Figure 1.
Generation of Pfkm−/− mice and the effect of Pfkm ablation on skeletal muscle glucose metabolism.
(A) Schematic representation of the wild-type Pfkm locus (top), targeting vector (middle) and targeted allele (bottom). The positions of HindIII (H) and XbaI (X) cleavage sites, the neoR (neo) and herpes simplex virus thymidine kinase (HSV-tk) genes, and the location of PCR primers used to detect wild-type (PFK-Fw and PFK-Rev) and targeted (Neo and PFK-Rev) alleles are shown. (B) PCR analysis of DNA from wild type (+/+), Pfkm+/− (+/−) and Pfkm−/− (−/−) mice using the primers shown in (A). The 0.6 Kb band corresponds to the wild-type allele and the 0.7 Kb band to the mutant allele. (C) Expression of Pfkm in skeletal muscle. Total RNA was obtained from gastrocnemius muscle and analyzed by Northern blot. A representative Northern blot hybridized with a Pfkm probe is shown. (D) PFK activity was determined in skeletal muscle as indicated in Materials and Methods. Basal PFK activity in wild-type mice was 36±2.4 U/g tissue. (E–G) Glucose-6-phosphate (E), glucose (F) and glycogen (G) concentrations were determined in perchloric extracts of skeletal muscle from 2–3 month-old wild-type (+/+) and and Pfkm−/− (−/−) mice, in rest and after exercise (5 min), as indicated in Materials and Methods. (H) Serum lactate levels in wild-type (+/+) and Pfkm−/− (−/−) mice, in rest and after exercise (5 min). Results in D-H are mean±SEM of five to eight mice per group. *P<0.05, **P<0.01 vs. wild-type.
Figure 2.
Pfkm−/− mice develop skeletal muscle glycogenosis and exercise intolerance.
(A) Glycogen storage evidenced by PAS staining in skeletal muscle sections from wild-type (WT) and Pfkm−/− mice. Scale bar 50 µm. (B) Transmission electron microscopic analysis of skeletal muscle. Arrows show glycogen storage and asterisks point to mitochondria. Scale bar 1 µm. (C) Pfkm−/− mice showing severe muscle cramps after exercise (5 min). (D) ATP and ADP content was determined in perchloric extracts of skeletal muscle from wild-type (+/+) and and Pfkm−/− (−/−) mice, in rest and after exercise (5 min), as described in Materials and Methods. Results are mean±SEM of five mice per group. *P<0.05 vs. wild-type.
Figure 3.
Effects of PFKM deficiency in skeletal muscle markers.
(A) Expression of key genes in oxidative metabolism in skeletal muscle of wild-type and Pfkm−/− mice: Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptor δ (PPARδ), carnitine palmytoiltransferase-1 (M-CPT-1), citrate sinthase (CS) and uncoupling protein 2 (UCP-2). (B) Histochemical staining for succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) activities in skeletal muscle of wild-type and Pfkm−/− mice. Scale bar 25 µm. (C) Expression of myosin heavy chains in skeletal muscle of wild-type and Pfkm−/− mice: Type I, IIa ,and IIb myosin heavy chains (MyHC-I, MyHC-IIa, MyHC-IIb). (D) Expression of the key genes in skeletal muscle glucose uptake, glucose transporter 4 (GLUT4) and hexokinase-II (HKII), in wild-type and Pfkm−/− mice. (E) Expression of pentose phosphate pathway genes, transaldolase (TALDO1) and transketolase (TK), in skeletal muscle of wild-type and Pfkm−/− mice. Relative expression in A, C, D and E was determined by quantitative PCR analysis of total RNA from skeletal muscle, as indicated in Materials and Methods. Results are mean±SEM of five mice per group. *P<0.05 vs. wild-type.
Figure 4.
Effect of Pfkm ablation in diaphragm glucose metabolism and in respiratory muscle glycogen storage.
