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Mol Cell Biol. Nov 2006; 26(21): 8042–8051.
Published online Aug 21, 2006. doi:  10.1128/MCB.00722-06
PMCID: PMC1636753

Life with a Single Isoform of Akt: Mice Lacking Akt2 and Akt3 Are Viable but Display Impaired Glucose Homeostasis and Growth Deficiencies[down-pointing small open triangle]

Abstract

To address the issues of isoform redundancy and isoform specificity of the Akt family of protein kinases in vivo, we generated mice deficient in both Akt2 and Akt3. In these mice, only the Akt1 isoform remains to perform essential Akt functions, such as glucose homeostasis, proliferation, differentiation, and early development. Surprisingly, we found that Akt2−/− Akt3−/− and even Akt1+/− Akt2−/− Akt3−/− mice developed normally and survived with minimal dysfunctions, despite a dramatic reduction of total Akt levels in all tissues. A single functional allele of Akt1 appears to be sufficient for successful embryonic development and postnatal survival. This is in sharp contrast to the previously described lethal phenotypes of Akt1−/− Akt2−/− mice and Akt1−/− Akt3−/− mice. However, Akt2−/− Akt3−/− mice were glucose and insulin intolerant and exhibited an ~25% reduction in body weight compared to wild-type mice. In addition, we found substantial reductions in relative size and weight of the brain and testis in Akt2−/− Akt3−/− mice, demonstrating an in vivo role for both Akt2 and Akt3 in the determination of whole animal size and individual organ sizes.

The coordination between signal specificity and kinome redundancy is a fundamental issue in cell biology that is not yet fully understood. Although most of the major kinase families are conserved among metazoans, the vertebrate genome contains distinctly more genes encoding protein kinases than do those of worms or flies (24). Duplications of gene loci in vertebrates have led to the expansion of individual kinases into several homologous isoforms and brought about a concomitant increase in signaling complexity. It has been proposed that such isoforms are uniquely adapted to transmit distinct biological signals.

The Akt protein signaling kinase (also known as protein kinase B [PKB]) has a high level of evolutionary conservation and plays a key role in the conserved phosphoinositide 3-kinase (PI3K) signaling pathway (27). In mammals, Akt is implicated in the regulation of widely divergent cellular processes, such as metabolism, differentiation, proliferation, and apoptosis (3, 20). Accordingly, activation of Akt is promoted by numerous stimuli, including growth factors, hormones, and cytokines. There are three isoforms of Akt in mammals, termed Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ. These isoforms are products of distinct genes but are highly related, exhibiting >80% protein sequence identity and sharing the same structural organization. To understand the specific physiological functions of the individual isoforms, animal models deficient in Akt1, Akt2, or Akt3 have been generated. Mice lacking Akt1 demonstrate increased perinatal mortality and reductions in body weight of 20 to 30% (6, 8, 38). In contrast, Akt2-deficient mice are born in the expected Mendelian ratio and display normal growth, but they exhibit a diabetes-like syndrome with an elevated fasting plasma glucose level, elevated hepatic glucose output, and peripheral insulin resistance (7, 15). Akt3-deficient mice exhibit a reduction in brain weight resulting from decreases in both cell size and cell number but maintain normal glucose homeostasis and body weights (13, 33). These observations indicate that the three Akt isoforms have some differential, nonredundant physiological functions. The relatively subtle phenotypes of mice lacking individual Akt isoforms as well as the viability of the animals suggest, however, that for many functions Akt isoforms are able to compensate for each other. To address the issue of isoform redundancy, mice with combined Akt deficiencies have been generated. Mice lacking both Akt1 and Akt2 develop to term but die shortly after birth and display multiple defects. They exhibit a severe growth deficiency (body weights at birth are ~50% of normal weights), skeletal muscle atrophy, impaired skin development, and a delay in ossification (25). Mice mutant in both Akt1 and Akt3 die around embryonic day 12, with severe impairments in growth, cardiovascular development, and organization of the nervous system (37). Such data obtained from double knockout mice argue strongly for partially overlapping functions of Akt isoforms in vivo. Certain physiological functions of Akt are thus revealed only when total Akt levels are below a critical threshold in particular cell types and tissues. Here we report the generation of Akt2−/− Akt3−/− mice to determine the combined roles of these isoforms in Akt-related physiological processes, such as glucose metabolism, development, and growth. It was surprising to us that compound Akt2−/− Akt3−/− mice and even mice retaining only one functional allele of Akt1 (Akt1+/− Akt2−/− Akt3−/−) were viable and did not display any gross abnormalities. The body size of Akt2−/− Akt3−/− mice was reduced at birth and throughout postnatal life, and they exhibited severe glucose and insulin intolerance.

