Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene

Abstract

The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Here we report the disruption of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt1(-/-) mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt1(-/-) mice, upon exposure to genotoxic stress, is shorter. However, Akt1(-/-) mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt1(-/-) male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt1(-/-) mice, and Akt1(-/-) thymocytes are more sensitive to apoptosis induced by gamma-irradiation and dexamethasone. Finally, Akt1(-/-) mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal.

Figure 1

Generation of Akt1^−/− mice. (A) Targeted disruption strategy to delete the akt1 gene. Shown from top to bottom, the wild-type akt1 allele with indicated exons, the targeting vector, and the disrupted allele. The locations of the PCR primers used to screen for homologous recombination are depicted by small arrows. A short bar spanning the region between exons 4 and 5 depicts the DNA fragment used as a DNA probe for Southern blots. (B) Mouse genotyping by Southern blot analysis. Genomic DNA extracted from mouse tails was digested by NcoI and hybridized with a DNA fragment spanning the region between exons 4 and 5. The wild-type fragment is ∼10 kb, whereas the deleted akt1 allele is ∼8.5 kb. (C) Western blot analysis of proteins extracted from mouse embryo fibroblasts (MEFs) isolated from wild-type embryos (+/+), heterozygous (+/−), and homozygous mutant embryos (−/−). (Top panel) Western blot analysis of twofold concentrations of extract using Akt1-specific antibodies (UBI). (Bottom panel) Western blot analysis with Akt1 pleckstrin homology (PH) domain-specific antibodies (UBI). Blots were probed with anti-β-actin as a control. (D) Side view of 5-week-old mice from the same littermate. Wild-type Akt1 (+/+) and Akt1 homozygous deletion (−/−) mice are shown. (E) Relative body weight of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Akt1 mutant mice 30 d after birth. The weight of each mouse is expressed relative to the mean weight of sex-matched, wild-type littermates in each of seven different litters.

Figure 1

Generation of Akt1^−/− mice. (A) Targeted disruption strategy to delete the akt1 gene. Shown from top to bottom, the wild-type akt1 allele with indicated exons, the targeting vector, and the disrupted allele. The locations of the PCR primers used to screen for homologous recombination are depicted by small arrows. A short bar spanning the region between exons 4 and 5 depicts the DNA fragment used as a DNA probe for Southern blots. (B) Mouse genotyping by Southern blot analysis. Genomic DNA extracted from mouse tails was digested by NcoI and hybridized with a DNA fragment spanning the region between exons 4 and 5. The wild-type fragment is ∼10 kb, whereas the deleted akt1 allele is ∼8.5 kb. (C) Western blot analysis of proteins extracted from mouse embryo fibroblasts (MEFs) isolated from wild-type embryos (+/+), heterozygous (+/−), and homozygous mutant embryos (−/−). (Top panel) Western blot analysis of twofold concentrations of extract using Akt1-specific antibodies (UBI). (Bottom panel) Western blot analysis with Akt1 pleckstrin homology (PH) domain-specific antibodies (UBI). Blots were probed with anti-β-actin as a control. (D) Side view of 5-week-old mice from the same littermate. Wild-type Akt1 (+/+) and Akt1 homozygous deletion (−/−) mice are shown. (E) Relative body weight of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Akt1 mutant mice 30 d after birth. The weight of each mouse is expressed relative to the mean weight of sex-matched, wild-type littermates in each of seven different litters.

Figure 1

Generation of Akt1^−/− mice. (A) Targeted disruption strategy to delete the akt1 gene. Shown from top to bottom, the wild-type akt1 allele with indicated exons, the targeting vector, and the disrupted allele. The locations of the PCR primers used to screen for homologous recombination are depicted by small arrows. A short bar spanning the region between exons 4 and 5 depicts the DNA fragment used as a DNA probe for Southern blots. (B) Mouse genotyping by Southern blot analysis. Genomic DNA extracted from mouse tails was digested by NcoI and hybridized with a DNA fragment spanning the region between exons 4 and 5. The wild-type fragment is ∼10 kb, whereas the deleted akt1 allele is ∼8.5 kb. (C) Western blot analysis of proteins extracted from mouse embryo fibroblasts (MEFs) isolated from wild-type embryos (+/+), heterozygous (+/−), and homozygous mutant embryos (−/−). (Top panel) Western blot analysis of twofold concentrations of extract using Akt1-specific antibodies (UBI). (Bottom panel) Western blot analysis with Akt1 pleckstrin homology (PH) domain-specific antibodies (UBI). Blots were probed with anti-β-actin as a control. (D) Side view of 5-week-old mice from the same littermate. Wild-type Akt1 (+/+) and Akt1 homozygous deletion (−/−) mice are shown. (E) Relative body weight of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) Akt1 mutant mice 30 d after birth. The weight of each mouse is expressed relative to the mean weight of sex-matched, wild-type littermates in each of seven different litters.

