Mice Deficient in Ribosomal Protein S6 Phosphorylation Suffer from Muscle Weakness that Reflects a Growth Defect and Energy Deficit

BACKGROUND: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6(P-/-)), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic beta-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests. METHODOLOGY/PRINCIPAL FINDINGS: A large variety of experimental methodologies, including morphometric measurements of histological preparations, high throughput proteomic analysis, positron emission tomography (PET) and numerous biochemical assays, were used in an attempt to establish the mechanism underlying the relative weakness of rpS6(P-/-) muscles. Collectively, these experiments have demonstrated that the physical inferiority appears to result from two defects: a) a decrease in total muscle mass that reflects impaired growth, rather than aberrant differentiation of myofibers, as well as a diminished abundance of contractile proteins; and b) a reduced content of ATP and phosphocreatine, two readily available energy sources. The abundance of three mitochondrial proteins has been shown to diminish in the knockin mouse. However, the apparent energy deficiency in this genotype does not result from a lower mitochondrial mass or compromised activity of enzymes of the oxidative phosphorylation, nor does it reflect a decline in insulin-dependent glucose uptake, or diminution in storage of glycogen or triacylglycerol (TG) in the muscle. CONCLUSIONS/SIGNIFICANCE: This study establishes rpS6 phosphorylation as a determinant of muscle strength through its role in regulation of myofiber growth and energy content. Interestingly, a similar role has been assigned for ribosomal protein S6 kinase 1, even though it regulates myoblast growth in an rpS6 phosphorylation-independent fashion

Mice deficient in ribosomal protein S6 phosphorylation suffer from muscle weakness that reflects a growth defect and energy deficit. PLoS One

Abstract Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6 P2/2 ), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic b-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests

Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis

The regulated phosphorylation of ribosomal protein (rp) S6 has attracted much attention since its discovery in 1974, yet its physiological role has remained obscure. To directly address this issue, we have established viable and fertile knock-in mice, whose rpS6 contains alanine substitutions at all five phosphorylatable serine residues (rpS6(P-/-)). Here we show that contrary to the widely accepted model, this mutation does not affect the translational control of TOP mRNAs. rpS6(P-/-) mouse embryo fibroblasts (MEFs) display an increased rate of protein synthesis and accelerated cell division, and they are significantly smaller than rpS6(P+/+) MEFs. This small size reflects a growth defect, rather than a by-product of their faster cell division. Moreover, the size of rpS6(P-/-) MEFs, unlike wild-type MEFs, is not further decreased upon rapamycin treatment, implying that the rpS6 is a critical downstream effector of mTOR in regulation of cell size. The small cell phenotype is not confined to embryonal cells, as it also selectively characterizes pancreatic β-cells in adult rpS6(P-/-) mice. These mice suffer from diminished levels of pancreatic insulin, hypoinsulinemia, and impaired glucose tolerance

Anonymized Full data set-CA72-4-111012

<p><strong>endoscopic findings:</strong><br> normal=1, inflammatory=2, mucosal tumor (polyp/carc)=3, 4=other<br> <strong>histological findings:</strong><br> 1=normal, 2=chronic gastritis, 3=acute gastritis, 4=intestinal metaplasia, 5=gastric cancer, 6=other<br> <strong>smoking status:</strong><br> 0=never, 1= active or ex<br> <strong>alcohol intake</strong><br> 0=never, 1= active or ex<br> <strong>Endoscopic evidence of gastric bleeding</strong><br> 0=no, 1=yes<br> <strong>PPI dose</strong><br> muliples of standard dose<br> <strong>Hp-status</strong><br> 0=negative, 1=positive</p

The amount of mitochondria and the activity of complexes of the oxidative phosphorylation system are unchanged in rpS6^P−/− muscle.

<p>Mitochondria were isolated from hind limb muscle of four age-matched rpS6^P+/+ (WT) and rpS6^P−/− (−/−) male mice and were assayed for citrate synthase (A), cytochrome C oxidase (B), as well as complexes I (C), II (D), II+III (E) and IV (F) of the oxidative phosphorylation system. The activity of the different complexes was normalized to that of citrate synthase and the results are presented as relative activities. Vertical bars represent SEM (n = 4 age- matched male mice for each genotype).</p

Muscle strength is impaired in rpS6^P−/− mice.