MATERIALS AND METHODS

Mice.

Akt3 mutant mice were generated as described previously (33). For the generation of Akt2 mutant mice, an ~9.4-kb mouse genomic DNA fragment containing exons 3 and 4 of the Akt2 gene was subcloned into pBluescript, and a NotI site was generated in exon 4 by PCR. An ~5-kb IRES-lacZ-Neor cassette was inserted into the NotI site, resulting in partial deletion of exon 4 and a frameshift in the open reading frame of the Akt2 gene. The resulting targeting vector was linearized with SalI and electroporated into 129/Ola embryonic stem (ES) cells. Screening of ES cell clones was performed by Southern blotting. DNAs were digested with EcoRI and probed with an external probe (see Fig. S2a, probe A, in the supplemental material). An internal probe (see Fig. S2a, probe B, in the supplemental material) was then used on BamHI-digested DNAs for further characterization of ES cell clones positive for homologous recombination. Correctly targeted ES cells were used to generate chimeras. Male chimeras were mated with wild-type C57BL/6 females to obtain Akt2+/− mice, which were intercrossed to produce Akt2 homozygous mutants. Progeny were genotyped for the presence of a targeted Akt2 allele by multiplex PCR. The following primers were used for genotyping: P1-as, 5′-CTCAGGGACACCCATGTGTGGCTGC-3′; P2/KO-s, 5′-GCTGCCTCGTCCTGCAGTTCATTC-3′; and P3/WT-s, 5′-CCACAGGCAGCAGAAAGGAA-3′. One primer set amplifies a 600-bp fragment from the targeted allele, and the second primer set amplifies a 360-bp fragment from the wild-type allele. Akt2−/− Akt3+/+ mice were crossed with Akt2+/+Akt3−/− mice to obtain Akt2+/− Akt3+/− offspring, which were mated to generate Akt2−/− Akt3−/− mice as well as the wild-type, Akt2−/−, and Akt3−/− littermates used in the study. Both starting strains were on a mixed 129 Ola and C57BL/6 background. For the generation of Akt1+/− Akt2−/− Akt3−/− mice, Akt2−/− Akt3−/− mice were mated with Akt1+/− Akt3−/− mice (37), and resulting Akt1+/− Akt2+/− Akt3−/− progeny were intercrossed. Mice were housed according to the Swiss Animal Protection Laws in groups with 12-h dark-light cycles and with free access to food and water. All procedures were conducted with the relevant approval of the appropriate authorities.

Cell culture.

Cerebellar granule cells were isolated and cultured as previously described (28). Cells were counted and plated on poly-d-lysine-coated six-well plates in culture medium (basal Eagle medium containing 10% fetal bovine serum, 100 U of penicillin-streptomycin, 2 mM glutamine, and 25 mM KCl). After 24 h, 10 μM cytosine arabinoside was added to the medium to inhibit the proliferation of nonneuronal cells. After 2 days in culture, cells were starved by serum deprivation for 12 h and then stimulated with 100 nM insulin.

Western blot analysis.

For Western blot analysis, protein lysates were prepared by homogenization of various organs in lysis buffer (50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 1% NP-40, 40 mM β-glycerophosphate, 10% glycerol, 4 μM leupeptin, 0.05 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 50 mM NaF, 1 mM Na3VO4, 5 mM EDTA, 1 μM Microcystin LR). Homogenates were centrifuged twice (13,000 rpm for 10 min at 4°C) to remove cell debris. Protein concentrations were determined using the Bradford assay. Proteins (50 μg per sample) were separated by 15%, 10%, or 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore). Akt isoform-specific antibodies were obtained by immunizing rabbits with isoform-specific peptides as previously described (38). Antibodies against total Akt, phospho-Akt (Ser473), phospho-glycogen synthase kinase 3α/β (phospho-GSK3α/β [Ser21/9]), phospho-GSK3β (Ser9), total GSK3β, phospho-TSC2 (Thr1462), phospho-p70 S6K (Thr389), and total 4E-BP1 were purchased from Cell Signaling Technologies. Anti-Foxo3a and anti-phospho-Foxo3a (Thr32) antibodies were obtained from Upstate Biotechnology. A rat monoclonal anti-α-tubulin (YL1/2)-producing hybridoma cell line was obtained from the American Type Culture Collection. Anti-TSC2 antibody was generated as previously described (35) and kindly provided by K. Molle (Biozentrum, University of Basel, Switzerland). For quantitation, Western blots were scanned using a GS-800 Bio-Rad densitometer with a resolution of 63.5 μm by 63.5 μm, and bands were quantified using Proteomweaver 4.0.0.5 (Bio-Rad). As shown in Fig. S1 in the supplemental material, the anti-total Akt and anti-phospho-Akt (Ser473) antibodies utilized in this study show equal affinities towards Akt1 and Akt3 but a lower affinity towards Akt2. As a consequence, the total Akt and phospho-Akt levels that were detected in our study with these antibodies may slightly understate the magnitude of the differences between wild-type and Akt2−/− Akt3−/− mice.

