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. 2004 Nov;24(21):9295-304.
doi: 10.1128/MCB.24.21.9295-9304.2004.

Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy

Ka-Man V Lai  1 , Michael Gonzalez, William T Poueymirou, William O Kline, Erqian Na, Elizabeth Zlotchenko, Trevor N Stitt, Aris N Economides, George D Yancopoulos, David J Glass
Affiliations

Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy

Ka-Man V Lai et al. Mol Cell Biol. 2004 Nov.

Abstract

Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage.

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Figures

FIG. 1.
FIG. 1.
Constitutive activation of Akt in transgenic chimeras (AktTg) results in skeletal muscle hypertrophy. (A) Strategy for generation of AktTg animals. A partial map of the WT mouse ROSA-26 locus, including exon 2 (E2) is shown. Upon homologous recombination, the targeting vector inserted a total of 7.4 kb—including a neomycin resistance cassette (NEO), the HSA promoter, and a constitutively active form of Akt fused to EGFP (c.a.Akt-EGFP)—into the ROSA-26 locus. WT and targeted loci are shown. Abbreviations: A, AvrII; B, BamHI; RV, EcoRV; N, NotI. (B) AktTg chimeras displayed a hypertrophic skeletal muscle phenotype. Increased skeletal muscle size is evident when we compared an AktTg animal to a WT littermate. Double-headed arrows point to a muscle that is significantly larger in the AktTg. The single arrow points to a fat pad in the WT that is absent in the AktTg chimeric animals. (C) The transgenic c.a.Akt-EGFP fusion protein is phosphorylated and is expressed at high levels in skeletal muscle in an immunoblot of total protein lysates isolated from AktTg and WT muscles. Both endogenous Akt and the c.a.Akt-EGFP fusion protein are evident in muscle from the AktTg, whereas only endogenous Akt is seen in muscle from the WT animal (top panel). The c.a.Akt-EGFP protein is phosphorylated on Serine-473. In the WT animal, only muscle obtained from a CH model is phosphorylated (bottom panel). Lanes (muscle abbreviations): BW, abdominal body wall; TA, tibialis anterior; GA, gastrocnemius; Quad, quadriceps; PL, plantaris. (D) Transverse sections of muscles immunostained with an anti-laminin antibody to outline the perimeters of muscle fibers. Enlarged fibers are evident in both TA and MG (medial gastrocnemius) muscles of AktTg but not in the WT. (E) Mean cross-sectional area of muscle fibers. TA and MG muscle fibers are significantly (✽) larger in the AktTg compared to WT. Significance assessed by using the Student t test (P < 0.05). (F) Distribution of mean cross-sectional areas of muscle fibers. Individual TA and MG muscle fiber sizes from the AktTg (red bars) are shifted to the right side of the curve in comparison to those from the WT (gray bars).
FIG. 2.
FIG. 2.
Postnatal induction of muscle-specific c.a.Akt-EGFP (AktInd.Tg) expression in transgenic mice results in rapid skeletal muscle hypertrophy. (A) Strategy for the generation of AktInd.Tg animals. The tamoxifen-inducible HSA.CreERt2.AktInd.Tg targeting cassette is generated by inserting a floxed Cre-ERt2 and stop sequence 5′ to c.a.Akt-EGFP of the HSA-c.a.Akt-EGFP transgenic construct (Fig. 1A). Upon homologous recombination, the new targeting vector inserted a total of 11 kb into the ROSA-26 locus (targeted locus). Tamoxifen induces the excision of the floxed Cre-ERt2 cassette. The c.a.Akt-EGFP cassette is transcribed by the HSA promoter as a result of DNA recombination (recombined locus). (B) Southern analysis of targeted ROSA-26 locus. Genotyping of AvrII-digested genomic DNA by Southern blot hybridization with a probe specific to the ROSA-26 locus. Arrows indicate the 5.3-kb WT fragment and the 8.7-kb targeted AktInd.Tg allele. (C) Determination of tamoxifen-induced recombination of the AktInd.Tg allele by Southern blot analysis. An EGFP probe was used to distinguish the 4.2-kb recombined AktInd.Tg allele from the 7.5-kb targeted ROSA-26 allele from AvrII-digested genomic DNA. Recombination was only detected in DNA isolated from tamoxifen-treated muscles of AktInd.Tg mice. Lanes (muscle abbreviations): TA, tibialis anterior; GA, gastrocnemius; Quad, quadriceps. An arrow points to the 4.2-kb recombined allele. (D) PCR analysis of tamoxifen-induced recombination of AktInd.Tg allele. Specific oligonucleotides were used to amplify the 473-bp PCR product from the AktInd.Tg targeted allele and the 253-bp band from the recombined locus (arrows). DNA recombination of the AktInd.Tg cassette is evident only in DNA from muscle (and not in genomic DNA obtained from tails) that has been treated with tamoxifen. (E) Tamoxifen-induced hypertrophy in the AktInd.Tg mice. Photographs of skinned WT and AktInd.Tg mice obtained 1 week after daily injection with tamoxifen (Tam) for 14 consecutive days. The induced AktInd.Tg mice displayed a noticeable size difference in all skeletal muscles. (F) Tamoxifen-dependent gain in AktInd.Tg muscle weights. Muscle weights from GA muscles were taken from either untreated (−Tam) or tamoxifen-treated (+Tam) WT and AktInd.Tg mice. A significant increase in muscle weight was observed only in the Tam-treated AktInd.Tg animal in both males and females. An asterisk indicates a significant difference in AktInd.Tg weights compared to all of other control groups (P < 0.0001). The mean ± the standard error of the mean is given for each group.
