Role for Akt3/Protein Kinase B{gamma} in Attainment of Normal Brain Size

Studies of Drosophila and mammals have revealed the importanceof insulin signaling through phosphatidylinositol 3-kinase andthe serine/threonine kinase Akt/protein kinase B for the regulationof cell, organ, and organismal growth. In mammals, three highlyconserved 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 carbohydratemetabolism but is essential for the attainment of normal organsize. However, in contrast to Akt1^–^/^– mice, whichdisplay a proportional decrease in the sizes of all organs,Akt3^–/– mice present a selective 20% decrease inbrain size. Moreover, although Akt1- and Akt3-deficient brainsare reduced in size to approximately the same degree, the absenceof Akt1 leads to a reduction in cell number, whereas the lackof Akt3 results in smaller and fewer cells. Finally, mammaliantarget of rapamycin signaling is attenuated in the brains ofAkt3^–/– but not Akt1^–/– mice, suggestingthat differential regulation of this pathway contributes toan isoform-specific regulation of cell growth.

INTRODUCTION

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Introduction

While complex organisms grow toward determinate final sizes,there must be precise regulation within each tissue as wellas coordination among organs to reach these final sizes (18,24). The regulation of both cell number and size contributesto the establishment of organ size, whereas cell number appearsto be predominant in determining differences between species.Several factors, including circulating hormones and metabolitesas well as cell-autonomous signaling cascades, control theseprocesses (31). One of the key extracellular effectors determiningorganismal size is insulin-like growth factor 1 (IGF1). As demonstratedby genetic studies with mice, IGF1 is required for normal embryonicand postnatal growth (4, 43, 44, 59). In addition, IGF1 controlsthe sizes of individual organs (43, 59). For example, normalbrain growth requires IGF1 (6, 43), as IGF1-deficient brainsare reduced in size secondary to a decrease in both cell numberand cell size (6, 15). Similarly, humans with IGF1 deficiencydisplay severe growth retardation and suffer from mental retardation(75).

In addition to extracellular factors, the intracellular signalingpathways determining growth are being uncovered. IGF1 acts throughthe type 1 IGF receptor to modulate an evolutionarily conservedpathway of molecules involved in the regulation of growth andmetabolism (38, 53). For many hormones, including IGF1 and insulin,binding to a receptor stimulates its protein tyrosine kinaseactivity, leading to the phosphorylation of scaffold proteinsof the insulin receptor substrate (IRS) family. IRS proteinsassemble complexes that include a number of potential signalingproteins, of which the lipid kinase phosphatidylinositol 3-kinase(PI3K) appears to be the most critical for the maintenance ofcell size and proliferation (10). PI3K catalyzes the generationof phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which bindsto several signaling molecules, in most cases via a canonicalpleckstrin homology domain. One of these proteins is the phosphoinositide-dependentkinase 1, which, in cooperation with the poorly defined phosphoinositide-dependentkinase 2, activates Akt/protein kinase B (Akt/PKB) by phosphorylationat two critical residues (8). The lipid phosphatase PTEN negativelyregulates this pathway by converting PIP₃ to phosphatidylinositol4,5-bisphosphate (PIP₂). There is now evidence that the IRS/PI3K/Aktpathway is involved in the control of organ size in severalmodel systems (28, 67). Initially, this pathway was establishedin the fruit fly Drosophila melanogaster, but it is now clearthat it is also operational in mammals (7, 12, 50, 56, 71).Tissue-specific activation of this pathway, either by the overexpressionof active PI3K or Akt or by a deletion of PTEN, results in anincrease in the sizes of several organs, including the heart(19, 48, 64-66), the endocrine pancreas (70), the brain (2,30, 37), lymphoid tissue (27), the prostate (45, 73), and mammaryglands (23, 39). In all of these cases, the enlargement in organsize is due at least in part, and often exclusively, to an increasein cell size. When the activation of Akt occurs early in braindevelopment, the brain's larger size is secondary to increasesin cell size and number, with the latter resulting from theaugmented proliferation of neuronal stem cells (30). However,the activation of this pathway later in development only affectscell size, and this increase requires the activation of a proteindownstream of IRS/PI3K/Akt, the mammalian target of rapamycin(mTOR) (36, 37). Nutrients as well as growth factors regulatemTOR, which therefore serves as a critical element in the coordinationof growth with the energy supply. mTOR activation results inthe phosphorylation of downstream targets such as p70 S6 kinaseand 4E-BP1, which regulate protein translation (55).