Figure 2

(A) Akt1^−/− mice are sensitized to genotoxic stress. Survival of Akt1^+/+ and Akt1^−/− mice was scored for 21 d after exposure to 10 Gy of γ-irradiation. (B) Oral glucose tolerance test (OGTT). Plasma glucose levels of 3-month-old Akt1^−/− male mice and their wild-type littermates were measured at 15, 30, 60, and 120 min after oral glucose load. The values are expressed as the mean ±SE (n = 4–6 for each genotype). Similar results were obtained in three independent experiments.

Figure 3

Spontaneous apoptosis in the testes and thymus of Akt1^−/− mice. (A) Morphology and histology of testes from Akt1^+/+ and Akt1^−/−. Testes were fixed with 4% paraformaldehyde, processed with paraffin, sectioned, and stained with hematoxylin/eosin. (Top panel) Seminiferous tubules of Akt null (−/−); (bottom panel) seminiferous tubules of wild-type (+/+) littermate (200× magnification). Arrows indicate multinucleated giant cells. (B) Spontaneous apoptosis in tubules from Akt1^−/− mice. Comparison of TUNEL-positive cells in tubules from Akt1^−/− mice (top panel) and from wild-type littermate (bottom panel). Cells with dark stained nuclei are apoptotic cells (200× magnification). (C) Sperm count of Akt1^+/+ male mice (n = 8, ±SE) and Akt1^−/− male mice (n = 10, ±SE; P < 0.001). (D) Spontaneous apoptosis is observed throughout the thymi of Akt1^−/− mice. TUNEL assays were performed on sections from thymi of Akt1^−/− (top panel) and wild-type (bottom panel) mice. Cells with dark stained nuclei are apoptotic cells. Magnification, 400×. (E) Percentage of apoptotic cells as measured by TUNEL-positive cells in sections from thymi of age-matched (1–2 mo old), wild-type, and Akt1^−/− mice as measured by counting 10 fields of each section (P < 0.001).

Figure 3

Spontaneous apoptosis in the testes and thymus of Akt1^−/− mice. (A) Morphology and histology of testes from Akt1^+/+ and Akt1^−/−. Testes were fixed with 4% paraformaldehyde, processed with paraffin, sectioned, and stained with hematoxylin/eosin. (Top panel) Seminiferous tubules of Akt null (−/−); (bottom panel) seminiferous tubules of wild-type (+/+) littermate (200× magnification). Arrows indicate multinucleated giant cells. (B) Spontaneous apoptosis in tubules from Akt1^−/− mice. Comparison of TUNEL-positive cells in tubules from Akt1^−/− mice (top panel) and from wild-type littermate (bottom panel). Cells with dark stained nuclei are apoptotic cells (200× magnification). (C) Sperm count of Akt1^+/+ male mice (n = 8, ±SE) and Akt1^−/− male mice (n = 10, ±SE; P < 0.001). (D) Spontaneous apoptosis is observed throughout the thymi of Akt1^−/− mice. TUNEL assays were performed on sections from thymi of Akt1^−/− (top panel) and wild-type (bottom panel) mice. Cells with dark stained nuclei are apoptotic cells. Magnification, 400×. (E) Percentage of apoptotic cells as measured by TUNEL-positive cells in sections from thymi of age-matched (1–2 mo old), wild-type, and Akt1^−/− mice as measured by counting 10 fields of each section (P < 0.001).