FIG. 3.
FIG. 3.
Analysis of skeletal muscle fibers from AktInd.Tg animals. (A) Comparison of teased muscle fibers by fluorescent confocal microscopy. A high level of EGFP is consistently correlated to enlarged muscle fibers isolated from the quadriceps muscles of tamoxifen-treated AktInd.Tg mice (AktInd.Tg + Tam). EGFP fluorescence is not observed in tamoxifen-treated WT (WT+Tam) mice. (B) Immunohistochemical analysis of teased muscle fibers by confocal microscopic imaging. An antibody-specific to alpha-actinin (α-actinin) demonstrates that the hypertrophic fibers isolated from the quadriceps muscle of AktInd.Tg mice (AktInd.Tg+Tam, right panels) maintain structural integrity, as also observed in the WT (WT+Tam, left panels). (C) Immunostaining of skeletal muscle cross-sections obtained from tamoxifen-treated WT (WT+Tam), tamoxifen-treated AktInd.Tg (AktInd.Tg+Tam), and oil-injected AktInd.Tg (AktInd.Tg+Oil) mice. Anti-laminin antibody (top panels in green) and anti-EGFP antibody (middle panels in red) were utilized to outline the borders of muscle fibers and to detect c.a.Akt.EGFP expressing fibers of the GA muscles, respectively. Overlapped images of the sections demonstrated a strong correlation between enlarged fibers (outlines in green) and c.a.Akt.EGFP-positive fibers (red), which were only observed in the cross-sections obtained from AktInd.Tg+Tam muscles. (D) Mean cross-sectional area of muscle fibers. GA muscle fibers obtained from the tamoxifen-treated AktInd.Tg mice (AktInd.Tg+Tam) have a larger mean area compared to WT muscle or uninduced AktInd.Tg muscle. An asterisk indicates a statistically significant difference in AktInd.Tg fiber size compared to each of the control groups (P < 0.0001). (E) Analysis of fiber size distribution of uninduced WT and AktInd.Tg mice. Individual plantaris muscle fibers from uninduced AktInd.Tg (AktInd.Tg−Tam) mice (red bars) were similar in size to fibers from untreated WT (WT−Tam) animals. (F) Distribution of muscle fiber size of AktInd.Tg mice with or without tamoxifen. Tamoxifen induces (+Tam) a dramatic increase in AktInd.Tg muscle fiber size and significantly shifts the frequency distribution curve to the right (green bars) in comparison to the distribution displayed by the uninduced AktInd.Tg muscles (red bars).
FIG. 4.
FIG. 4.
Tamoxifen-induced c.a.Akt-EGFP transgene expression and biochemistry. (A) Northern analysis of total RNA isolated from control (−) or tamoxifen-treated (+) WT and AktInd.Tg quadriceps muscles. The c.a.Akt-EGFP transcript was barely detectable in two of the untreated muscle samples (lanes 2 and 3), whereas a high level of the c.a.Akt-EGFP transcript was observed after tamoxifen treatment (the arrow points to the single 3-kb band that corresponds to the c.a.Akt-EGFP transcript). (B) The transgenic c.a.Akt-EGFP fusion protein is rapidly produced and phosphorylated upon induction with tamoxifen. Tamoxifen-induced c.a.Akt-EGFP expression is prominent by 7 days of tamoxifen treatment in the AktInd.Tg quadriceps muscles (top panel, 7 days). The protein level is further induced after 14 days of treatment (top panel, 14 days), and the AktInd.Tg locus continues to express the c.a.Akt-EGFP protein 7 days after tamoxifen treatment was halted (top panel, 14 + 7 days). This protein is undetectable in both the untreated AktInd.Tg (day 0) and WT mice (top panel, lanes 1 to 4). Comparable amount of endogenous Akt is detected in all muscles (top panel). The induced c.a.Akt-EGFP protein is phosphorylated on serine-473. In the WT animal, only muscle obtained from a CH model is phosphorylated (bottom panel). (C) The transgenic Akt-EGFP fusion protein is produced only in skeletal muscle, as shown by Western immunoblot analysis. Upon treatment with tamoxifen, the c.a.Akt-EGFP fusion protein is evident in TA and GA muscles from the AktInd.Tg mice but not in WT muscle. The c.a.Akt-EGFP fusion protein is not evident in liver (LI) even after tamoxifen treatment. Endogenous Akt is seen in muscle from both WT and AktInd.Tg animals (compare top panel). Upon treatment with tamoxifen, the induced c.a.Akt-EGFP protein is phosphorylated on serine-473. In the WT animal, only plantaris muscle (PL) obtained from a CH model is phosphorylated (bottom panel). (D) Induction of the transgenic Akt-EGFP fusion protein leads to the activation of endogenous p70S6 kinase. Equal amounts of protein extracts from treated (+) or control (−) AktInd.Tg and WT quadriceps muscles were immunoblotted. A phospho-specific antibody for p70S6K demonstrates that p70S6 kinase is only activated in tamoxifen-treated AktInd.Tg muscles (top panel). Activation of p70S6 kinase was also visualized by monitoring the total p70S6K protein in a gel shift assay; the upper bands are the phosphorylated, activated form of the protein (bottom panel).
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