In contrast to overexpression studies, only a few reports havedescribed the effects of reduction in the activity of the IRS/PI3K/Aktpathway in specific organs (51). In some cases, the deletionof a gene from all of the tissues of an animal results in aselective decrease in the size of a single organ. Examples ofthis include IRS2 and p70 S6 kinase, two proteins whose eliminationleads to a reduction in the brain and the aggregate mass ofthe ß cells of the pancreas, respectively (57, 63).Regarding the former, IRS2-deficient brains are 38% smallerthan normal primarily due to decreased cell proliferation, ascell size and apoptosis are unaffected (63).

Understanding the requirement of Akt for the determination ofcompartment size in mammals is complicated by the existenceof three isoforms encoded by independent genes, namely, Akt1/PKB ${alpha}$ ,Akt2/PKBß, and Akt3/PKB ${gamma}$ . Despite their significanthomology, Akt1 and Akt2 have distinct functions in the controlof growth and metabolism, respectively (13, 16, 17). Akt1-deficientmice display impaired overall growth, whereas Akt2 null miceare insulin intolerant, demonstrating a diabetes-like syndrome(13, 16, 17). However, the function of Akt3 has not yet beenassessed by the generation and characterization of Akt3^–/–mice. The distribution of Akt3 mRNA is more limited than thatof either Akt1 or Akt2 (76). Akt3 mRNA is most highly expressedin the brain and testes, but it is also detected in fat, lungs,and mammary glands (76). In human tissue, Northern blot analyseshave demonstrated the highest expression in adult brains, lungs,and kidneys as well as in fetal hearts, livers, and brains (9).Thus, a critical question to be addressed is whether Akt3 isimportant for growth and/or metabolism and, if so, whether itexerts its effect on particular cell types.

We report here the generation of mice lacking Akt3, in whichwe have observed a selective reduction in brain size. Moreover,by comparing brains from Akt3^–/– mice to brainsfrom Akt1^–/– mice, we demonstrate isoform-specificmechanisms for the regulation of brain size.

MATERIALS AND METHODS

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Materials and Methods

Generation of Akt3-deficient mice. Using a mouse Akt3 cDNA probe, we isolated overlapping clonesfrom a 129 mouse genomic library (Stratagene, La Jolla, Calif.).To introduce a null mutation, we deleted exon 3 (correspondingto nucleotides 209 to 320 of the mouse cDNA), which introduceda frame shift in the open reading frame and a truncation ofthe protein product.

The targeting construct (Fig. 1A) consisted of a 1.9-kb 5' genomicfragment (NotI-BglII), a loxP-flanked phosphoglycerate kinase-hygromycin-poly(A)cassette in the opposite transcriptional orientation (SalI-BamHIfragment), and a 2.7-kb 3' homology region (SphI-EcoRI). The5' homology fragment (NotI-BglII) and the loxP-flanked phosphoglyceratekinase-hygromycin-poly(A) cassette (SalI-BamHI) were clonedinto pBKSKII that had been opened with NotI and SalI. The 3'homology fragment (SphI-EcoRI) was subcloned into pSP73. The5' homology selection fragment was released with NotI and SalIand cloned with the 3' homology fragment (XhoI-EcoRI) into avector containing the herpes simplex virus thymidine kinasegene cassette (for negative selection). The targeting constructwas linearized with NotI. Genomic DNAs from recombinant cloneswere digested with EcoRI and screened by Southern blot analysis.The probe recognized a 10.4-kb fragment in the wild-type alleleand a 5.9-kb fragment in the targeted allele (Fig. 1B).

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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.

Mice were housed in a facility on a 12-h light-dark cycle withfree access to food and water. All procedures were performedin accordance with the policies of the Institutional AnimalCare and Use Committee at the University of Pennsylvania.

Mice were genotyped by PCR. Primers 1 (5'-GCA TCC ATC CTT GTTCAC GC) and 2 (5'-GGG AGA GAG AAG TGC CAT TGT ATT G) producedan $~$ 650-bp band from DNAs of wild-type mice. Primers 1 and 3(5'-GTG GGG TGG GAT TAG ATA AAT GC) produced an $~$ 700-bp bandfrom DNAs isolated from Akt3^–^/^– mice (Fig. 1C).

An antibody to Akt3 was produced by use of a glutathione S-transferasefusion protein, with amino acids 1 to 147 of AKT3 as the antigen.Antisera were produced in rabbits and affinity purified by chromatographywith the glutathione S-transferase fusion protein. Western blotanalyses with this antibody did not detect any of the proteinin brain extracts from Akt3 knockout animals (Fig. 2A).

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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.

