a marked increase of blue-stained iron deposits in spleen sections of AMPKα1^−/− mice. F, Giemsa-stained blood smears from wild-type and AMPKα1^−/− mice.

FIGURE 2.

Erythropoiesis is increased in AMPKα1^−/− mice. A, Ter119-PE and CD71-FITC staining of splenocytes from wild-type and AMPKα1^−/− mice. B, bone marrow cells were stained with Ter119-PE and CD71-FITC. R1, proerythroblasts; R2, basophilic erythroblasts; R3, late basophilic and polychromatophilic erythroblasts; R4, orthochromatic erythroblasts.

FIGURE 3.

Reticulocyte counts and plasma EPO levels in AMPKα1^−/− mice. A and B, thiazole orange staining followed by flow cytometry and statistical analysis. Data were expressed as means ± S.D. (**, p < 0.01 versus WT, n = 6 in each group). C, plasma EPO level determined by enzyme-linked immunosorbent assay. Data were expressed as mean ± S.D. (**, p < 0.01 versus WT, n = 10 in each group).

FIGURE 4.

Effects of blood infusion on erythrocyte life span and clearance in AMPKα1^−/− and wild-type mice. A, representative of two independent experiments showing decreased life span of erythrocytes in AMPKα1^−/− mice (n = 3 in each group). The dashed line indicates the time for the 50% clearance of N-succinimidyl- biotin-labeled red blood cells in mice. B, clearance of reinfused biotin-labeled blood cells. WT-WT, blood from wild-type donor infused into wild-type recipients; KO-WT, blood from AMPKα1^−/− donor infused into wild-type recipients; WT-KO, blood from wild-type donor infused into AMPKα1^−/− recipients; KO-KO, blood from AMPKα1^−/− donor infused into AMPKα1^−/− recipients (n = 5–7 in each group).

FIGURE 5.

Osmotic fragility and survival rate in wild-type and AMPKα1^−/− mice following PHZ treatment. A, decreased osmotic fragility in erythrocytes from AMPKα1^−/− mice (n = 5 in each group). The dashed line indicates the concentration of NaCl solution in which 50% of red blood cells were lysed. B, survival was markedly decreased in AMPKα1^−/− mice compared with wild-type littermate controls after PHZ treatment (n = 6 in each group).

FIGURE 6.

Increased ROS levels and protein oxidation in erythrocytes of AMPKα1^−/− mice. A, erythrocyte intracellular ROS levels were determined by flow cytometry using a 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (H₂DCFDA) probe without or with addition of exogenous H₂O₂ (50 μm). B, increased protein oxidation in lysates of erythrocytes from AMPKα1^−/− mice. A representative results from three independent experiments showing detection of oxidized proteins using 2,4-dinitrophenylhydrazine-derived carbonyl immunochemistry staining. C, quantification of the quantitative analysis of the oxidative status of wild-type and AMPKα1^−/− erythrocytes by comparison of signal intensity of lanes in B using AlphaEaseFC software version 4.0.0 (Alpha Innotech Co.). Data were expressed as mean ± S.D. (**, p < 0.01 versus WT, n = 3 in each group).

FIGURE 7.

Chronic administration of tempol prolongs the life span of erythrocytes in AMPKα1^−/− mice. A, reticulocyte index was measured after 5 weeks in all groups. NS, not significant. B, in vivo tempol therapy improves the life span of erythrocytes in AMPKα1^−/− mice. Wild-type and AMPKα1^−/− mice were provided normal drinking water or water containing tempol (1 mm) for 5 weeks, and the life span of in vivo biotinylated erythrocytes was measured. Tempol was administered continuously during the experimental period. C, selected antioxidant genes were monitored in wild-type and AMPKα1^−/− erythroblast cells by quantitative real time RT-PCR. Gene expression was normalized to β-actin and is expressed as a percentage of the control values. Data were expressed as means ± S.D. (*, p < 0.05; **, p < 0.01, n = 4 in each group). D, spleen indexes after the mice were provided tempol (1 mm) in drinking water for 4 months, (**, p < 0.01, n = 6 in each group). E, proposed mechanism illustrating that AMPKα1 is essential for normal erythrocyte life span.