AMPKα1 Deletion Shortens Erythrocyte Life Span in Mice

Reagents and Animals

Rabbit anti-AMPKα antibody was obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-AMPKα1 and rabbit anti-AMPKα2 antibodies were obtained from Bethyl Laboratories, Inc. (Montgomery, TX). Mouse anti-glyceraldehyde-3-phosphate dehydrogenase antibody was from Abcam Inc. (Cambridge, MA). Other chemicals and organic solvents of the highest available grade were obtained from Sigma. AMPKα1^−/− mice and AMPKα2^−/− mice were described elsewhere (18, 19). Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy) was provided to the animals for the indicated times (see “Results”) at a concentration of 1 mm in drinking water. Mice were handled in accordance with study protocols approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Science Center.

Western Blot

Freshly heparinized blood was collected from wild-type, AMPKα1^−/−, and AMPKα2^−/− mice. Blood samples were centrifuged at 1,000 × g for 10 min. The plasma and buffy coat were removed by aspiration. The pellets were suspended in three volumes of phosphate-buffered saline, pH 7.4, and then passed through a column of α-cellulose and microcrystalline cellulose (1:l, w/w) to remove platelets and white blood cells, as described previously (20). Washed RBCs were then collected and lysed in 25 mm Tris·HCl, pH 7.6, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, supplemented with 0.25 mg/ml phenylmethylsulfonyl fluoride, 1× protease inhibitor, and 1× phosphatase inhibitor mixtures (Calbiochem) (0.15 ml of lysis solution per 1 × 10⁷ cells). An equal volume of 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 112.5 mm NaCl, 37.5 mm Tris·HCl, pH 7.4, was added, and cleared extracts were denatured, electrophoresed (25 μg), and blotted. For chemiluminescence, horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and SuperSignal West Dura reagent (Pierce) were used.

Histological and Hematological Analyses

10–12-Week-old male and female mice were weighed and sacrificed. Selected organs, including spleen, liver, kidney, heart, and femur, were removed and weighed to calculate an organ index (organ weight/body weight × 100). Tissues processed for microscopic evaluation were fixed in 10% neutrally buffered formalin, embedded in paraffin, sectioned, mounted on slides, and stained with hematoxylin and eosin or Prussian blue. Bones were decalcified before fixing in formalin. Tissue sections were examined with an Olympus AX70 microscope with UPlanFI 20×/0.50 or 40×/0.85 lens, blood smears with oil immersion UPlanApO 100×/1.30 lens, and an Qimaging Retiga Exi digital CCD camera (QImaging Co.) using QCapture software (version 2.98.0). Peripheral blood samples were harvested in EDTA-coated microtubes (Aktiengesellschaft Co., Germany) by retro-orbital sinus bleeding and analyzed with a HEMAVET 950 Veterinary Hematology Analyzer (Drew Scientific Inc.) using the mouse species program. Blood smears were stained with Giemsa or new methylene blue and analyzed microscopically.

Flow Cytometry Analysis

Mice spleens were mechanically dissociated through a 70-μm strainer and washed with cold phosphate-buffered saline containing 2% fetal calf serum. Splenocyte single cell suspensions were double-stained with antibodies against fluorescein isothiocyanate-conjugated CD71 (CD71-FITC) and phycoerythrin-conjugated erythroid antigen (Ter119-PE; BD Biosciences). Flow cytometry was performed using a FACSCalibur (BD Biosciences), and FACS data were analyzed using Summit version 4.3 software. Reticulocytes were assayed using a Retic-Count reticulocyte reagent system (BD Biosciences).

Erythrocyte Life Span Determination

In vivo biotinylation followed by FACS analysis were performed according to Suzuki and Dale (21) with modifications (22). Briefly, RBCs were labeled in vivo by intravenous injection of 30 mg/kg N-succinimidyl-6-[biotinamido] hexanoate (Thermo Fisher Scientific). Small samples (5 μl) of peripheral blood were collected from tail vein 1 h after biotin labeling and stained using streptavidin-phycoerythrin. Staining was followed by FACS analysis to ensure that at least 95% of red cells were labeled with biotin. Blood samples were analyzed at 7-day intervals to quantify biotin-labeled cells remaining in the circulation.

Erythropoietin (EPO) Enzyme-linked Immunosorbent Assay

EPO level in blood plasma was determined using the Quantikine mouse EPO enzyme-linked immunosorbent assay kit from R & D Systems, Inc. (Minneapolis, MN) according to the manufacturer's protocol.