<p>(A to C) Age-matched (4 and 7 month) male mice (n = 13 for WT and 12 for rpS6^P−/− mice) were subjected to screening, according to the SHIRPA behavioral protocol (see details in “Experimental Procedures”). (A) Grip strength. Mice were allowed to grip a grid and a gentle horizontal backwards pull through their tail was applied. Higher scores indicate greater grip strength. An unbiased observer, blinded to the genotype, performed the experiment in a blind fashion; (B) Wire maneuver. Results represent time in seconds required for a mouse that is hung from a wire with its forearms to elevate its hind limbs and grip the wire. (C) Rota-rod performance. Mice were placed on a moving cylinder, which was gradually accelerated from an initial speed of 4 rpm to a maximum of 40 rpm. Latency to fall from the rota-rod is presented in seconds. Motor performance was measured in three 10 min sessions (time 0, 1 h and 24 h). In each trial, the time in seconds until falling off was recorded. *<i>P</i><0.0005 for each trial versus rpS6^P+/+ mice. (D) Endurance test. 5 rpS6^p+/+ and 4 S6^P−/− age-matched (7–9 weeks) male mice were allowed to run on the treadmill set with a slope of 12.5 degree and a speed of 20 m/min. The results represent the total running time with two attempts to pause. Results of all experiments are presented as average±SEM, and the absence of SEM bars for some measurements simply reflects a value close to zero that is graphically invisible.</p

Insulin-induced signaling and glucose uptake are similar in rpS6^P−/− and rpS6^p+/+ muscles.

<p>(A) Soleus muscles were excised from rpS6^P−/− (−/−) and rpS6^p+/+ (WT) 2- to 3-mo-old male mice following 16 h starvation and intraperitoneal injection of saline (−) or 2.5 U of insulin/kg of body weight (+) for 5 min. Cytoplasmic extracts were subjected to Western blot analysis with the indicated antibodies. Note the enrichment for the upper band of 4E-BP1 (hyperphosphorylated form) upon insulin treatment. (B) Insulin-induced uptake of glucose into isolated muscle. Right and left soleus muscles were isolated from the hind limbs of 16 h starved rpS6^P−/− (−/−) and rpS6^p+/+ (WT) male mice. 2-Deoxy glucose uptake is presented as µmol per gram tissue per h. The data are presented as average±SEM for 5 rpS6^P−/− and 4 S6^p+/+ age-matched (7–9 weeks) male mice. (C) Mice were injected intraperitoneally with 0.25 U insulin/kg body weight and within seconds with 10–24 Ci [¹⁸F]fluoro-2-deoxyglucose (FDG) into the tail vein. The radioactivity concentration in hind limb muscles was estimated at 40 to 45 min after FDG injection by positron emission tomography. The concentration of radioactivity is presented as % injected dose (ID) per gm body weight per ml tissue. The data are shown as average±SEM for 5 male mice of each genotype.</p

Newly formed myofibers are smaller in a rpS6^P−/− muscle.

<p>(A) Sections of tibialis anterior of 2-mo-old rpS6^P+/+ and rpS6^P−/− mice were prepared 5 days after cryoinjury, and were immunostained for eMHC. One representative section is presented. (B) Enlargement of the framed area in (A). (C) Digital images of the immunostained sections were used for measurement of CSA of individual eMHC-positive myofibers, as described in “Experimental Procedures”. The average CSA of rpS6^P−/− myofibers (n = 327) was normalized to that of their wild type counterparts (n = 210), which was arbitrarily set at 1. The results are presented as average±SEM. *, p<0.0001.</p

Stores of glycogen and triacylglycerol are larger and similar, respectively, in rpS6^P−/− muscle.

<p>(A) Soleus glycogen content. Soleus muscles were excised from 20 rpS6^P+/+ (WT) and 15 age-matched rpS6^P−/− (−/−) male mice and their glycogen content was measured. (B) Glycogen synthase (GS) and (C) glycogen phosphorylase (GP) activities were assayed in the same extracts (n = 4 age-matched male mice for each genotype). (D) and (E) TG content in soleus and gastrocnemious, respectively. 5 soleus (left and right muscles were pooled) and 10 individual gastrocnemious muscles were excised from 5 rpS6^P+/+ (WT) and 5 age-matched rpS6^P−/− (−/−) male mice and their TG content was measured. All results are presented as average±SEM.</p