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

Impaired glucose tolerance in rats fed low-carbohydrate, high-fat diets

Moderate low-carbohydrate/high-fat (LC-HF) diets are widely used to induce weight loss in overweight subjects, whereas extreme ketogenic LC-HF diets are used to treat neurological disorders like pediatric epilepsy. Usage of LC-HF diets for improvement of glucose metabolism is highly controversial; some studies suggest that LC-HF diets ameliorate glucose tolerance, whereas other investigations could not identify positive effects of these diets or reported impaired insulin sensitivity. Here, we investigate the effects of LC-HF diets on glucose and insulin metabolism in a well-characterized animal model. Male rats were fed isoenergetic or hypocaloric amounts of standard control diet, a high-protein “Atkins-style” LC-HF diet, or a low-protein, ketogenic, LC-HF diet. Both LC-HF diets induced lower fasting glucose and insulin levels associated with lower pancreatic β-cell volumes. However, dynamic challenge tests (oral and intraperitoneal glucose tolerance tests, insulin-tolerance tests, and hyperinsulinemic euglycemic clamps) revealed that LC-HF pair-fed rats exhibited impaired glucose tolerance and impaired hepatic and peripheral tissue insulin sensitivity, the latter potentially being mediated by elevated intramyocellular lipids. Adjusting visceral fat mass in LC-HF groups to that of controls by reducing the intake of LC-HF diets to 80% of the pair-fed groups did not prevent glucose intolerance. Taken together, these data show that lack of dietary carbohydrates leads to glucose intolerance and insulin resistance in rats despite causing a reduction in fasting glucose and insulin concentrations. Our results argue against a beneficial effect of LC-HF diets on glucose and insulin metabolism, at least under physiological conditions. Therefore, use of LC-HF diets for weight loss or other therapeutic purposes should be balanced against potentially harmful metabolic side effects.</jats:p

Induction of ketosis in rats fed low-carbohydrate, high fat diets depends on the relative abundance of dietary fat and protein

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis, however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Methods: Male Wistar rats were pair-fed either a standard chow diet or one of 3 different LC-HFDs for 4 weeks, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by NMR, analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 weeks on the respective diets. Results: Compared to chow, rats fed with LC-75/10, LC-65/20 and LC-55/30 gained significantly less bodyweight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about 16-fold higher with LC-75/10, only (

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

A: Serum IGF-I levels in female Fzt:DU and DU6P mice over life time (n = 8 per age group).

<p>B: Expression of IGF-II precursor (23 kDa) in muscle tissues from 11-, 29- and 54-week female Fzt:DU and DU6P mice (n = 10 per age group). The results are normalized for the signal intensities as detected by Coomassie blue staining of the membranes used for Western immuno detection. Data are expressed as percent of female 11-week Fzt:DU mice (100%). 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 signal transduction in muscle lysates from 11-, 29- and 54-week female DU6P and Fzt:DU mice.

<p>The Western blot inserts show phosphorylated (Ser-473) and total expression of AKT from one representative experiment, whereby all samples were studied on the identical membrane. Each Western blots were performed three times. Thus a total of 9 different animals was included in the bar chart for each timepoint. Specific activation was calculated from the ratios of phosphorylated versus total AKT (n = 9 per age group). Coomassie blue staining of the membranes used for Western immuno detection was used as loading control. The error bars represent SEM.</p

In mid-aged female DU6P mice inhibition of PTEN by means of reduced PTEN expression and increased PTEN phosphorylation correlates with higher levels of AKT phosphorylation at Ser-473, inactivation of GSK3ß on one hand and higher levels of S6K on the other.

<p>An increase of muscle mass is due to higher levels of muscle glycogen and an increased rate of protein synthesis in female 29-week DU6P mice.</p