Western blot analysis. Brains were homogenized in lysis buffer (50 mM Tris [pH 8],250 mM NaCl, 2 mM EDTA, 1% IGEPAL, 200 mM NaF, 1 mM Na₃VO₄,complete protease inhibitor cocktail [Roche, Mannheim, Germany]).Homogenates were clarified by centrifugation, the supernatantswere collected, and the protein concentration was determinedby a bicinchoninic acid assay (BCA) (Pierce, Rockford, Ill.).For examinations of the phosphorylation of the ribosomal proteinS6, 25 µg of protein was resolved by sodium dodecyl sulfate-15%polyacrylamide gel electrophoresis (SDS-15% PAGE). Blots wereprobed with anti-phospho-S6 (68) and anti-total S6 (Cell SignalingTechnology, Beverly, Mass.). For the quantification of Akt isoforms,recombinant Akt1, Akt2, and Akt3 proteins were purchased fromUpstate USA, Inc. (Charlottesville, Va.), and the concentrationof each recombinant protein was determined by analysis of aCoomassie blue-stained gel by use of the Odyssey system (LI-CORBiosciences, Lincoln, Nebr.) and a bovine serum albumin standard.A standard curve for each recombinant Akt protein and dilutionsof total brain lysates were resolved by SDS-10% PAGE, transferredto nitrocellulose, and probed with isoform-specific antibodies(for Akt1 [Upstate USA, Inc.], Akt2 [68], and Akt3 [describedabove]). Brain lysates from each isoform-specific knockout animalwere also included on the blot to ensure the specificities ofthe antibodies. Secondary antibodies were labeled with IR Dye800 (Rockland Immunochemicals, Inc., Gilbertsville, Pa.) forquantification by the Odyssey system. The total amount of Aktprotein was determined as the sum of the amounts of the individualisoforms, and the amount of each isoform was expressed as apercentage of the total amount of Akt protein.

Myelin assay. The myelin content in Akt3-deficient brains was measured asdescribed by Cheng et al. (14). Briefly, brains from controland mutant mice were homogenized in 0.32 M sucrose. The brainhomogenates were layered over an equal volume of 0.85 M sucroseand centrifuged at 75,000 x g for 30 min. Myelin was collectedfrom the interface, osmotically shocked with water on ice for30 min, and then sedimented by centrifugation at 12,000 x gfor 20 min. The precipitate was resuspended in 10 mM Tris-HCl(pH 8.0)-1 mM EDTA, and the protein concentration was determinedby a BCA protein assay.

Metabolic analysis. For glucose tolerance tests, glucose (1 g/kg of body weight)was administered by intraperitoneal injection to fasted consciousmice, and glucose concentrations were determined by use of aglucometer (Glucometer Elite XL; Bayer, Tarrytown, N.Y.) fromwhole blood collected from transversely sectioned tails. Forinsulin tolerance tests, porcine insulin (1 U/kg) was administeredby intraperitoneal injection to fasted conscious mice, and glucoseconcentrations were determined by use of a glucometer from wholeblood collected from transversely sectioned tails. Insulin andfree fatty acid concentrations were measured as previously described,and insulin assays were performed by the Radioimmunoassay CoreFacility at the Penn Center for Diabetes (17). A NEFA C kit(Wako Chemicals USA, Inc., Richmond, Va.) was used to determinefree fatty acid levels.

DNA and protein content. For analyses of DNA and protein content, organs dissected fromage-matched mutant and control animals were weighed and thenhomogenized in TNE buffer (10 mM Tris base, 10 mM EDTA, 200mM NaCl, pH 7.4). An aliquot of each sample was diluted in TNEbuffer containing 0.1 µg of Hoechst 33258/ml for measurementof the DNA content by use of a TD-700 fluorometer (Turner Designs,Sunnydale, Calif.) at an excitation wavelength of 365 nm andan emission wavelength of 460 nm. A standard curve using calfthymus DNA was used to calibrate the fluorometer. NP-40 (Sigma,St. Louis, Mo.) was added to the remaining brain homogenateto a final concentration of 1%, and the lysate was clarifiedby centrifugation at 16,000 x g at 4°C. The protein concentrationof the supernatant was determined by a BCA protein assay. Dataare expressed as percentages of the mean wild-type value, presentedas means ± standard deviations (SD) for one experimentwith littermate controls. Experiments were repeated at leastthree times.