Histology and TUNEL assays.

For histological analysis, organs were dissected and fixed in 4% paraformaldehyde-phosphate-buffered saline overnight at 4°C. After dehydration in ethanol, samples were embedded in paraffin. Tissues were cut into 6-μm-thick sections and stored for staining. For hematoxylin-eosin (Sigma) and cresyl violet (Sigma) staining, sections were deparaffinized and stained. A terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was performed as previously described (37). For quantitation of apoptosis in testes, sections of testes derived from wild-type and Akt2−/− Akt3−/− mice were subjected to TUNEL assay, and the number of TUNEL-positive cells per area was counted (for each mouse, an area of ~20 to 40 mm2 of stained testis sections was analyzed).

Testosterone measurement.

Blood samples were collected from tail veins of 10-week-old male mice. Samples were allowed to clot at room temperature, and serum was separated by centrifugation. To account for the pulsatile secretion of testosterone, three separate samples were taken from each mouse on the same day (spaced 3 h apart) and pooled. A commercial enzyme-linked immunosorbent assay kit (DRG Instruments GmbH, Marburg, Germany) was used to measure testosterone in serum following the manufacturer's recommendations.

Glucose and insulin tolerance test.

All mice used for glucose and insulin tolerance tests were offspring from doubly heterozygous parents. Note that glucose and insulin tolerance was not affected in doubly heterozygous mice, and an influence of the mother on the diabetic phenotype of the offspring could therefore be excluded. Three-month-old mice of the selected genotypes were made to fast overnight; for glucose tolerance tests, glucose (2 g/kg of body weight) (d-(+)-glucose anhydrous; Fluka) was given orally, and for insulin tolerance tests, insulin (1 U/kg) (human recombinant insulin; Sigma) was administered by intraperitoneal injection. Blood samples were collected at the indicated times from tail veins, and glucose levels were determined using Glucometer Elite (Bayer). Similarly, random-fed and fasting blood glucose levels were determined for 3-month-old mice by the collection of blood samples from tail veins, with glucose levels determined as described above. Blood insulin levels were measured with an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (Immunodiagnostic Systems).

In vivo insulin stimulation.

In vivo insulin stimulation was performed on ~8-week-old male mice. Following an overnight fast, a bolus of insulin (1 mU/g or 10 mU/g, as indicated) (human recombinant insulin; Sigma) or saline solution was injected via the inferior vena cava into terminally anesthetized mice. White adipose tissue, liver, skeletal muscle, and whole brain were harvested after 12 min of stimulation and immediately snap frozen in liquid nitrogen. Tissues were homogenized thereafter and lysed as described above.

Statistics.

Data are provided as arithmetic means ± standard errors of the means (SEM), and n represents the number of independent experiments. All data were tested for significance using one-way analysis of variance (ANOVA) and a paired or unpaired Student t test, as applicable. Only results with P values of <0.05 were considered statistically significant.

RESULTS

A single functional allele of Akt1 is sufficient for successful embryonic development and postnatal survival in mice lacking Akt2 and Akt3.

