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

Lifelong Obesity in a Polygenic Mouse Model Prevents Age- and Diet-Induced Glucose Intolerance– Obesity Is No Road to Late-Onset Diabetes in Mice

AIMS/HYPOTHESIS: Visceral obesity holds a central position in the concept of the metabolic syndrome characterized by glucose intolerance in humans. However, until now it is unclear if obesity by itself is responsible for the development of glucose intolerance. METHODS: We have used a novel polygenic mouse model characterized by genetically fixed obesity (DU6) and addressed age- and high fat diet-dependent glucose tolerance. RESULTS: Phenotype selection over 146 generations increased body weight by about 2.7-fold in male 12-week DU6 mice (P<0.0001) if compared to unselected controls (Fzt:DU). Absolute epididymal fat mass was particularly responsive to weight selection and increased by more than 5-fold (P<0.0001) in male DU6 mice. At an age of 6 weeks DU6 mice consumed about twice as much food if compared to unselected controls (P<0.001). Absolute food consumption was higher at all time points measured in DU6 mice than in Fzt:DU mice. Between 6 and 12 weeks of age, absolute food intake was reduced by 15% in DU6 mice (P<0.001) but not in Fzt:DU mice. In both mouse lines feeding of the high fat diet elevated body mass if compared to the control diet (P<0.05). In contrast to controls, DU6 mice did not display high fat diet-induced increases of epididymal and renal fat. Control mice progressively developed glucose intolerance with advancing age and even more in response to the high fat diet. In contrast, obese DU6 mice did neither develop a glucose intolerant phenotype with progressive age nor when challenged with a high fat diet. CONCLUSIONS/INTERPRETATION: Our results from a polygenic mouse model demonstrate that genetically pre-determined and life-long obesity is no precondition of glucose intolerance later in life

Longitudinal study of fasting glucose levels in male DU6 mice fed chow (A) or a high fat (B) diet (n_week3 = 4_; n_week27>13; *: P<0.05).

<p>Longitudinal study of fasting glucose levels in male DU6 mice fed chow (A) or a high fat (B) diet (n_week3 = 4_; n_week27>13; *: P<0.05).</p

Establishment and basic features of the polygenic DU6 mouse model.

<p>Body weight selection was performed over 146 generations beginning in 1976 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079788#pone-0079788-g001" target="_blank">Fig 1A</a>). Epididymal fat mass (Fig. 1B) in male DU6 mice from 7 to 39 weeks of age (n = 15; *: P<0.01; in 39-week DU6 mice: n = 9). Longitudinal consumption of chow food (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079788#pone-0079788-g001" target="_blank">Fig 1C</a>) was assessed in male mice in 4 different age groups over a period of 7 days (6, 12, 18 and 24 weeks of age; n>12; *: P<0.01).</p

Compositions of Altromin control chow and high fat diet (HFD; C 1080) used in the present study.

<p>Ether extracts from soybean oil and lard were used for fat isolation. Corn starch, maltodextrin, dextrose and sucrose were used for the preparation of the carbohydrate fraction. Casein was the source for the protein fraction and cellulose was used for crude fiber production.</p

Longitudinal glucose tolerance tests in male DU6 and Fzt:DU mice under standard chow or HFD (n>14; *: significant interaction of line, diet, age and repeated measures of glucose analysis with P<0.05; §: significant interaction of line and age with P<0.001).

<p>Longitudinal glucose tolerance tests in male DU6 and Fzt:DU mice under standard chow or HFD (n>14; *: significant interaction of line, diet, age and repeated measures of glucose analysis with P<0.05; §: significant interaction of line and age with P<0.001).</p

Changes in body and organ mass over life-time under chow and HFD.

<p>Effects of a HFD on body weight in male mice (Fig. 2A; until week 27: n>14; week 33 and 39: n>7; week 33: P<0.05). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079788#pone-0079788-g002" target="_blank">Fig 2B:</a> Effects of a HFD on tissues masses in male mice at an age of 39 weeks (n>7; *: significant effect of line*diet P<0.01 vs. chow diet (100%); °: significant effect of diet as a main effect in both lines: P<0.05 (epidid. fat: epididymal fat mass; Musc. r.f.: Musculus rectus femoris, gastr: gastrocnemius, t.a.: tibialis anterior; sol.: soleus).</p

Higher levels of muscle glycogen in 29-week female DU6P mice.

<p>Top: Biochemical analysis of glycogen content in muscle tissue of 29-week female Fzt:DU (n = 6) and DU6P (n = 12) mice. Lower: PAS staining of cryosectioned muscle of 29-week Fzt:DU and DU6P female mice. The images correspond to an area of about 1.3×0.88 mm in the histological sections. The error bars represent SEM.</p

Long-term phenotype selection for high protein mass in mice.

<p>A: Phenotypes of female Fzt:DU and DU6P mice at the age of 11, 29 and 42 weeks. B: Success for the selected trait (protein mass) in the course of long-term selection in DU6P mice versus unselected control mice (Fzt:DU). C: Dissected <i>M. quadriceps femoris</i> of 11, 29 and 42-week female Fzt:DU and DU6P mice. D: Longitudinal body weights in female DU6P mice versus Fzt:DU mice. E and F: Relative weights of <i>M. quadriceps femoris</i> and carcasses from female Fzt:DU and DU6P mice (n = 15; p<0.03 for all comparisons of age-matched mouse lines). The sample number (n) depicts the number of samples per age group, the error bars represent SEM.</p

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