Erythrocyte Reinfusion and Clearance Test

In vivo RBC tracking was performed as reported previously (23, 24) with modifications. Briefly, RBCs of donor mice were biotin-labeled in vivo by tail vein injection of N-succinimidyl-6-[biotinamido] hexanoate, as described above. One hour after biotin infusion, blood was collected from donor mice and placed in tubes containing heparin as an anticoagulant. A small aliquot of labeled RBCs was incubated with streptavidin-phycoerythrin and analyzed by flow cytometry to ensure that at least 95% of the blood cells were labeled. Blood was washed and resuspended in sterile saline, and 100 μl of RBC suspension was infused into each recipient mouse by tail vein injection. Initial postinfusion blood samples were obtained after 30 min and analyzed by flow cytometry. The typical initial percentage of biotin-labeled RBCs (recorded as day 0%) was greater than 5% in recipient mice. Seven days after blood reinfusion, the remaining labeled red cells were determined (recorded as day 7%). The red cell clearance rate was calculated as follows: clearance rate = ((day 7% − day 0%)/day 0%) × 100%.

Protein Oxidation Detection

The Oxyblot^TM protein oxidation detection kit (Millipore, MA), based on a previously described method (25), was used as described by the manufacturer. Briefly, RBC lysates from wild-type mice and AMPKα1^−/− mice were derivatized with dinitrophenylhydrazine, and derivatized samples (20 μg of protein/sample) were separated by SDS-PAGE on 12% gels. Proteins were transferred to nitrocellulose membranes, and membranes were blocked and immunoblotted using OxyBlot kit methods and reagents. Bands were visualized with chemiluminescence (captured on film) and analyzed densitometrically. Membranes stained with Red Ponceau S (Sigma) were used as loading controls.

Intracellular ROS Measurement

Red cell intracellular ROS measurement was performed essentially as reported previously (8). RBCs were washed and resuspended in staining buffer (BD Biosciences) and loaded with 5 μm 5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (Invitrogen), in the dark for 30 min at 37 °C in a 5% CO₂ atmosphere. Intracellular fluorescent products were measured immediately by flow cytometry.

Osmotic Fragility Assay

The osmotic fragility of erythrocytes in freshly collected blood from AMPKα1^−/− and wild-type mice was measured with modifications (26). Briefly, RBCs were harvested into heparin-coated tubes and suspended in varying concentrations of NaCl, and samples were incubated at room temperature for 10 min and then centrifuged at 1500 × g for 10 min to sediment unlysed cells and stroma. 200 μl of supernatant was collected, and the hemoglobin concentration of each sample was measured by Synergy HT multidetection microplate reader (BioTek Instrument, Inc) at 540 nm, with appropriate controls. The lyses percentage of RBCs was calculated from the absorbance, and a fragility curve was generated by plotting varying salt concentrations versus hemolysis.

Fluorescence-activated Cell Sorting (FACS) and Quantitative Reverse Transcription (RT)-PCR

Bone marrow cell suspensions were stained with antibodies against CD71-FITC and Ter119-PE (BD Biosciences) before sorting on an Influx Cell Sorter. Total RNA in sorted cells was extracted using an RNeasy mini kit (Qiagen), and cDNA was synthesized from total RNA using an iScript cDNA synthesis kit (Bio-Rad), as described by the respective manufacturers. Real time quantitative RT-PCR was performed using iQ SYBR Green SuperMix and the MyiQ single color real time PCR detection system (Bio-Rad). Primer sequences are presented in the supplemental material. Quantitative RT-PCR results were analyzed according to the 2^−ΔΔCT method (27) using expression of the β-actin gene as a housekeeping control.

Statistical Analysis

Two-tailed Student's t test was conducted, or two-way analysis of variance followed by Bonferroni post hoc analyses was used to determine statistical differences between various experimental and control groups. p value < 0.05 was considered statistically significant.

AMPKα1 Is the Predominant Isoform in Murine Erythrocytes

We found that AMPKα1 is the predominant isoform of AMPKα in murine erythrocytes, as demonstrated by Western blots. Using an antibody specific for AMPKα1, we confirmed the expression of AMPKα1 in mice RBCs (Fig. 1A). Because no recognizable bands were seen on the blot with the same loading with an antibody specific for AMPKα2, we proceeded to use an antibody that recognizes both AMPKα1 and AMPKα2 isoforms. As shown in Fig. 1A, AMPKα was largely absent in erythrocytes from AMPKα1^−/− mice, whereas there was no significant difference of the expression of total AMPKα between AMPKα2^−/− and wild-type erythrocytes.