Measurements of cardiomyocyte size. Hearts dissected from age- and sex-matched mutant and wild-typeanimals were fixed overnight in 10% neutral buffered formalin(NBF) (Sigma). After fixation, hearts were cut sagittally, embeddedin paraffin, and sectioned. Five-micrometer sections were deparaffinized,stained with fluorescein isothiocyanate (FITC)-conjugated wheatgerm agglutinin (Vector Laboratories Inc., Burlingame, Calif.),counterstained with propidium iodide, and then analyzed by microscopywith a Nikon Eclipse TE300 microscope (34). Two to eight 20xfields were analyzed per animal. The myocyte cross-sectionalarea was quantified by using the FITC-demarcated outline ofthe cell and Metamorph software (Universal Imaging Corporation,Downingtown, Pa.).

Analysis of cell density. Animals were anesthetized with pentobarbital and then perfusedwith phosphate-buffered saline containing 10 U of heparin/mlfollowed by 10% NBF. Brains were dissected, fixed overnightin 10% NBF, and transferred to 50 mM Tris-150 mM NaCl, pH 7.6,prior to being processed and embedded in paraffin. Six-micrometersections were cut by use of a rotary microtome. Every othersection was stained with cresyl violet. Sections were matchedby the use of anatomic landmarks and a mouse brain atlas. Sectionsbetween 0.14 and 0.21 mm anterior to the bregma were deparaffinizedand stained with 10 µg of Hoechst 33258/ml (Sigma). Quantificationof the number of nuclei per defined area of motor cortex wasperformed with the Metamorph image analysis suite, and the meancell area was expressed as the field area per number of cells.Three areas per slide were evaluated. Four to seven slides wereevaluated per animal. Micrographs of motor cortexes were obtainedwith a cooled charge-coupled device camera (Princeton Instruments).

Livers isolated from Akt1- and Akt3-null mice were fixed in10% NBF overnight, rinsed in phosphate-buffered saline, andthen embedded in paraffin. Six-micrometer sections were cutby use of a rotary microtome. Sections were deparaffinized andstained with Hoechst 33258. The density was measured and themean cell area was calculated as described above.

Statistical analysis. For comparisons between mutant and wild-type groups, Student'stwo-tailed t test was utilized and significant P values areindicated. The representative experiments shown were repeatedat least three times.

RESULTS

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Results

Akt3 expression and generation of Akt3-deficient mice. Although the phenotypes of mice deficient in Akt1 and Akt2 arenow well established, little is known about the role of Akt3in mammals. For this reason, we generated mice in which theexpression of the Akt3 gene was eliminated by standard targetingin embryonic stem cells (Fig. 1). As a consequence of this mutation,the Akt3 protein was undetectable in brains of knockout animals(Fig. 2A). Akt3 nullizygous animals were born at a Mendelianfrequency and did not exhibit any overt abnormalities for atleast 1 year.

To search for potential phenotypic consequences of the Akt3elimination that were not immediately obvious, we reevaluatedthe tissue distributions of Akt isoforms in the adult mouse,keeping in mind that mRNA and protein levels are not alwaysin correlation, as, for example, in the case of Akt1 (76). Ofall the mouse tissues examined, Akt3 expression was highestin the brain, which was consistent with the mRNA expressionpattern (Fig. 2A). However, when we compared relative levelsof mRNA and protein in different organs, protein expression,unlike mRNA expression, was observed in the heart, ovaries,and spleen (76).

To evaluate the relative expression of each of the Akt proteinsin the brain, we developed an assay using isoform-specific antibodiesand recombinant protein standards to quantify the absolute amountsof Akt1, -2, and -3. In adult brains, Akt3 was the predominantisoform, representing about one-half of the total Akt protein,whereas Akt1 accounted for approximately 30% of the total andAkt2 made up the rest (Fig. 2B). In postnatal day 1 brains,Akt1 was more abundant, accounting for 47% of the total Aktprotein, whereas the relative levels of Akt2 and Akt3 were 14and 39%, respectively (data not shown). Akt3 was present inall regions of the adult mouse brain and was the predominantisoform in the cortex and hippocampus. On the other hand, Akt2was the most expressed isoform in the cerebellum, whereas Akt1was the major isoform in the spinal cord (Fig. 2B).

