Role for Akt3/protein kinase Bgamma in attainment of normal brain size

Abstract

Studies of Drosophila and mammals have revealed the importance of insulin signaling through phosphatidylinositol 3-kinase and the serine/threonine kinase Akt/protein kinase B for the regulation of cell, organ, and organismal growth. In mammals, three highly conserved proteins, Akt1, Akt2, and Akt3, comprise the Akt family, of which the first two are required for normal growth and metabolism, respectively. Here we address the function of Akt3. Like Akt1, Akt3 is not required for the maintenance of normal carbohydrate metabolism but is essential for the attainment of normal organ size. However, in contrast to Akt1-/- mice, which display a proportional decrease in the sizes of all organs, Akt3-/- mice present a selective 20% decrease in brain size. Moreover, although Akt1- and Akt3-deficient brains are reduced in size to approximately the same degree, the absence of Akt1 leads to a reduction in cell number, whereas the lack of Akt3 results in smaller and fewer cells. Finally, mammalian target of rapamycin signaling is attenuated in the brains of Akt3-/- but not Akt1-/- mice, suggesting that differential regulation of this pathway contributes to an isoform-specific regulation of cell growth.

FIG. 1.

Generation of Akt3-deficient mice. (A) Diagram of the Akt3 genomic locus and the targeting vector. The positions of the Southern probe (probe) and PCR primers (1, 2, and 3) are indicated. (B) Genomic DNAs isolated from wild-type (+/+), Akt3 heterozygous (+/−), and Akt3-null (−/−) mice were digested with EcoRI and analyzed by Southern blotting. (C) PCR analysis of genomic DNAs from wild-type (+/+), Akt3 heterozygous (+/−), and Akt3-null (−/−) mice. Primers 1 and 2 generate a 650-bp band from wild-type DNA, and primers 1 and 3 generate a 700-bp band from DNA with a targeted Akt3 allele.

FIG. 2.

Akt protein expression. (A) Tissues from three adult female wild-type mice were isolated for protein analysis. The amount of Akt3 protein in each tissue relative to the amount in the wild-type brain is depicted, and a representative Western blot is shown. Data for brains isolated from adult Akt1 (1KO)-, Akt2 (2KO)-, and Akt3 (3KO)-deficient mice are shown for comparison. The graph represents means ± SD for three blots. (B) Brains were dissected from three adult wild-type C57BL/6 mice, and the cortices, hippocampusi, and cerebella were isolated and homogenized for Akt protein measurements. Two dilutions (10 μg [a] and 20 μg [b]) from each sample were subjected to Western blot analysis. A standard curve of the recombinant protein for each isoform was used to quantify the amount of Akt in the lysate (as detailed in Materials and Methods). The mean amounts of protein in the two dilutions and the standard deviations are depicted by the bar graph, and a representative Western blot is shown below.

FIG. 3.

Selective reduction of brain size in Akt3-deficient mice. (A) Brains were dissected from 30-week-old wild-type (WT) and Akt3-deficient (3KO) mice. (B) Plot of adult brain weights from Akt3 knockout mice and littermate controls (N = 6 or 7). The mean value is shown by a horizontal red bar. The asterisk indicates that P < 0.001 for wild-type versus Akt3-deficient brains. Male (C) and female (D) mice were weighed periodically during the first 8 weeks of life. Filled circles represent wild-type mice; open circles depict Akt3-deficient mice. Values are means ± standard errors of the means (SEM) (N = 15 to 20).

FIG. 4.

Allometric plot. A log-log plot with first-order regression of the brain weights (BR) of wild-type and Akt3 knockout mice versus their corresponding body weights (BW) determined at postnatal days 3, 7, 12, 16, 20, 21, and 23 is shown. The results of the corresponding regression analyses were as follows: for wild-type mice, BR = 0.08BW^0.75 (r = 0.97); for Akt3 knockout mice, BR = 0.09BW^0.61 (r = 0.98).

FIG. 5.

Cell size in the Akt3-deficient brain. (A and B) Sections of cortex stained with Hoechst 33258. Bar = 100 μm. (C) Mean cell areas based on nuclear densities. *, P < 0.001 for Akt3 knockout (KO) versus wild-type (WT) brains.

FIG. 6.

Metabolism in Akt3-deficient mice. (A and B) Glucose tolerance tests were performed with male (A) and female (B) mice as described in Materials and Methods. Values represent means ± SEM (N = 8 to 10). Filled squares represent wild-type mice; open squares depict Akt3-deficient mice. (C and D) Insulin tolerance tests were performed with male (C) and female (D) mice as described in Materials and Methods. Values represent means ± SEM (N = 8 to 10). Filled squares represent wild-type mice; open squares depict Akt3-deficient mice.

FIG. 7.

Cell size in Akt1-deficient brains and hearts. (A and B) Sections of cortex stained with Hoechst 33258. Bar = 50 μm. (C) Mean cell areas based on nuclear densities. (D and E) Micrographs of sections of hearts from Akt1 knockout mice (E) and littermate controls (D) were stained with FITC-conjugated wheat germ agglutinin (green) and propidium iodide (red). (F) The mean cell area of cardiomyocytes is represented as a percentage of the mean wild-type control value. Values are means ± SD (N = 3). *, P < 0.01 for Akt1 knockout myocyte size versus wild-type control size.

FIG. 8.

Phosphorylation of ribosomal protein S6 in brains from Akt-deficient mice. (A and C) Lysates (25 μg) from postnatal day 1 brains were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed for phosphorylated ribosomal protein S6 (P-S6) or total S6 ribosomal protein as indicated. Each lane represents a lysate from an individual animal. (B and D) The intensities of phosphorylated ribosomal protein S6 from several experiments were normalized to that of the total S6 ribosomal protein and expressed as percentages of the mean wild-type value. The data shown are means ± SD (N = 3 to 6). *, P < 0.01 for Akt3 knockout versus wild-type littermates.