Considering the lethal phenotypes and multiple developmental defects of Akt1−/− Akt2−/− and Akt1−/− Akt3−/− mice, Akt isoforms appear to play essential roles in various aspects of embryonic development (25, 37). Moreover, the Akt genes seem to compensate functionally for one another in vivo, as no developmental deficiencies have been observed in any mice lacking only one isoform of the Akt family (6-8, 13, 15, 33). We wanted to evaluate the impact of combined Akt2 and Akt3 deficiencies on the viability and embryonic development of mice. Akt2−/− mice were generated by targeted disruption of exon 4 of the Akt2 gene (see Fig. S2a in the supplemental material). In mice homozygous for the targeted allele, expression of the Akt2 protein was not detectable with isoform-specific antisera (see Fig. S2c in the supplemental material). Thus, the targeted disruption resulted in a functional null allele. Akt2−/− Akt3+/+ mice were crossed with Akt2+/+ Akt3−/− mice to obtain Akt2+/− Akt3+/− mice. These doubly heterozygous mice were intercrossed to generate Akt2−/− Akt3−/− mice. Surprisingly, Akt2−/− Akt3−/− offspring were viable and showed no obvious abnormalities, except for their smaller size (Fig. (Fig.1A).1A). Examination of >500 pups from matings between Akt2+/− Akt3+/− mice showed the expected Mendelian distribution for Akt2−/− Akt3−/− progeny and the other resulting genotypes (see Table S1 in the supplemental material). We investigated whether upregulation of the remaining Akt isoform in these mice, Akt1, could account for the mild phenotype of Akt2−/− Akt3−/− mice. Immunoblotting of protein extracts from various tissues of 4-day-old Akt2−/− Akt3−/− mice with an isoform-specific antibody against Akt1 revealed no significant increase in Akt1 protein levels compared to those in wild-type littermates (Fig. (Fig.1B).1B). Since we had previously observed that in neonates the tissue distribution of Akt isoforms differs from that in adult mice (37), we also examined Akt1 expression levels in tissues of adult Akt2−/− Akt3−/− mice. As in neonates, Akt1 was not upregulated in tissues of adult Akt2−/− Akt3−/− mice (data not shown). We therefore excluded the possibility that a compensatory upregulation of Akt1 could influence the phenotype of Akt2−/− Akt3−/− mice.

FIG. 1.
(A) Top view of wild-type (WT), Akt2−/− Akt3−/− (DKO), and Akt1+/− Akt2−/− Akt3−/− (Akt1+/−DKO) mice (12-week-old male mice; the WT and DKO mice were from ...

Next, we asked the question of whether Akt1 affects mouse development in a dose-dependent fashion. To evaluate the impact of a decrease in the Akt1 dose, we crossed our Akt2−/− Akt3−/− mice with Akt1+/− Akt3−/− mice (38). Resulting Akt1+/− Akt2+/− Akt3−/− mice were subsequently intercrossed to obtain mice containing only one functional allele of Akt1 (Akt1+/− Akt2−/− Akt3−/− mice). We did not obtain any Akt1−/− Akt2−/− Akt3−/− mice, as a combined deficiency of Akt1 and Akt3 is lethal at embryonic stage E12 (37). However, we obtained viable Akt1+/− Akt2−/− Akt3−/− mice from these crosses (Fig. (Fig.1A;1A; see Table S2 in the supplemental material). Approximately 14% of the mice in the analyzed cohort (n = 105) were Akt1+/− Akt2−/− Akt3−/− mutants. As shown in Fig. Fig.1C,1C, a stepwise reduction in functional Akt alleles was reflected by a concomitant reduction in total Akt protein levels. For instance, in both brain and liver, there was an ~3-fold reduction of total Akt levels in Akt2−/− Akt3−/− mice and an ~10-fold reduction of total Akt levels in Akt1+/− Akt2−/− Akt3−/− mice compared to wild-type levels (Fig. (Fig.1C).1C). Akt1+/− Akt2−/− Akt3−/− mice developed normally, with no obvious defects except for a severe growth deficiency, with body weights at the age of 12 weeks reduced ~40% compared to those of wild-type mice (17.1 ± 1.7 g versus 29.1 ± 0.7 g, respectively [n = 5]; P < 0.01). Taken together, these findings demonstrate that Akt1 (even in the case where only one allele is present) is sufficient to enable successful embryonic development and postnatal survival in mice lacking Akt2 and Akt3.

Growth deficiency and substantially reduced brain and testis mass in Akt2−/− Akt3−/− mice.