Genetic Deletion of AMPKα1, but Not AMPKα2, in Mice Causes Splenomegaly and Moderate Anemia

Next, we determined if genetic deletion of AMPKα1 or AMPKα2 affected the pathology of organs, including hearts, kidneys, and spleens. AMPKα1^−/− mice exhibited splenomegaly (Fig. 1, B and C), whereas body weights and the weights of other major organs were not significantly changed (supplemental Fig. 1); this phenotype was not observed in AMPKα2^−/− mice. Hematological tests showed moderate anemia in AMPKα1^−/− mice, as evidenced by low erythrocyte number, low hemoglobin content, and increased red cell distribution width compared with wild-type mice. Leukocyte and platelet counts were not significantly changed in AMPKα1^−/− mice compared with wild-type littermates (Table 1). In AMPKα2^−/− mice, there were no significant differences in erythrocyte or platelet parameters compared with wild-type mice (supplemental Table 1). Accordingly, subsequent experiments focused on AMPKα1^−/− mice. Hematoxylin and eosin-stained sections of spleens from AMPKα1^−/− mice revealed expanded red pulps and an increase in erythroid precursor cells (Fig. 1D), indicating a compensatory reaction to anemia. Prussian blue staining showed elevated iron deposits in spleen sections from AMPKα1^−/− mice (Fig. 1E), indicating increased destruction of erythrocytes in the spleen, and Giemsa staining of blood smears indicated polychromasia, combined macrocytosis and microcytosis, and reticulocytosis in AMPKα1^−/− mice (Fig. 1F). Collectively, these data indicate that a deficiency of AMPKα1 caused moderate anemia, characterized by a significant decrease in red cell count, hematocrit, and hemoglobin in peripheral blood relative to wild-type littermate controls. The phenotypic differences between AMPKα1^−/− mice and AMPKα2^−/− mice likely stem from the distinctively different expression levels of these two isoforms in erythrocytes.

Erythropoiesis Is Increased in AMPKα1^−/− Mice

The anemia observed in AMPKα1^−/− mice could be due to defective red cell maturation (ineffective erythropoiesis), increased red cell destruction (hemolysis), or a combination of both processes. We first analyzed splenocytes by flow cytometry. Erythroid cells (Ter119⁺) in the spleens of AMPKα1^−/− mice were increased compared with those of wild-type mice (supplemental Fig. 2A), whereas the relative abundance of other cell types in the spleen was not increased (supplemental Fig. 2, B–E). A subpopulation of Ter119^high cells was distinguished based on their expression of the transferrin receptor (CD71), which decreases with erythroblast maturation. Combining Ter119 and CD71 expression distinguishes four subpopulations of erythroid cells, Ter119^medCD71^high, Ter119^highCD71^high, Ter119^highCD71^med, and Ter119^highCD71^low, corresponding to increasingly mature erythroblasts (28). Using this approach, we found a dramatic expansion of the early erythroblast population (Ter119^highCD71^high) and increased proerythroblast population (Ter119^mediumCD71^high) in AMPKα1^−/− mice (Fig. 2A). Further analysis of the Ter119^high erythroblasts was arbitrarily based on the forward scatter (FSC) parameter and CD71 expression level. Ter119^high erythroblasts can be subdivided into three groups as follows: Ery.A (Ter119^highCD71^highFSC^high) are basophilic; Ery.B (Ter119^highCD71^highFSC^low) are late basophilic and polychromatic; and Ery.C (Ter119^highCD71^lowFSC^low) are orthochromatic erythroblasts and reticulocytes. Increasingly mature erythroblasts, according to the reports (29, 30), indicated that the major elevated population was late basophilic and polychromatic cells (supplemental Fig. 4).

Next, using new methylene blue-stained blood smears (data not shown) and flow cytometry with thiazole orange staining (Fig. 3A), we determined the number of reticulocytes and found that the reticulocyte count in AMPKα1^−/− mice was 3–4-fold higher than that in wild-type mice (Fig. 3B). The increased reticulocyte levels were correlated with elevated plasma levels of EPO (Fig. 3C). These data indicate that erythropoiesis was elevated in AMPKα1^−/− mice, possibly reflecting a compensatory reaction to the moderate anemia observed in these mice.

Erythrocyte Life Span Is Shortened in AMPKα1^−/− Mice

The normal life span of circulating RBCs, which is determined by their clearance from the peripheral circulation (predominantly by the spleen), has been reported to be ∼120 days and 40 days in humans and mice, respectively (31). To determine more directly whether the observed anemia was due to decreased production of mature RBCs or increased destruction of RBC in the circulation, we measured erythrocyte life span using direct in vivo biotin labeling. To this end, wild-type and AMPKα1^−/− mice were injected with N-succinimidyl-6-[biotinamido] hexanoate to label RBCs, and cell life span was determined by flow cytometric analysis of circulating, biotinylated RBCs. The time required for 50% of labeled erythrocytes to be lost from wild-type mice (half-life) was about 24 days, consistent with previous reports (5, 22). However, the half-life of labeled erythrocytes decreased to about 14 days in AMPKα1^−/− mice (Fig. 4A).

