Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice

Analysis of body weight, contents of DNA, RNA and protein in <i>M. quadriceps femoris</i> of Fzt:DU (29 weeks) and DU6P (11 and 29 weeks) female mice (n = 5).

<p>a,b - different superscripts indicate significant differences (p<0.05);</p>*<p>- significantly different if compared to 11-week DU6P or Fzt:DU, respectively as evaluated using the Wilcoxon-signed rank test.</p><p>Furthermore the non-polysomal and polysomal RNA fraction in <i>M. quadriceps femoris</i> of Fzt:DU (29 weeks) and DU6P (11 and 29 weeks) female mice (n = 4) was analysed.</p

Analysis of signal transduction in muscle lysates from 11, 29 and 54-week female DU6P and Fzt:DU mice.

<p>Western blot identified phosphorylated and total expression of the respective signaling molecule. Specific activation was calculated from the ratios of phosphorylated versus total protein. Coomassie blue staining of the membranes used for Western immuno detection was used as loading control. A: IGF-1Rß in membrane fractions (n = 3); B: ILK (n = 9); C: GDF-8, 26 kDa band (n = 11) and D: PTEN (n = 9). The inserts provide representative experiments, whereby all samples were studied on the identical membrane. Sample numbers (n) depict the number of samples per age group, the error bars represent SEM.</p