(A) PFK activity was determined in diaphragm extracts as indicated in Materials and Methods. Basal PFK activity in wild-type mice was 26.5±4.2 U/g tissue. (B,C) Glucose-6-phosphate (B) and glycogen concentrations (C) were determined in diaphragm perchloric extracts from wild-type (+/+) and Pfkm−/− (−/−) mice, as indicated in Materials and Methods. Results are mean±SEM of five mice per group. *P<0.05, **P<0.01 vs. wild-type. (D,E) Glycogen storage evidenced by PAS staining in diaphragm sections (D) from wild-type (wt) and Pfkm−/− mice (scale bar 50 µm) and in intercostal muscle sections (E) from Pfkm−/− mice (scale bar 300 µm). IC, intercostal muscles; R, rib.
Figure 5.
Pfkm−/− mice show altered heart glucose metabolism and develop cardiomegaly with age.
(A) PFK activity was determined in heart extracts. Basal PFK activity in wild-type mice was 27.4±5.4 U/g tissue. (B,C) Glucose-6-phosphate (B) and glycogen concentrations (C) were determined in heart perchloric extracts from 2-month-old wild-type (+/+) and Pfkm−/− (−/−) mice. Results are mean±SEM of five mice per group. *P<0.05, **P<0.01 vs. wild-type. (D) Transmission electron microscopic analysis of cardiac muscle. Arrows show glycogen storage. Scale bar 2 µm. (E,F) One-year-old Pfkm−/− mice develop cardiac hypertrophy, evidenced by hematoxilin-eosin staining of heart sections (scale bar 1 mm) (E) and cardiomegaly (F). (G) Longitudinal sections of heart from 3-month-old mice stained with Masson trichromic reagent (scale bar 1 mm). Inset shows septum sections (scale bar 50 µm).
Figure 6.
Reduction of erythrocyte PFK activity leads to hemolysis, reticulocytosis, and splenomegaly.
(A) PFK activity was determined in blood cell lysates from wild-type (+/+) and Pfkm−/− (−/−) mice as indicated in Materials and Methods. (B,C) Glucose-6-phosphate (B) and 2,3-bisphosphoglycerate (2,3-BPG) (C) concentrations were determined in blood cell perchloric extracts as indicated in Materials and Methods. (D–F) Pfkm−/− show high serum bilirubin levels (D) and reticulocyte number (E,F). New methylene blue stained blood samples were extended on slices (E) and counted (F). Arrows indicate reticulocytes. Scale bar 15 µm. (G,H) Splenomegaly in Pfkm−/− mice. A high increase in spleen size (G) and weight (H) was observed. Scale bar 5 mm. (I,J) Hematopoietic precursors in cultured cells from spleen (I) and femur (J) from wild-type (+/+) and Pfkm−/− (−/−) mice. Results are mean±SEM of five to eight mice per group. **P<0.01 vs. wild-type.
Figure 7.
Pfkm−/− mice show increased skeletal muscle hypoxic markers, vascularization, and fiber necrosis.
(A) The expression of the hypoxia-induced factor (HIF-1α), pyruvate kinase (PK-M), lactate dehydrogenase (LDH), and glucose transporter-1 (GLUT-1) in skeletal muscle of Pfkm−/− mice was determined by quantitative PCR analysis, as indicated in Materials and Methods. Results are mean±SEM of four mice per group. *P<0.05, **P<0.01 vs. wild-type. (B) Skeletal muscle sections showed increased immunostaining for VEGF, leading to hypervascularization, as evidenced by greater immunostaining for endothelial cell marker PECAM-1 (scale bar 25 µm) and collagen IV (scale bar 10 µm). Arrows show blood vessels around muscle fiber. (C) Fiber necrosis in skeletal muscle sections of Pfkm−/− mice. Arrows indicate cell infiltration of necrotic fibers (scale bar 25 µm). (D) Muscle fiber regeneration is evidenced by multiple centrally located nuclei (arrows) (scale bar 25 µm).