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.

Two high-fertility mouse lines show differences in component fertility traits after long-term selection.

UNLABELLED: Two selected high-fertility mouse lines, namely FL1 and FL2, and a non-selected control (Fzt:DU), all derived from the same genetic pool, were analysed as an animal model for polytocous species to elucidate the effects of long-term selection and to identify relevant component traits that may be responsible for fertility performance. The index trait used for breeding selection was largely increased by 104% and 142% in the FL1 and FL2 lines, respectively, resulting in an average litter size of 17.3 pups and 18.7 pups per litter in the FL1 and FL2 lines, respectively, compared with a litter size of 11.0 pups per litter in the control (Fzt:DU). In addition, different component fertility traits were analysed in females of all three lines at different stages of the oestrous cycle and pregnancy. IN CONCLUSION: (1) early embryonic development was accelerated in the FL1 and FL2 lines compared with control; (2) plasma progesterone levels were not correlated with fertility performance; (3) a largely increased ovulation number (i.e. number of corpora lutea) was responsible for high prolificacy in both lines; however, (4) the number of ova shed, as well as the rate of loss of ova and pre- and postimplantation conceptuses, was very different in the FL1 and FL2 lines, suggesting that different genetic components may be responsible for the high prolificacy in both high-fertility lines.

Long-term growth selection of mice changes the intrinsic susceptibility of myogenic cells to apoptosis.

Myogenic cells were derived from mice long-term selected over 70 generations for high 6-weeks body weight (DU-6) and from unselected control mice (DU-Ks). The cells were grown in medium with 10% foetal bovine serum (FBS) for 8 days or transferred to low serum conditions (1% FBS) at days 4 and 6 of cultivation, respectively, and maintained for two further days. In both cell lines, serum reduction induced decreases in DNA and protein contents, and in DNA synthesis rate. It also triggered apoptotic cell death as demonstrated by increased DNA strand breaks and expression of active caspase-3. Concomitantly, the anti-apoptotic protein bcl-2 was enhanced. The basal frequency of apoptotic cells decreased with time of cultivation in both lines and was lower in DU-6 than in DU-Ks cells. However, the increase in apoptosis induced by serum reduction was more pronounced in DU-6 than in DU-Ks cells and did not differ between the time points of serum reduction. The results suggest that growth selection decreases the basal apoptosis frequency of muscle satellite cells under normal supply, but enhances the intrinsic susceptibility to growth factor withdrawal by serum deprivation as a severe apoptotic stimulus. Furthermore, the apoptotic response to growth factor withdrawal seems to be largely independent of the stage of myogenic development.

Susceptibility to apoptosis in different murine muscle cell lines.

Objective of the study was to investigate growth characteristics and susceptibility to apoptosis in different murine muscle cell lines (selected for high body weight, DU-6; randomly mated control, DU-Ks; immortalized myoblast cell line, C2C12). Apoptosis was induced by serum deprivation. At days 4, 5, and 6 of cultivation, protein, DNA and the frequency of apoptotic cells were determined. Until day 4, C2C12 accumulated more DNA and protein compared with DU-Ks and DU-6, while exhibiting a lower percentage of apoptotic cells. Serum deprivation impaired the growth of each cell line. C2C12 continued to accumulate DNA and protein after serum deprivation, whereas reductions, indicative of cell death, were apparent in DU-Ks and DU-6. Serum deprivation did not enhance apoptosis in C2C12. Higher percentages of apoptosis were observed in DU-Ks and DU-6 after 2 days of serum deprivation with greater responsiveness of DU-6 to serum deprivation. The results suggest that cell loss in response to serum deprivation is in part due to induction of apoptosis. C2C12 are less sensitive to sub-optimal culture conditions compared with DU-Ks and DU-6 which are at a closer distance to the in vivo status. Moreover, long-term selection for growth decreases the basic frequency of apoptosis of muscle satellite cells, but increases their susceptibility to apoptosis induction.