Regulation of brain size by Akt3. Considering that Akt is required for the attainment of normalorgan and organismal size in D. melanogaster and mice (13, 17,61, 71) and that Akt3 is highly expressed in the mouse brain,we measured the brain weights of mutants lacking Akt3. Akt3-deficientmice demonstrated a reduction in brain size, as brains fromadult nullizygotes were 25% smaller than those of their wild-typelittermates, expressed either in absolute terms (349 ±14.0 mg versus 464 ± 11.7 mg; N = 6 or 7; P < 0.001)(Fig. 3A and B) or as a ratio to body weight (Table 1). However,mouse size was unaffected by the Akt3 deficiency (Fig. 3C and D),and all organs examined other than the brain were normallysized (Table 2). Even the testes, which, like ovaries, had thesecond highest level of Akt3 protein expression, were normalin size in Akt3-deficient male mice (data not shown). Thus,the reduction in organ size in Akt3^–^/^– mice wasspecific to the brain and was not proportional to body size(Table 2). This abnormality was present at birth, with postnatalday 1 mice displaying a 15% reduction in brain size comparedto wild-type mice (68 ± 7.7 mg versus 80 ± 7.5mg; N = 13 to 24; P < 0.00), despite their normal body size(1.4 ± 0.14 g for Akt3^+/+ animals versus 1.4 ±0.17 g for Akt3^–/– animals; N = 13 to 24) (Table1). The relative reduction in brain size compared to the wildtype continued to increase during the first 3 weeks of life,reaching the same degree as that seen in adults by the end ofthe third week (394 ± 19.1 mg for Akt3^+/+ animals versus302 ± 21.7 mg for Akt3^–/– animals; N = 4or 5; P < 0.01).

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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).

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TABLE 1. Ratios of brain weights to body weights in wild-type and Akt3^–/– mice

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TABLE 2. Comparison of organ-to-body-weight ratios for wild-type (Akt3^+/+) and Akt3-deficient (Akt3^–/–) mice^b

To obtain a more quantitative estimate of the relationship betweenbrain size and body weight, we performed an allometric analysis.Thus, we plotted the corresponding weight values for wild-typeand Akt3 knockout mice as a logarithmic transformation of thescaling equation y = ax^b and performed a linear regression analysis.This allowed us to determine the slope of the regression line,or allometric exponent b, which represents a formal quantitativemeasure of the relationship between x and y, in this case bodyweight and brain weight (Fig. 4). For wild-type mice, b = 0.75,which is in close approximation to that previously reportedfor mammals (47). In contrast, the value of b (0.61) for Akt3-deficientmice indicated that the brain size for a given body weight wassignificantly reduced.

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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).

Next, we considered possible effects of the Akt3 mutation onmyelination, which begins during the first week of postnatalbrain development and peaks within the third to fourth weekof life (33, 52). IGF1 has been implicated in the control ofthis process, as its overexpression increases myelination (11).Moreover, IGF1-deficient brains have decreased myelination,which is probably proportional to the reduction in cell number(6, 14). The total myelin content was reduced by 20% in Akt3-deficientbrains (3.8 ± 0.27 mg; N = 7) compared to wild-type brains(4.7 ± 0.32 mg; P = 0.005; N = 4), which was commensuratewith the decrease in brain size. Thus, when the myelin contentwas corrected for the brain weight, there was no significantdifference between brains from Akt3-deficient (10.7 ±0.73 µg of myelin/mg of brain) and wild-type (9.8 ±0.78 µg of myelin/mg of brain) mice. We concluded, therefore,that a reduction in myelin alone does not account for the decreasein brain size in Akt3 knockout animals.

In Drosophila, the Akt homolog Dakt1 regulates organ size primarilyby the control of cell growth, although the cell number canalso influence organ volume (29, 71). To clarify how Akt3 controlsmammalian brain size, we measured the DNA and protein contentin brains from Akt3-null mice. In the absence of polyploidy,the total DNA content reflects the cell number, whereas theprotein-to-DNA ratio conveys information about cell size. Forexample, if an organ were smaller because the cell number wasreduced, then the total DNA content would be decreased but theprotein-to-DNA ratio would remain unchanged. If cell size alonewere decreased, then the protein content would be reduced butthe total DNA content would be unaffected, thereby producinga decrease in the protein-to-DNA ratio.

In Akt3-deficient mice, the brain had a 13% decrease in totalDNA content and an 11% reduction in the protein-to-DNA ratio(Table 3). This suggested that the 25% size reduction in theAkt3-deficient brain was secondary to both a decrease in cellnumber and a reduction in cell size. To obtain an independentmeasure of cell size, we measured the cell density in the adultcortex of Akt3-deficient brains and calculated the mean cellarea. Using Hoechst 33258, we labeled nuclei in serial sectionsfrom matched regions of cerebral cortex and counted the numberof nuclei in a defined area in both wild-type and knockout brains.In Akt3-deficient brains, the number of nuclei in the definedarea was increased, indicative of a smaller mean cell area (Fig.5). This reduction in cell size may be secondary to a changein the cell body size or to decreases in the number and sizeof dendrites. In neuronal cultures, inhibitory Akt constructsresult in both diminished axon elongation and diminished somasize (46). In organs that were unaffected by the Akt3 deficiency,such as the liver and the heart (Table 2), which retained theirnormal sizes, no change in cell size was observed (data notshown), while the total DNA content and the protein-to-DNA ratioremained at wild-type levels (Table 3). We noted that the densityof hepatocytes and the mean area of cardiomyocytes were normalin Akt3-deficient mice.

