α-MSH (100 nM) stimulated 2-Deoxy Glucose uptake and TBC1D1 S237, T596 and S700 phosphorylation +/- H89 in dissected soleus explants from WT mice.

<p>A: Soleus muscle was dissected, stimulated and 2-DG was measured as described (n indicated in the individual bars). B: Phosphorylation of TBC1D1 was measured in soleus muscle using WB as described. TBC1D1 S237, T596 and S700 phosphorylation is normalized to total TBC1D1. Findings are shown as a representative immunoblot and pooled data quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001). Data generated in the experiment are only obtained from experiment day 4.</p

α-MSH (100 nM) stimulated TBC1D1 S237 and T596 phosphorylation in WT and AMPK KD mice.

<p>TBC1D1 S237 and T596 phosphorylation sites were measured in soleus muscle using WB as described (n indicated in the individual bars). Phosphorylation of TBC1D1 S237 and T596 is normalized to total TBC1D1. Findings are shown as representative immunoblots and pooled data is quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH induced phosphorylation of AMPK in differentiated L6 myotubes and in soleus explants dissected from lean and diet-induced obese (DIO) mice.

<p>A: Phosphorylation of AMPK in lean and DIO mice in basal conditions in soleus muscle explants (n=4). 2.B: α-MSH-phosphorylation of AMPK in lean and DIO soleus muscle explants (n=6). 2.C: Phosphorylation of AMPK after α-MSH-stimulation (100 nM) in differentiated L6 myotubes (n=4). AMPK phosphorylation is normalized to total AMPK. Unpaired one-tailed t-test was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle)</p

Overview of cellular signaling induced by α-MSH in skeletal muscle.

<p>α-MSH stimulates glucose uptake by an unknown mechanism, which acts independently of AMPK, TBC1D1 and GLUT4 translocation. α-MSH stimulates phosphorylation of TBC1D1 independently of PKA and AMPK. α-MSH stimulates phosphorylation of AMPK independently of PKA.</p

α-MSH stimulated 2-Deoxy Glucose uptake in soleus and extensor digitorum longus (EDL) muscle explants.

<p>3.A: soleus explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). Data is presented as the mean ± SEM of pooled data from a series of experiments (see individual bars). 3.B: EDL explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). 3.C: Phosphorylation of Akt was measured in soleus explants after α-MSH-stimulation (100 nM). Data is presented as the mean ± SEM. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p