Fig. 4

Silencing of IL32 influences differentiation capacity and insulin signaling whereas IL32 overexpression in mice impairs insulin sensitivity. a Array data of mRNA expression and DNA methylation (only significant sites) of IL32 before versus after differentiation of primary human myoblasts from non-obese subjects (n = 13, *q < 0.05). b Protein expression of IL-32 in primary human myoblasts (0 h) and after 3 and 7 days of differentiation. Stain-free total protein staining was used for normalization. A representative blot is shown above the bars (n = 7). c Increased protein level of IL-32 found during differentiation was significantly blocked with siRNA after 3 and 7 days of differentiation of myoblasts. The average of Si-SCR is set to 1 at both time points. Stain-free total protein staining was used for normalization. Representative blots are shown above the bars. d Significantly enriched gene sets in Gene Set Enrichment Analysis (GSEA) based on differential gene expression in IL-32 deficient myotubes (day 7) versus control (si-SCR). Lower panels show expression of genes contributing to the gene sets Hypertrophic Cardiomyopathy HCM and Pentose Phosphate Pathway (n = 5, *q < 0.05). e A number of genes involved in myogenesis and metabolism with significantly different expression between IL-32 deficient myotubes (day 7) and control (n = 5, *q < 0.05). Silencing of IL32 was associated with increased levels of ATP (f) and increased insulin-stimulated AKT phosphorylation at Ser473 and Thr308 (n = 3) (g) in differentiated myotubes (day 7). h The expression of IL32 in skeletal muscle biopsies obtained from 27 adult men correlated positively with HOMA-IR (Pearson correlation). i Experimental set-up of the mouse study with IL32tg mice showing the duration of HFD, including time points for different analyses. j There was no difference in body weight between IL32tg and WT mice after 18 weeks on a HFD. IL32tg mice had significantly lighter tibialis anterior in absolute value (k) and in relation to body weight (l) compared to WT after 18 weeks on a HFD. IL32tg mice had lower glucose levels in the fasted state and higher levels 20 minutes after intravenous insulin administration compared with WT (m), resulting in a decreased insulin response (n). o Significantly enriched gene sets in GSEA based on differential gene expression in tibialis anterior from IL32tg versus WT mice after 18 weeks on a HFD (n = 6). p Soleus from IL32tg mice have decreased Akt phosphorylation at Thr308 after 30 minutes in vitro incubation with or without insulin (WT n = 4, IL32tg n = 5). Data are presented as mean ± SEM, n = 4 for cell experiments, n = 10 for WT mice, and n = 9 for IL32tg mice if nothing else stated. WT, Wild type mice (control); IL32tg, IL32 transgenic mice; HFD, High fat diet; * P < 0.05, ** P < 0.01, *** P < 0.001 for figure b and e–o. Statistics were calculated with paired t-test for cell experiments and Mann–Whitney U test for mice data