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TABLE 3. DNA and protein levels in organs from Akt knockout mice^a

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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.

Analysis of metabolism in Akt3-deficient mice. In addition to regulating growth, the PI3K/Akt pathway is alsorequired for normal metabolism. For example, Akt2 regulatesglucose homeostasis, as mice deficient in this isoform are hyperinsulinemicand hyperglycemic (16, 29). Akt3 is expressed in cultured adipocytesand its activity is increased by insulin, raising the possibilitythat Akt3 is also important for the regulation of glucose orfat metabolism (5). Nevertheless, serum insulin, glucose, andfree fatty acids were normal in Akt3-null mice (Table 4). Inaddition, adult Akt3-deficient mice exhibited glucose and insulintolerance that was indistinguishable from that of wild-typemice (Fig. 6). Thus, we were unable to detect abnormalitiesin carbohydrate metabolism in Akt3^–/– mice.

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TABLE 4. Circulating metabolites in Akt3 knockout mice

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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.

Comparison of Akt3 mutants to Akt1- and Akt2-null mice. Akt1-deficient mice exhibited a 14% reduction in brain sizecompared to littermate controls (383 ± 32.9 mg versus446 ± 8.6 mg; N = 5; P = 0.01) that was proportionalto the reduction in body size. An analogous maintenance of normalorgan size for body size was also observed for the livers andhearts of Akt1 nullizygotes (data not shown). Akt2-null micedisplayed no change in brain size compared to control animals(431 ± 19.3 mg versus 439 ± 20.6 mg; N = 4).

Unlike Akt3, Akt1 was required for the size control of otherorgans, such as the heart and liver, in addition to the brain.In Akt1-deficient brains, the total DNA content was reducedby 21% and there was a trend towards an increased protein/DNAratio that did not achieve statistical significance (Table 3).These data suggest that Akt1 is required mainly for the determinationof the proper cell number. As performed with Akt3-deficientbrains, we labeled nuclei in serial sections from matched regionsof the cerebral cortex and counted the number of nuclei in adefined area in both wild-type and Akt1 knockout brains. Wefound no difference in the numbers of nuclei per area in Akt1-deficientcortices compared to wild-type controls (Fig. 7A to C). WhileAkt1 affected the cell number in the brain, Akt1-deficient heartshad a normal cell number, as indicated by an unchanged totalDNA content compared to wild-type hearts (Table 3). However,hearts from Akt1^–/– mice displayed a 21% reductionin the protein-to-DNA ratio, suggesting that the cells weresmaller (Table 3). This was confirmed by direct measurementsof myocyte cross-sectional areas by light microscopy (Fig. 7D to F).Akt1-deficient cardiomyocytes were 19% smaller than wild-typeheart cells (202 ± 12.7 µm² versus 249 ±13.6 µm²; N = 5; P < 0.01) (Fig. 7D to F). The sameresult was obtained when the number of nuclei was counted ineach field and when the mean cell size was computed as performedfor brains (data not shown).

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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.

Unlike the heart, the Akt1-deficient liver revealed a normalprotein-to-DNA ratio, suggesting an appropriate cell size, buthad a 25% reduction in the total DNA content, which was representativeof a decrease in cell number (Table 3). As described above forthe heart and brain, liver sections from Akt1-deficient andAkt1 wild-type mice were stained with Hoechst 33258, and thenumbers of nuclei were determined for defined fields. The celldensity did not differ between Akt1 knockout and wild-type liversections (1,745 ± 44.6 cells/mm² versus 1,755 ±41.5 cells/mm²; N = 3), indicating that there was no changein the mean cell area (473 ± 35.5 µm² for Akt1^–/–animals versus 536 ± 43.5 µm² for Akt1^+/+ animals).Thus, livers from Akt1^–/– mice were smaller dueto an overall decrease in the number of cells.

