Activation of AMPK & p38MAPK Pathways with a Novel Agonistic Compound in LHCN-M2 Skeletal Muscle Myotubes

Introduction: Pharmacological targeting of skeletal muscle to enhance metabolism through signalling pathways independent of insulin could be effective in treating metabolic diseases such as obesity and type 2 diabetes. BI-9774 is an allosteric agonist which has been shown to have high potency in enhancing 5’ AMP-activated protein kinase (AMPK) activity. The purpose of these experiments was to establish the effects of BI-9774 on AMPK signalling and mitochondrial capacity in LHCN-M2 human skeletal muscle cells.

Methods: LHCN-M2 human skeletal muscle cells were cultured and differentiated into multinucleated myotubes. Following 10 days of differentiation, time (0-12 h) and dose response curves (0-1µnM) were conducted to establish phosphorylation of AMPK (Thr172), Acetyl-CoA carboxylase (ACC) (Ser79) and p38 MAPK Kinase (Thr180/Tyr182) in response to BI-9774.

Results: Time course experiments showed an increase in the phosphorylation of AMPK at 15, 30 and 60 minutes and phosphorylation of ACC at 30 (P= 0.006), 60 (P= 0.004), 300 (P= 0.010) minutes when stimulated with 10nM of BI-9774. Despite being described as a selective AMPK agonist, there was an increase in p38 MAPK phosphorylation at 15 minutes of stimulation at 10nM (P= 0.015). Dose response curves showed a significant increase in AMPK and ACC phosphorylation at concentrations of equal to and greater than 10nM (P<0.05) and 1nM (P<0.05), respectively. Preliminary seahorse data suggests that concentrations of BI-9774 up to 1µM induce no changes to basal or maximal oxygen consumption rates.

Conclusions: These experiments show for the first time that BI-9774 can induce phosphorylation of the AMPK-ACC and p38 MAPK signalling pathways in human skeletal muscle myotubes. Further experiments are required to investigate the effects of BI-9774 on enhancing skeletal muscle metabolism as a potential therapeutic for treating metabolic disease.