To further demonstrate the destruction of erythrocytes in AMPKα1^−/− mice, we performed a blood cross-transfusion experiment. Wild-type mice received either biotin-labeled wild-type erythrocytes (WT-WT) or biotin-labeled AMPKα1^−/− erythrocytes (KO-WT), and AMPKα1^−/− mice received either biotin-labeled wild-type erythrocytes (WT-KO) or biotin-labeled AMPKα1^−/− erythrocytes (KO-KO). The result demonstrated that the KO-WT group had a higher clearance rate of infused erythrocytes than did the WT-WT group, and similar results were observed when compared KO-KO with WT-KO group (Fig. 4B), suggesting accelerated clearance of erythrocytes in AMPKα1^−/− mice. Consistent with these results, Prussian blue-stained spleen sections from AMPKα1^−/− mice showed a marked increase in iron deposits.

Osmotic Fragility Is Decreased in Erythrocytes from AMPKα1^−/− Mice

To uncover the possible cause of why erythrocytes from AMPKα1^−/− mice had an increased clearance rate, we first determined the osmotic fragility of erythrocytes. As depicted in Fig. 5A, AMPKα1^−/−-deficient erythrocytes were significantly more resistant to hypotonic lysis than were wild-type red cells (Fig. 5A), suggesting that they had a more rigid and less deformable cell membrane.

Phenylhydrazine-induced Mortality Is Increased in AMPKα1^−/− Mice

Phenylhydrazine (PHZ), a potent inducer of oxidative stress in vivo, is capable of denaturing hemoglobin and hemolyzing red cells (32). To test PHZ tolerance, we injected wild-type and AMPKα1^−/− mice with 100 mg/kg PHZ. As shown in Fig. 5B, five of six PHZ-treated AMPKα1^−/− mice died within 24 h, and the 6th died within 48 h after treatment. In contrast, all six wild-type mice receiving PHZ survived (Fig. 5B). These results imply that AMPKα1^−/− mice are hypersensitive to PHZ-induced oxidative stress.

Intracellular ROS Levels and Protein Oxidation Are Increased in AMPKα1^−/− Erythrocytes

Next, we determined if increased levels of protein aggregates were due to increased oxidative stress in erythrocytes. Protein oxidation can be measured indirectly by Western blotting for carbonyls that result from the reaction of side chains of lysine, proline, threonine, or arginine with ROS (25, 33). Intracellular ROS concentrations measured under base-line conditions and after challenging with exogenous H₂O₂ (50 μm) showed that ROS levels were elevated in erythrocytes from AMPKα1^−/− mice compared with wild-type mice under both conditions (Fig. 6A). In agreement with the observed elevation in ROS concentrations, oxidized protein levels in erythrocytes from AMPKα1^−/− mice were significantly increased (∼7-fold) as measured by 2,4-dinitrophenylhydrazine-derivatized carbonyl immunochemical staining (Fig. 6, B and C).

Chronic Administration of Tempol Prolongs the Life Span of Erythrocytes in AMPKα1^−/− Mice

To further evaluate the potential effect of ROS on shortened red cell survival, we fed tempol, a potent antioxidant, to wild-type and AMPKα1^−/− mice. Tempol treatment had no effect on reticulocyte index or erythrocyte life span in wild-type mice. However, it significantly increased the life span of erythrocytes in AMPKα1^−/− mice but decreased the reticulocyte index (Fig. 7, A and B). 4 months after tempol treatment, the spleen index was significantly decreased in treatment groups compared with no treatment AMPKα1^−/− controls (Fig. 7C).

Expression of the Foxo3 Transcription Factor and Antioxidant Genes Is Decreased in Erythroblast Cells from AMPKα1^−/− Mice

Finally, we investigated the molecular basis of increased oxidative stress in the erythrocytes from AMPKα1^−/− mice by measuring the mRNA levels of several antioxidant genes known to play a key role in erythrocytes. Total RNA was extracted from sorted CD71^highTer119^high erythroblast cells from wild-type and AMPKα1^−/− mice and analyzed by quantitative real time RT-PCR. As shown in Fig. 7D, the expression of the Foxo3 transcription factor was significantly reduced in the erythroblast cells from AMPKα1^−/− mice compared with that in wild-type mice; the mRNA levels of Sod2, catalase, and glutathione peroxidase (GPx-1) were also reduced.