Additional file 1: Table S1. of Investigating mechanisms underpinning the detrimental impact of a high-fat diet in the developing and adult hypermuscular myostatin null mouse

Blood parameters. Values from minimum of six male mice. (DOCX 13 kb

Additional file 3: Figure S2. of Investigating mechanisms underpinning the detrimental impact of a high-fat diet in the developing and adult hypermuscular myostatin null mouse

Effect of high fat diet on Gastrocnemius lipid content. All values are fraction of total lipid content. ANOVA; (*) P<0.05 vs. WT ND; WT HF; (**) P<0.05 vs. Mstn-/- ND. N=6 male mice per group

Expression of SGK1 in wild-type and <i>Mstn</i>^{<i>−/−</i>} Tibialis Anterior (TA) before and after acute 24 starvation.

<p>(A) Western blot and scanning densitometry analysis of 54kD band. (B) (D) Altered profile of SGK1 expression in IIB fibres of <i>Mstn</i>^{<i>−/−</i>} TA muscle. (C) Immunochemistry for the expression of MCH IIB and SGK1 (Scale bar = 100μm). (E) Western blot analysis of phosphorylated FoxO3a at residues T32 and S253 from the TA muscles and the influence of starvation. Phosphorylation at T32 indicated by arrow marking the upper band. (F) Quantification of pFoxO3a-T32/ FoxO3a (Two-way ANOVA; *p<0.05).</p

Molecular and protein analysis of the Tibialis Anterior (TA) for catabolic and anabolic markers in response to acute starvation.

<p>(A) qPCR analysis of changes in the expression of master regulator of catabolism, <i>Foxo1</i>. (B-C) E3 class of ubiquitin ligases, <i>MuRF1</i> and <i>atrogin</i>. Regulators of autophagy (D) <i>Bnip3</i>, (E) <i>Beclin</i>, ((F) <i>LC3b</i>, (G) <i>ATF4</i>, (H) <i>CathepsinL</i> (I) <i>Vsp34</i> (J) <i>p62</i>, (K) <i>Gadd34</i>. (L) mRNA expression of <i>Mul1</i> a mitophagy regulator. All qPCR levels normalised to levels of G6PDH. (M) Quantification of LC3-II in relation to LC3 at protein level. (N) Quantification of pAkt at residue S473 in relation total Akt at protein level. (O) Western blot analysis of regulators of autophagy (LC3 and p62), and protein synthesis (Akt-total and phosphorylated versions at S473 and T308, 4ebp1 and S6) with tubulin serving as loading control. (Two-way ANOVA; *p<0.05).</p

Pair-wise comparisons of the metabolic profiles obtained from gastrocnemius muscle from wild-type and <i>Mstn</i>^−/− mice under fed and starved states.

<p>PCA scores plots (PC1 vs PC2) comparing (A) wild-type fed vs <i>Mstn</i>^−/− fed; (B) wild-type fed vs wild-type starved; (C) <i>Mstn</i>^−/− fed vs <i>Mstn</i>^−/− starved; (D) wild-type starved vs <i>Mstn</i>^−/− starved; (E) wild-type fed vs <i>Mstn</i>^−/− starved; (F) wild-type fed vs <i>Mstn</i>^−/− fed vs <i>Mstn</i>^−/− starved (% variance explained in the parenthesis). Colour loadings plots shown for (G) PC2 of the model comparing wild-type fed vs <i>Mstn</i>^−/− fed and (H) PC1 of the model comparing wild-type fed vs <i>Mstn</i>^−/− starved. Product of PC loadings with standard deviation of the entire data set coloured by the square of the PC loading.</p

Changes in body mass in wild-type (WT) and <i>Mstn</i>^{<i>−/−</i>} (Mstn) at 12 and 24 hours after complete food withdrawal (acute starvation).

<p>(A) Wild-type mice showed a significant decrease after 24h starvation. In contrast <i>Mstn</i>^{<i>−/−</i>} lost mass in the first and second 12h of starvation. (B) Changes in body weight expressed as a % of normal body weight. Weight changes of (C) EDL, (D) soleus, (E) TA and (F) Gastrocnemius at 12 and 24h after acute starvation. Wild-type animals showed no significant muscle mass reduction in contrast to all muscles examined from <i>Mstn</i>^{<i>−/−</i>}. (Two-way ANOVA; *p<0.05)</p

Intra-muscular bodies and muscle function.

<p>(A) Immunohistochemical analysis of p62 expression in the EDL of wild-type and <i>Mstn</i>^{<i>−/−</i>} mice. (AI) Wild-type mice on normal diet show very few punctate p62 positive bodies in any fibre whereas many appear after starvation (AII). (AIII) Normal diet <i>Mstn</i>^{<i>−/−</i>} EDL fibres show large number of punctate p62 bodies predominantly in the large fibres which almost all disappear after starvation (IV) (Scale bar 75μm). Inset images shows high magnification images of p62 staining (scale bar 10μm). (B) Quantification of (B) twitch tension and (C) specific tension of the EDL from wild-type and <i>Mstn</i>^{<i>−/−</i>} before and after starvation. (Two-way ANOVA; *p<0.05).</p

Fibre type profiling after acute starvation.

<p>MHC fibre cross sectional area of Wild-type and <i>Mstn</i>^{<i>−/−</i>} EDL (A-C), Soleus (D-G) and Tibialis Anterior (TA) (H-J) before and 24h after starvation of wild-type and <i>Mstn</i>^{<i>−/−</i>} mice. (K) Percentage of SDH positive (oxidative) and SDH negative fibres from the EDL of wild-type and <i>Mstn</i>^{<i>−/−</i>} mice. (L) Changes in fibre diameter of SDH negative fibres of the EDL in wild-type and <i>Mstn</i>^{<i>−/−</i>} mice before and after starvation. (M) Changes in fibre diameter of SDH positive fibres of the EDL in wild-type and Mstn mice before and after starvation. (N) Percentage of SDH positive (oxidative) and SDH negative fibres from the Soleus of wild-type and <i>Mstn</i>^{<i>−/−</i>} mice. (O) Changes in fibre diameter of SDH negative fibres of the soleus in wild-type and <i>Mstn</i>^{<i>−/−</i>} mice before and after starvation. (P) Changes in fibre diameter of SDH positive fibres of the soleus in wild-type and Mstn mice before and after starvation. (Two-way ANOVA; *p<0.05).</p

Quantitative and qualitative analysis of protein synthesis in wild-type and <i>Mstn</i>^{<i>−/−</i>} Tibialis anterior and gastrocnemius muscle using SUnSET.

<p>(A-B) Slot-blot western analysis for quantification of protein synthesis in the gastrocnemius and TA muscle. (C) Western blot showing total protein distribution and puromycin labelled polypeptides from wild-type and <i>Mstn</i>^{<i>−/−</i>} TA muscle. Note that some bands remain of similar intensity whereas others decrease. (D) Altered profile of high levels of protein synthesis in IIB fibres of <i>Mstn</i>^{<i>−/−</i>} TA muscle. (E) Immunochistochemistry for the expression of MHC IIB and high levels of protein synthesis determined by puromycin incorporation. (Scale bar = 100μm). (Two-way ANOVA; *p<0.05)</p