Differential activation of downstream targets by Akt isoforms. One explanation for the distinct effects of Akt1 and Akt3 inthe brain may be that the different isoforms of Akt regulateunique downstream targets. The mTOR-p70 S6 kinase pathway, whichis downstream of Akt, regulates cell size (55) and is activeduring the period of brain growth throughout the first two postnatalweeks (69). mTOR is also required for the growth induced byPTEN activation (36). Therefore, we evaluated the phosphorylationof ribosomal protein S6, a downstream target of mTOR, in brainsisolated from postnatal day 1 mice, in which p70 S6 kinase demonstratespeak activity (69). In Akt3 knockout brains, ribosomal proteinS6 phosphorylation was decreased almost 50%. However, no reductionwas observed in Akt1-null brains (Fig. 8). Similarly, the phosphorylationof p70 S6 kinase at serine 389 was reduced by 30% in Akt3-deficientbrains, while no significant alteration was observed in Akt1mutant brains (data not shown). Thus, Akt3 is required for thenormal phosphorylation of p70 S6 kinase and its target, ribosomalprotein S6, during postnatal brain growth.

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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.

DISCUSSION

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Discussion

Our analysis of the Akt3 null phenotype has provided the firstevidence of a nonredundant function for this protein kinasein mammals. Similar to the closely related isoform Akt1, Akt3does not appear to contribute significantly to the maintenanceof normal metabolism but is critical for the attainment of normalorgan size. However, in marked contrast to Akt1, Akt3 does notregulate organismal size but controls exclusively the mass ofthe mouse brain by influencing both cell size and number, mostlikely, at least in part, through the selective activation ofdownstream effectors in the mTOR pathway.

Now that experiments with fruit flies and mice have convincinglydemonstrated a role for the PI3K/Akt pathway in the controlof cell and compartment size, the question arises of how specificityis achieved given the large number of regulatory processes forwhich Akt appears to function as an essential intermediary (62).Although some of the previous studies that considered this issuerelied on knockout strategies like ours, most reports have beenbased on overexpression studies of wild-type, constitutivelyactive, or dominant-negative mutants (20, 22, 46, 66). We havereported previously that at least some of the specificity ofAkt signaling, when growth versus metabolism is considered,is conferred by dissimilarities between Akt1 and Akt2 and thatdifferences intrinsic to the Akt proteins contribute to selectivesignaling (3, 16, 17). The present study extends those observationsby providing the unexpected result that two Akt isoforms, Akt1and Akt3, are both essential for normal growth control but thateach functions in a distinct manner. Whereas Akt1 is importantfor global, organismal growth, the requirement for Akt3 is remarkablyorgan specific. It is likely that the mechanism is cell autonomous,because fibroblasts immortalized from Akt1-null mice also demonstratea reduced size (4, 59; K. Roovers and M. J. Birnbaum, unpublishedobservations). Thus, growth control may be exerted by each Aktisoform operating as a signaling intermediate within the targetcell. However, evidence that Akt1 and Akt3 have dissimilar signalingcapabilities and do not simply function interchangeably is providedby the observation that the loss of each of the isoforms leadsto a decreased brain size by distinct mechanisms, i.e, a reductionin only the cell number for Akt1^–^/^– mice and inboth cell size and number in Akt3^–/– mutants. Aprobable explanation is that the activation of isoform-specificsignaling pathways accounts for much of the difference, whichis further modulated by external, developmentally regulatedprocesses. For example, Akt3, but not Akt1, is required forappropriate ribosomal protein S6 phosphorylation during thepostnatal period of brain growth (Fig. 8). However, these differencesmay be tissue specific, and Akt1 may be important for ribosomalprotein S6 phosphorylation in the heart, where Akt1 controlscell size. In addition, selectivity in the ability of proximalsignaling pathways to regulate Akt isoforms probably contributesto specificity. The elimination of the gene encoding IRS2, ascaffolding protein thought to lie upstream of Akt in a linearsignaling pathway, results in an impairment of brain growthsecondary to a decrease in neuronal proliferation (63). Interestingly,no change in cell survival or cell size was observed for IRS2-nullbrains, suggesting that IRS2 might preferentially regulate Akt1in the brain, which would be surprising given the similaritiesbetween the roles of IRS2 and Akt2 in the control of peripheralmetabolism (16, 29, 74). In addition, other induced mutationsleading to organismal growth deficiencies, such as an IRS1 deficiency,cause a decrease in brain size and more closely resemble thephenotype of Akt1-null mice (1, 13, 17, 35, 76). Interestingly,in contrast to the case for Akt1^–/– mice, for modelsbased on a loss of IRS1, the diminution in brain size is notas severe as the reduction in body size (6, 63). This may occurbecause the IRS1-deficient animals are smaller than Akt1 knockoutanimals, and a commensurate reduction in brain size may notbe compatible with life.

