Exercise regulates a diverse array of phosphorylation networks thought to promote numerous health benefits and therefore hold great promise as novel therapeutic targets. Recently, we performed phosphoproteomic analysis of human skeletal muscles subject to endurance, sprint, and resistance exercise to identify canonical signalling pathways during and after exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was Ser-67 on the uncharacterized protein C18ORF25 which we validate as an exercise-regulated AMPK substrate. Interestingly, integration with human genome-wide association studies linked genetic variants of C18ORF25 with glycated haemoglobin and type II diabetes.
To functionally characterise C18ORF25, we generated a whole-body knockout (KO) mouse model. KO mice gained similar weight on a chow diet compared to wild-type (WT) littermates, but we observed a striking increase in adiposity and subtle decrease in lean mass. Interestingly, KO mice on a chow or high-fat diet displayed no major differences in whole body glucose tolerance or skeletal muscle insulin sensitivity as assessed by ex vivo insulin-stimulated glucose uptake. Also, forced treadmill exercise revealed KO mice fatigue quicker than WT mice. These data prompted us to further investigate skeletal muscle function revealing KO mice have reduced Soleus force production, increased fatigability and recover slower than WT siblings. Histological analysis revealed no difference in muscle fibre-type but a drastic reduction in fibre cross sectional area.
Moreover, proteomic analysis of tibialis anterior muscles from KO mice revealed increased extracellular matrix proteins. In contrast, loss of C18ORF25 resulted in a reduction of proteins associated with translation, pyruvate and branched-chain amino acid metabolism, NEDDylation and several mitochondrial metabolic pathways. Interestingly, the most significantly down-regulated protein in KO muscles was cAMP-dependent protein kinase catalytic subunit beta. Phosphoproteomic analysis of KO soleus muscles subject to ex vivo contraction revealed elevated phosphorylation of substrates downstream of MEK and LCK while substrates of PKA, ERK, MK2 and GSK3 displayed attenuated contraction-induced phosphorylation.
Taken together, our data suggest C18ORF25 plays a vital role in AMPK-mediated skeletal muscle adaptations to exercise and that loss of C18ORF25 attenuates several known exercise-induced signalling pathways and kinases including PKA that mediate skeletal muscle contractile function.