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. 2002 Apr;22(8):2799-809.
doi: 10.1128/MCB.22.8.2799-2809.2002.

Akt/protein kinase B promotes organ growth in transgenic mice

Tetsuo Shioi  1 , Julie R McMullen, Peter M Kang, Pamela S Douglas, Toshiyuki Obata, Thomas F Franke, Lewis C Cantley, Seigo Izumo
Affiliations

Akt/protein kinase B promotes organ growth in transgenic mice

Tetsuo Shioi et al. Mol Cell Biol. 2002 Apr.

Abstract

One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.

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Figures

FIG. 1.
FIG. 1.
Generation of Akt transgenic mice. (A) Expression of the transgene. To compare the amount of transgene with endogenous Akt, 1, 10, and 100 μg of cardiac tissue lysate from caAkt or kdAkt transgenic mice and 100 μg of protein from NTg mice were separated by SDS-PAGE and probed with an anti-Akt antibody. (B) Akt activity in the transgenic heart. One milligram of cardiac tissue lysate was immunoprecipitated, and the kinase activity was measured by an in vitro kinase assay. kdAkt transgenic mice or NTg mice were injected with saline or 0.5 mg of IGF1 per kg for 5 min to activate Akt. Each group represents three or four hearts. Total Akt activity was markedly increased in the heart tissue of caAkt mice. Akt activity was significantly decreased in kdAkt mice under both basal and IGF1-stimulated conditions. Symbols: ∗, P < 0.05 versus saline-injected NTg mice; †, P < 0.05 versus IGF1-injected NTg mice.
FIG. 2.
FIG. 2.
Activation of GSK-3β and S6K1 in Akt transgenic mouse hearts. (A) Phosphorylation of GSK-3β in the heart tissue of Akt transgenic mice. GSK-3β was immunoprecipitated and probed with an anti-phospho-GSK-3β (Ser9) antibody (top) or anti-GSK-3β antibody (middle). In caAkt mice, the amount of phosphorylated GSK-3β normalized to total GSK-3β was not different from that in NTg mice. In kdAkt mice, the amount of phosphorylated GSK-3β normalized to total GSK-3β was significantly decreased compared with that in NTg mice (bottom). (B) S6K1 activity in the heart tissue of Akt transgenic mice. S6K1 activity was measured by an immune complex kinase assay using GST-S6 as a substrate (top). The amount of immunoprecipitated (IP) S6K1 was analyzed by Western blotting (middle). S6K1 activity normalized to total S6K1 was increased in the hearts of caAkt mice and decreased in the hearts of kdAkt mice (bottom). Faster-migrating bands were obtained from kdAkt mice, which suggests that S6K1 was less phosphorylated (middle, ∗).(C) Phosphorylation of ribosomal S6 protein in the heart tissue of Akt transgenic mice. Phosphorylation of ribosomal S6 protein was examined by Western blotting using an anti-phospho-S6 antibody (top). The amount of protein loading was normalized to the amount of GAPDH (middle). The amount of phosphorylated S6 normalized to GAPDH was increased 1.8-fold in caAkt mice, and it was decreased to 36% of that in NTg mice in kdAkt mice (bottom). Each group represents four hearts. ∗, P < 0.05 versus NTg mice.
FIG. 3.
FIG. 3.
PKCζ activity in kdAkt transgenic mice. PKCζ activity in the heart tissue of NTg mice or kdAkt transgenic mice was measured by an in vitro kinase assay using a synthetic peptide as a substrate (top). PKCζ was immunoprecipitated from 1 mg of heart tissue lysate. GST-PKCζ was used as a positive control for the kinase assay. PKCζ activity in the hearts of kdAkt transgenic mice was not different from that in the hearts of NTg mice. Each group represents three hearts. Immunoprecipitated (IP) PKCζ or GST-PKCζ was separated by SDS-PAGE, blotted onto a PVDF membrane, and probed with an anti-PKCζ antibody (bottom).
FIG. 4.
FIG. 4.
Pathological analysis of Akt transgenic mice. (A) Macropathological analysis of Akt transgenic mice. In caAkt mice, there was a marked increase in size in all of the chambers and in ventricular wall thickness. The proportion of the sizes of the chambers was maintained. In kdAkt mice, heart size was not different from that in NTg mice. Bars represent 1 mm. (B) Histopathological analysis of Akt transgenic mice. Hypertrophy of myocytes was evident in caAkt mice. Extensive interstitial fibrosis was also observed (blue on Masson's trichrome stain). Histology was normal in kdAkt mice. H&E, hematoxylin and eosin stain; trichrome, Masson's trichrome stain. Bars represent 10 μm.
FIG. 5.
FIG. 5.
Analysis of Akt transgenic mouse cell size. Cardiac myocyte size was determined by using isolated adult myocytes. The mean value for each mouse was calculated by using the measurements of 100 cells isolated from that mouse. Next, the mean value (± SE) for each experimental group was calculated on the basis of the mean values of the individual mice, and this value is presented. The cell volume was increased 1.9-fold ± 0.2-fold in caAkt transgenic mice. n, number of hearts examined.
FIG. 6.
FIG. 6.
Genetic interaction of PI3K and Akt in the regulation of organ size. (A) The HW/BW ratios of double-transgenic mice expressing both the caPI3K and kdAkt transgenes were significantly lower than those of caPI3K mice (1.10 ± 0.01 versus 1.22 ± 0.03; P = 0.0096; NTg mice = 1.00). (B) The HW/BW ratios of double-transgenic mice having both kdPI3K and caAkt were similar to those of caAkt mice (2.24 ± 0.08 versus 2.31 ± 0.25; P = 0.7438; NTg mice = 1.00). n, number of mice. Symbols: ∗, P < 0.05 versus NTg mice: †, P < 0.05 versus caPI3K mice; #, P < 0.05 versus kdPI3K mice.
FIG. 7.
FIG. 7.
Effect of rapamycin on S6K1 activity in caAkt mice. NTg mice or caAkt mice were treated with vehicle (V) or rapamycin (R). S6K1 activity in heart tissue was measured by an immune complex kinase assay using GST-S6 as the substrate (top). The amount of immunoprecipitated (IP) S6K1 was analyzed by Western blotting (middle). The S6K1 activity/S6K1 protein ratio is shown at the bottom. Each group represents four mice. S6K1 activity was increased in the hearts of vehicle-treated caAkt mice compared with that in the hearts of vehicle-treated NTg mice. Rapamycin significantly reduced S6K1 activity in the hearts of NTg mice. S6K1 activity in the hearts of rapamycin-treated caAkt mice was comparable to that in the hearts of rapamycin-treated NTg mice. Symbols: ∗, P < 0.05 versus vehicle-treated NTg mice; †, P < 0.05 versus vehicle-treated mice of the same genotype. IgG (H), immunoglobulin heavy chain.
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