Previous studies have demonstrated that the activation of Aktby the deletion of PTEN affects brain size (30, 37). Since aPTEN deficiency modulates other targets in addition to Akt,it is important to demonstrate, as shown here, that this kinaseis indeed a critical member of the pathway regulating normalbrain growth. In mature neurons, the increase in brain sizesecondary to a targeted deletion of PTEN is a consequence ofthe increase in cell size and is dependent on the activationof mTOR (36). The deletion of PTEN at an earlier stage of neuronaldevelopment leads to both enlarged and more abundant cells (30).This suggests that the different phenotypes observed for Akt1^–/–and Akt3^–/– mice may be related to the stage ofneuronal development at which each isoform is active, with Akt3being more critical for the later periods of brain growth (21).The increased expression of the Akt3 protein in the adult brainis consistent with this hypothesis.

Several publications have reported experiments with brains andisolated neurons that implicate Akt in neuronal functions suchas survival (20, 22, 32), process formation (54), cell growth(46), and synaptic plasticity (72). Unfortunately, these studieshave relied on constitutively active or putative dominant-negativeforms of Akt, which, in addition to their inherent problemsof specificity, do not address Akt isoform selectivity. In thisstudy, we confirmed the role of Akt in cell growth by usinga loss-of-function strategy and further defined Akt1 and Akt3as being the most important isoforms for this process in thebrain. These genetic studies do not resolve, however, whetherthe reduction in cell number in the Akt3^–/– brainis due to a defect in proliferation or survival. It has beenargued that much of the macrocephaly in the brain-specific PTENknockout is due to an expansion of central nervous system stemcells via increased proliferation (30). Akt has also been stronglyimplicated as a major regulator of the progression through theG₁ and G₂ phases of the cell cycle (41). Whether the lack ofAkt1 and/or Akt3 decreases the cell number by affecting theproliferative capacity of stem cells will need to be evaluatedin future studies. Interestingly, the microcephaly and macrocephalyof humans that are observed in IGF1-deficient patients and patientswith the Lhermitte-Duclos PTEN deficiency syndrome, respectively,result in cognitive defects such as mental retardation (40,75). This suggests a link between Akt and higher cerebral functionthat has not yet been studied in detail. Recently, a decreasein the Akt1 protein in the brain has been linked to schizophrenia(25). This is supported by the observation that a mutation inAkt1 leading to a decreased amount of protein increased therisk of schizophrenia, while the antipsychotic drug haloperidolwas shown to activate Akt signaling (25). In rodents, the PI3Kpathway has been implicated in memory formation (42, 49), whichis often impaired in schizophrenic patients (26).

Finally, note that although the reduction in organ mass in Akt1-deficientmice was proportional to body size, the mechanism of this decreasedepended on the tissue. Some indication that this is the caseis provided by the observation that in Akt1^–/– Akt2^–/–mice, skeletal muscle atrophy is due to decreased myocyte size,whereas there is a reduced number of cells in the skin (58).These organ-specific differences in the modes of mass reductionmay reflect the growth potential of particular organs. For example,the heart enlarges in the postnatal period exclusively throughincreases in cell size, whereas the liver maintains a proliferativecapacity (60). It is perhaps for this reason that Akt1-deficientlivers have decreased cell numbers, whereas Akt1-deficient heartshave smaller cells. Previous studies in which Akt activity wasreduced in the heart have yielded contradictory results. Theexpression of a kinase-deficient Akt1 did not affect cardiacsize, whereas a dominant-negative PI3K reduced the heart masssignificantly (65, 66). This difference might be attributableto more attenuation in the total Akt activity produced by themutant PI3K or to nonspecific actions of the dominant inhibitoryform (19, 65). In this study, we have shown that Akt1 activityis required for the attainment of normal cardiomyocyte and heartsize. This finding further serves to emphasize the remarkablecapacity of the metazoan organism to coordinate the growth ofindividual organs and the critical role of Akt in this process.

ACKNOWLEDGMENTS

This work was partially funded by NIH grant R01 DK56886 to MorrisBirnbaum and NCI grant P01 CA97403 (project 2) to Argiris Efstratiadis.Rachael Easton and Kristin Roovers were supported by NIH grantT32 DK7314 and by an American Heart Association fellowship,respectively. Services were also provided by the RadioimmunoassayCore of the Penn Diabetes Center (NIH grant DK19525) and theMorphology Core of the Center of Molecular Studies in Digestiveand Liver Diseases (NIH grant DK50306).

FOOTNOTES

* Corresponding author. Mailing address: University of Pennsylvania School of Medicine, Clinical Research Building 322, 415 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-5001. Fax: (215) 573-9138. E-mail: birnbaum{at}mail.med.upenn.edu.

REFERENCES

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