Figure 1. Phosphoproteomics of insulin signalling in mouse skeletal muscle.

(a) Workflow for skeletal muscle phosphoproteomics of insulin signalling. (b) Quantification of skeletal muscle phosphoproteomics. (c) Volcano plot identifying insulin-regulated phosphopeptides. The greatest log2(insulin/unstimulated) fold change across strain-diet combinations is plotted against significance (insulin stimulation main effect, three-way ANOVA). Three phosphopeptides with -log10 q-values greater than 35 were removed for visual clarity. (d–g) Example insulin-regulated phosphopeptides. The protein and phosphorylated amino acid are indicated, as well as the number of phosphosites on the phosphopeptide (e.g. ‘P1’). n=4–6 biological replicates.

Figure 1—figure supplement 1. Genetics and diet alter morphometric and metabolic phenotypes.

(a) Mouse body weight was measured during a 6-week diet regimen. Two-sided t-tests were performed to compare HFD to CHOW within each strain after 6 weeks, following Benjamini-Hochberg p-value adjustment (*). (b–d) Measurement of (b) adiposity, (c) lean mass, (d) ground soleus mass, (e) fasting blood glucose, and (f) fasting blood insulin at the end of the diet regimen. (g) At the end of the diet regimen a glucose tolerance test was performed. (h) The area of the blood glucose curve (GTT AOC) was calculated. In (b–h), two-sided t-tests were performed to compare HFD to CHOW within each strain (*) or to compare each strain to C57Bl6J within either diet (#). p-Values were adjusted by the Benjamini-Hochberg procedure. Error bars indicate SEM. In (g) t-tests were only performed on 15 min blood insulin levels. No comparisons across strains on CHOW were significant. n=8–11 biological replicates. */#: 0.01≤p<0.05, **/##: 0.001≤p<0.01, ***/###: p<0.001.

Figure 1—figure supplement 2. Quality control analysis of phosphoproteomics data.

(a) The number of unique class I phosphopeptides quantified in each sample and in total. (b) Pearson’s correlation was performed between each pair of samples. Samples are ordered by hierarchical clustering. (c) Principal component analysis was performed on the phosphoproteome. The first two principal components (PC1 and PC2) are plotted for each sample and the percentage of overall variance explained by each principal component is indicated. ‘bas’: unstimulated, ‘ins’: insulin-stimulated. (d) Hierarchical clustering was performed on all samples.

Figure 1—figure supplement 3. Characterisation of the insulin-regulated phosphoproteome.

(a) The enrichment of Gene Ontology (GO) biological processes in genes containing insulin-regulated phosphopeptides relative to the entire phosphoproteome (one-sided Fisher’s exact test, Benjamini-Hochberg p-value adjustment). Only significant pathways are shown (adj. p<0.05). The pathway ‘negative regulation of vascular-associated smooth muscle cell differentiation’ is abbreviated. (b) The number of phosphosites regulated by insulin in this study or a previous phosphoproteomic study of human skeletal muscle (Needham et al., 2022). Only phosphosites quantified in both studies were considered. (c) The number of insulin-regulated phosphopeptides with prior annotation of insulin regulation in the PhosphositePlus database (Hornbeck et al., 2015). (d) The number of phosphosites regulated by insulin in this study or regulated by exercise in two human phosphoproteomics studies (Needham et al., 2022; Hoffman et al., 2015). Only phosphosites quantified in all three studies were considered. (e) A phosphopeptide where HFD-feeding enhanced insulin responses in BXH9 but suppressed insulin responses in C57Bl6J and CAST. A two-way ANOVA was performed on insulin response values followed by two-sided t-tests comparing HFD to CHOW within each strain (q-values: *). (f) Phosphopeptides with a Strain effect were examined to determine whether the effect was due to altered unstimulated phosphorylation (‘Unstimulated’; Strain/C57Bl6J fold change >1.3 in unstimulated samples), altered insulin-stimulated phosphorylation (‘Insulin’; Strain/C57Bl6J fold change >1.3 in insulin-stimulated samples), or both (‘Both’). A proportion of phosphopeptides passed neither of these filters (‘Undetermined’). (g) The same analysis was performed on Strain×Diet-affected phosphopeptides, using the HFD/CHOW fold changes in either unstimulated or insulin-stimulated samples for each strain. (h–i) The percentage of (h) Strain effects and (i) Diet effects (Uniform diet or Strain×Diet effect) among canonical or non-canonical insulin signalling proteins. p-Values indicate two-sided Fisher’s exact tests. The number of phosphopeptides in each group is shown. (j) The overlap of Strain and Diet effects.