Figure 3

Spontaneous apoptosis in the testes and thymus of Akt1^−/− mice. (A) Morphology and histology of testes from Akt1^+/+ and Akt1^−/−. Testes were fixed with 4% paraformaldehyde, processed with paraffin, sectioned, and stained with hematoxylin/eosin. (Top panel) Seminiferous tubules of Akt null (−/−); (bottom panel) seminiferous tubules of wild-type (+/+) littermate (200× magnification). Arrows indicate multinucleated giant cells. (B) Spontaneous apoptosis in tubules from Akt1^−/− mice. Comparison of TUNEL-positive cells in tubules from Akt1^−/− mice (top panel) and from wild-type littermate (bottom panel). Cells with dark stained nuclei are apoptotic cells (200× magnification). (C) Sperm count of Akt1^+/+ male mice (n = 8, ±SE) and Akt1^−/− male mice (n = 10, ±SE; P < 0.001). (D) Spontaneous apoptosis is observed throughout the thymi of Akt1^−/− mice. TUNEL assays were performed on sections from thymi of Akt1^−/− (top panel) and wild-type (bottom panel) mice. Cells with dark stained nuclei are apoptotic cells. Magnification, 400×. (E) Percentage of apoptotic cells as measured by TUNEL-positive cells in sections from thymi of age-matched (1–2 mo old), wild-type, and Akt1^−/− mice as measured by counting 10 fields of each section (P < 0.001).

Figure 3

Spontaneous apoptosis in the testes and thymus of Akt1^−/− mice. (A) Morphology and histology of testes from Akt1^+/+ and Akt1^−/−. Testes were fixed with 4% paraformaldehyde, processed with paraffin, sectioned, and stained with hematoxylin/eosin. (Top panel) Seminiferous tubules of Akt null (−/−); (bottom panel) seminiferous tubules of wild-type (+/+) littermate (200× magnification). Arrows indicate multinucleated giant cells. (B) Spontaneous apoptosis in tubules from Akt1^−/− mice. Comparison of TUNEL-positive cells in tubules from Akt1^−/− mice (top panel) and from wild-type littermate (bottom panel). Cells with dark stained nuclei are apoptotic cells (200× magnification). (C) Sperm count of Akt1^+/+ male mice (n = 8, ±SE) and Akt1^−/− male mice (n = 10, ±SE; P < 0.001). (D) Spontaneous apoptosis is observed throughout the thymi of Akt1^−/− mice. TUNEL assays were performed on sections from thymi of Akt1^−/− (top panel) and wild-type (bottom panel) mice. Cells with dark stained nuclei are apoptotic cells. Magnification, 400×. (E) Percentage of apoptotic cells as measured by TUNEL-positive cells in sections from thymi of age-matched (1–2 mo old), wild-type, and Akt1^−/− mice as measured by counting 10 fields of each section (P < 0.001).

Figure 4

Akt1^−/− thymocytes and MEFs are more susceptible to apoptosis. (A) Survival of thymocytes in the absence of treatment. (B) Survival of thymocytes following exposure to γ-irradiation (500 rad). (C) Survival of thymocytes upon treatment with 1 μM of dexamethasone. Values represent the average of four independent experiments of thymocytes derived from three mice of each genotype (±SE). (█) Percentage survival of wild-type thymocytes; (□) percentage survival of Akt1^−/− thymocytes as measured by trypan blue exclusion. (D) Akt1^−/− MEFs are sensitized to apoptosis by various apoptotic stimuli. MEFs derived from Akt1^+/+, Akt1^+/−, and Akt1^−/− embryos were treated with 1 μg/mL anti-Fas antibody (Pharmingen), 10 ng/mL TNFα (Life Technologies), 5 μg/mL cycloheximide CHX (Sigma), UV irradiation (60 or 120 J/m²) in the presence (20% FCS) or absence (0% FCS) of fetal calf serum in the medium and apoptosis was quantitated 24 h following treatment. Apoptosis induced just by serum deprivation (0% FCS) was quantitated 48 h following serum deprivation. Apoptosis was measured by quantitation of DNA fragmentation using the cell death detection ELISA method (Boehringer Mannheim). The average of 3 independent experiments (±SE) is shown (*, P < 0.01; **, P < 0.05).