It was previously shown that disruption of the Akt1 gene in mice leads to growth retardation (6, 8, 38), and Akt3-deficient mice were shown to have a reduction in brain size (13, 33). In progeny of Akt2+/− Akt3+/− intercrosses, we observed that the combined deletion of Akt2 and Akt3 resulted in a growth deficiency in both male and female Akt2−/− Akt3−/− mice. One-week-old male Akt2−/− Akt3−/− mice were ~28% smaller than their wild-type littermates (3.81 ± 0.21 g versus 5.26 ± 0.14 g; P < 0.001), and an average decrease in body weight of 25% persisted throughout the examined 3-month life period of male Akt2−/− Akt3−/− mice (Fig. (Fig.2).2). For female Akt2−/− Akt3−/− mice, a significant decrease in body weight was evident only from the fourth week of age onward, with an average decrease of 14% compared to wild-type littermates. Consistent with previous reports, single knockouts of either Akt2 or Akt3 did not significantly affect animal size compared to wild-type littermates (data not shown) (7, 33). Comparisons of selected organ weights (brain, heart, lung, thymus, liver, spleen, kidney, and testis) from 3-month-old Akt2−/− Akt3−/−, Akt2−/−, Akt3−/−, and wild-type littermate mice revealed an ~35% reduction in brain weights of Akt2−/− Akt3−/− mice (0.31 ± 0.01 g versus 0.49 ± 0.01 g; P < 0.001) and an ~40% reduction in testis weights (0.12 ± 0.01 g versus 0.20 ± 0.01 g; P < 0.001) compared to those of wild-type mice (Fig. 3A and B). While most organs of Akt2−/− Akt3−/− mice were decreased proportionally to the reduced body weights, the relative weights of brains and testes were reduced by 18.8% ± 1.7% and 28.5% ± 2.7%, respectively (Fig. 3A and B). The prominent size reduction in the testes prompted us to measure serum testosterone levels in 10-week-old male Akt2−/− Akt3−/− and wild-type mice. In Akt2−/− Akt3−/− mice, testosterone levels were ~70% lower than those measured in wild-type mice (1.4 ± 0.5 ng/ml versus 4.7 ± 1.2 ng/ml [n = 5]; P < 0.05). In addition, we compared ovary weights in 3-month-old Akt2−/− Akt3−/− and wild-type females. We observed an ~26% reduction in absolute weights of ovaries of Akt2−/− Akt3−/− mice (13.3 ± 1.3 mg versus 18.2 ± 1.0 mg [n = 5]; P < 0.05); however, there was no significant difference from the wild type in the organ/body weight ratio.

FIG. 2.
Growth deficiency in Akt2−/− Akt3−/− (DKO) mice. Body weights of male (upper panel) and female (lower panel) wild-type (filled triangles) and Akt2−/− Akt3−/− (open diamonds) littermate mice ...
FIG. 3.
Selective reduction of brain and testis size in Akt2−/− Akt3−/− mice. (A) Brains were dissected from 3-month-old male wild-type (WT) and Akt2−/− Akt3−/− (DKO) littermate mice (left). Mean ...

The PI3K/Akt pathway also plays a critical role in cell survival and apoptosis, and therefore we assessed whether an increased rate of spontaneous apoptosis could account for the smaller sizes of testes in Akt2−/− Akt3−/− mice. TUNEL assays were performed on testis sections from adult Akt2−/− Akt3−/− mice and their wild-type littermates. Generally, the rate of spontaneous apoptosis was low for both genotypes, but the testes of Akt2−/− Akt3−/− mice showed an ~1.5-fold increase in TUNEL-positive cells compared to wild-type littermates (Fig. (Fig.3D).3D). In addition, areas of seminiferous tubules were reduced ~24% in Akt2−/− Akt3−/− mice compared to those in wild-type littermates (0.76 ± 0.05 versus 1.0 ± 0.05 [n = 4]; P < 0.05). However, histopathological analysis of testes from Akt2−/− Akt3−/−, Akt2−/−, Akt3−/−, and wild-type littermate mice (3-month-old mice; n = 5 to 8 mice per genotype) revealed no morphological abnormalities (Fig. (Fig.3C).3C). Furthermore, both male and female Akt2−/− Akt3−/− mice were fertile, although litter sizes from such crossings were smaller (data not shown).

Akt1 is sufficient for phosphorylation of key downstream targets in brains and testes of Akt2−/− Akt3−/− mice.

We were interested to know how Akt signaling was affected in the brains and testes of Akt2−/− Akt3−/− mice. First, we assessed steady-state phosphorylation/activation levels of Akt in brains and testes of adult Akt2−/− Akt3−/− and wild-type mice by immunoblotting protein extracts with an antibody against phospho-Ser473. This phosphorylation site is a distinctive indicator of Akt activation status, and the antibody detects phosphorylation of this site in all three Akt isoforms. In the testes of Akt2−/− Akt3−/− mice, steady-state phosphorylation of Akt was reduced ~10-fold compared to that in wild-type littermate controls and represented residual Akt1 activity (Fig. 4A and B). Interestingly, we found that in vivo insulin stimulation could increase the phosphorylation of Akt in testes of wild-type mice, although this induction was less prominent than that in a classical insulin-responsive tissue, such as the liver, and was scarcely detectable in Akt2−/− Akt3−/− mice (see Fig. S3 in the supplemental material). Similarly, phosphorylation of Akt in the brain was severely reduced (Fig. (Fig.5C5C shows phospho-Akt under fasting and insulin-stimulated conditions; data for steady state not shown).

FIG. 4.
Phospho-Western blot analysis of downstream targets of Akt. (A) Representative blot showing steady-state phosphorylation of downstream targets of Akt in testes of wild-type (WT), Akt2−/−, Akt3−/−, and Akt2−/− ...
FIG. 5.
Glucose metabolism in Akt2−/− Akt3−/− mice. (A) A glucose tolerance test was performed on 12-week-old male (upper panel) and female (lower panel) mice of the indicated genotypes. The graph depicts arithmetic means ± ...

Next, we generated primary cultures of cerebellar granule cells to study inducible Akt activation in an in vitro cellular system. In wild-type cerebellar granule cells, the expression of all three Akt isoforms could be detected by isoform-specific antibodies (data not shown), and there was no compensatory upregulation of Akt1 in cells of Akt2−/− Akt3−/− mice (Fig. (Fig.4C).4C). Cerebellar granule cell cultures were deprived of serum for 12 h and then stimulated with 100 nM insulin. After serum deprivation, phospho-Akt levels were >10-fold lower in Akt2−/− Akt3−/− cells than in wild-type cells, and after stimulation, phospho-Akt levels increased in both wild-type and Akt2−/− Akt3−/− cells but remained ~2-fold lower in Akt2−/− Akt3−/− cells (Fig. (Fig.4C).4C). We then assessed the phosphorylation levels of several downstream targets of Akt. GSK3 is a key molecule implicated in neuronal survival, protein synthesis, and glycogen synthesis (10-12), whereas tuberous sclerosis complex 2 (TSC2) is a critical target of Akt in mediating cell growth (17, 23, 26) and p70 S6 kinase (S6K) is a downstream component of the PI3K/Akt/TSC pathway (19, 22). Both GSK3 and TSC2 are well-established Akt substrates. In contrast, the regulation of S6K is more complex, as both PI3K-dependent and -independent signaling pathways are involved in its activation (5, 9, 14). Upon insulin stimulation, a clear induction in phosphorylation of all three downstream targets could be observed in cerebellar granule cells, but interestingly, no differences in phosphorylation levels were apparent in Akt2−/− Akt3−/− cells compared to wild-type cells (Fig. (Fig.4C).4C). Similarly, no significant differences in phosphorylation levels of these downstream targets were apparent in the testis (Fig. (Fig.4B).4B). We also assessed phosphorylation of the Akt substrate Foxo3a and of the mammalian target of rapamycin (mTOR) substrate 4E-binding protein 1 in testes of Akt2−/− Akt3−/− mice but found no marked differences compared to the wild type (see Fig. S3 in the supplemental material). In summary, residual Akt1 activity appears be sufficient to phosphorylate these downstream targets under both steady-state and induced conditions in Akt2−/− Akt3−/− mice.

Perturbation of glucose metabolism in Akt2−/− Akt3−/− mice.

The Akt2 isoform of the Akt family has a major role in the regulation of glucose metabolism (36). It is the predominant isoform expressed in insulin-responsive tissues and is involved in the regulation of insulin-stimulated glucose transport, lipogenesis, and glycogen synthesis (1, 7, 15). To investigate glucose metabolism in Akt2−/− Akt3−/− mice, glucose tolerance and insulin tolerance tests were performed on 3-month-old mice obtained from doubly heterozygous intercrosses (Fig. 5A and B). For the glucose tolerance test, glucose (2 mg/g of body weight) was administered orally to fasted Akt2−/− Akt3−/−, Akt2−/−, Akt3−/−, and wild-type littermate mice. For all four genotypes, blood glucose levels peaked 15 to 30 min after glucose administration and returned to baseline after 3 h. Akt3−/− mice had similar glucose excursions as wild-type mice. However, in male Akt2−/− Akt3−/− mice, blood glucose levels rose faster, and at 30 min they were ~50% higher than those of wild-type mice (21.8 ± 1.3 mM versus 14.8 ± 1.2 mM; P < 0.01) (Fig. (Fig.5A).5A). Akt2−/− mice had similar blood glucose levels as Akt2−/− Akt3−/− mice at all time points. The susceptibility to glucose intolerance was gender dependent because in female mice of all four genotypes, blood glucose levels were not statistically different (P < 0.05). Generally, female mice manifest better glucose tolerance than do male mice (16). For assessment of insulin sensitivity, insulin (1 mU/g of body weight) was administered by intraperitoneal injection to fasted Akt2−/− Akt3−/−, Akt2−/−, Akt3−/−, and wild-type littermate mice. In wild-type and Akt3−/− mice, insulin elicited a >50% decrease in blood glucose levels 60 to 90 min after administration. After 90 min, glucose levels began to recover and reached baseline values 3 h after insulin administration. In contrast, blood glucose levels remained close to baseline levels at all measured time points in Akt2−/− Akt3−/− and Akt2−/− mice. The reduced insulin sensitivity was evident in both male and female mice of the Akt2−/− Akt3−/− and Akt2−/− genotypes.

To further explore the observed differences in glucose disposal, fasting and fed blood glucose and plasma insulin levels were examined in 3-month-old male Akt2−/− Akt3−/−, Akt2−/−, and wild-type littermate mice. Plasma insulin levels were significantly higher in Akt2−/− Akt3−/− mice than in the wild type, under both random feeding (4.6- ± 1.6-fold; P < 0.05) and fasting (1.9- ± 0.3-fold; P < 0.05) conditions (see Table S3 in the supplemental material). In addition, hyperglycemia was observed in Akt2−/− Akt3−/− mice under random feeding conditions (1.7-fold ± 0.2-fold increase; P < 0.05), whereas fasting blood glucose levels were not significantly different from the wild-type levels. We also assessed the percentage of nonenzymatically glycated hemoglobin (hemoglobin A1c [HbA1c]) in male mice of all four genotypes. This value provides an integrated measure of blood glucose levels during a period of approximately 1 month prior to sample removal. Despite the defects in glucose homeostasis, HbA1c levels were similar in both Akt2−/− and Akt2−/− Akt3−/− mice to those in wild-type mice (see Table S3 in the supplemental material). Next, we performed in vivo insulin stimulation in these mice to investigate the induction of Akt phosphorylation in insulin-responsive tissues. Wild-type and Akt2−/− Akt3−/− mice were made to fast overnight and then injected with a bolus of insulin (10 mU/g) or a saline control. Twelve minutes after injection, insulin-responsive tissues (liver, skeletal muscle, and white fat tissue) were collected. In wild-type mice, Akt became strongly phosphorylated upon insulin stimulation in all three tissues (Fig. (Fig.5C).5C). In Akt2−/− Akt3−/− mice, inducible Akt could also be detected, but phospho-Akt levels were ~10- to 20-fold lower than those in wild-type mice (see Fig. S4 in the supplemental material for quantitation). As a readout for Akt1 activity in Akt2−/− Akt3−/− mice, we assessed phospho-GSK3β levels in insulin-responsive tissues. Phosphorylation of GSK3β was induced robustly in the liver and skeletal muscle following insulin stimulation, and phosphorylation levels were similar in both Akt2−/− Akt3−/− and wild-type mice. There was no apparent induction of phospho-GSK3β in white fat in our experimental settings. Peripherally injected insulin was recently shown to also induce Akt phosphorylation in the brain, suggesting an in vivo regulation of the Akt signaling pathway in mouse brain in response to changes in glucose metabolism (29). However, within 12 min of intravenous insulin injection, we could not detect any induction of phospho-Akt in the brain (Fig. (Fig.5C),5C), although phospho-Akt levels were consistently lower in Akt2−/− Akt3−/− mice than in wild-type mice.

DISCUSSION

In order to fully evaluate the impact of the Akt kinase pathway on physiological processes, combined ablation of its isoforms is necessary. Complete inhibition of Akt activity appears to be incompatible with cell survival, but we were able to generate viable Akt mutant mice that contain only a single Akt isoform. Our results provide strong genetic evidence that the Akt1 isoform of the Akt family is sufficient to perform all essential Akt functions in embryonic development and postnatal survival. We observed that Akt2−/− Akt3−/− mice and even Akt1+/− Akt2−/− Akt3−/− mice, which retain only one functional allele of Akt1 and have total Akt levels that are up to 10-fold lower than those in wild-type mice, can develop normally and survive with minimal dysfunctions. This is in sharp contrast to the lethal phenotype of mice that contain only Akt3 (Akt1−/− Akt2−/− mice) or Akt2 (Akt1−/− Akt3−/− mice) (25, 37). These mice have multiple developmental defects that culminate in lethality at embryonic stage E12 (Akt1−/− Akt3−/− mice) or the neonatal stage (Akt1−/− Akt2−/− mice).

Significantly, combined deficiency of Akt2 and Akt3 affects the determination of whole animal size and individual organ sizes, indicating that Akt1 is not able to fully compensate for the lack of the other isoforms in growth signaling. Akt is a crucial downstream effector of IGF-1 signaling (2, 21). Consistent with a function in cell growth and proliferation, various reports have shown that the expression of activated Akt in specific mouse organs increases organ size (18, 30, 34), whereas ablation of Akt can cause reductions in whole animal growth and/or specific organ sizes (6, 8, 13, 15, 33). In Akt2−/− Akt3−/− mice, we observed a substantial reduction (25% in males) in body weights compared to those of wild-type mice. Interestingly, no growth deficiencies have been observed in either Akt2 or Akt3 single-knockout phenotypes for the same strain background. However, a mild growth deficiency was shown for Akt2−/− mice with a pure DBA/1lacj background (16%) (15). In addition, Akt2 and Akt3 appear to be involved in the regulation of brain and testis size, as the weights of both organs were severely reduced in Akt2−/− Akt3−/− mice. A testicular phenotype has also been described for Akt1−/− mice, which exhibit several morphological abnormalities in the testis (6). In contrast, we found no morphological abnormalities in testes of Akt2−/− Akt3−/− mice but observed a severe reduction in serum testosterone levels. Interestingly, relative to body weight, brain size was not further reduced in Akt2−/− Akt3−/− mice compared to Akt3-deficient mice, despite a high expression of Akt2 in the brain (25% of total Akt [13]) which is abrogated in Akt2−/− Akt3−/− mice. This argues for a selective influence of Akt3 on the regulation of brain size, rather than the size being a result of the total Akt dose in the tissue. Furthermore, Akt3 appears not to contribute to glucose homeostasis in mice, as a combined disruption of Akt2 and Akt3 did not noticeably exacerbate the glucose and insulin intolerance observed in Akt2−/− mice.

Detection of active Akt by a phospho-Ser473 antibody showed substantially reduced levels of total active Akt in various tissues of Akt2−/− Akt3−/− mice both at the steady state and when induced. Surprisingly, phosphorylation levels of critical downstream targets, such as GSK3, TSC2, and S6K, were similar in the Akt2−/− Akt3−/− mice to those in wild-type controls. Very low levels of active Akt1, the residual isoform, appear to be sufficient to phosphorylate certain downstream targets. However, we cannot formally exclude that these targets may be phosphorylated by another compensatory mechanism. For instance, S6K and p90 ribosomal S6 kinases (RSK) are two other insulin-stimulated protein kinases that can catalyze the phosphorylation of GSK3 in vitro (10, 31, 32). Although Akt appears to be the major kinase phosphorylating GSK3 in vivo upon insulin stimulation (4, 11), S6K and/or RSK may constitute a compensatory mechanism by which GSK3 becomes phosphorylated in Akt-deficient mice.

In conclusion, we provide genetic evidence for a dominant role of Akt1 in embryonic development and postnatal survival. We show that minimal amounts of Akt1 appear to be sufficient for full activation of many downstream targets. In addition, we identify redundant and nonredundant functions of the Akt2 and Akt3 isoforms in growth and glucose metabolism and provide insights into Akt isoform hierarchy.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank J. F. Spetz and P. Kopp for their assistance with ES cell culture and embryonic aggregation and R. Portmann for help with Western blot quantifications.

B.D. is supported by the Swiss Cancer League (KFS 1167-09-2001 and KFS 01002-02-2000), O.T. is supported by the Novartis Stiftung für Medizinisch-Biologische Forschung, and Z.Z.Y. is supported, in part, by a grant from Bundesamt für Bildung und Wissenschaft (BBW 98.0176). The Friedrich Miescher Institute for Biomedical Research is funded by the Novartis Research Foundation.

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 August 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

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