In-vitro evaluation of Adipo-AI on adiponectin signaling.
To screen the optimal concentration and to calculate the IC50 values, we exposed C2C12 myotubes to different doses of AdipoAI (0-10 mM) in comparison to AdipoRON (0-10 mM), an adiponectin agonist, upon 24hr of incubation (Fig 1A). Our CCK-8 experiment results showed decrease in cell viability for both AdipoAI and AdipoRON treated groups with an increase in their respective concentrations. The IC50 values of AdipoAI and AdipoRON obtained from CCK8 assay was 4.7mM and 6.22mM respectively (Fig.1A). Therefore, the approximate IC50 mean value of 5 mM was chosen to be at the end of the increasing scale, apart from the aforementioned value mentioned earlier (Qiu et al., 2021); for further in-vitro experiments.
It has been reported earlier that activation of adiponectin receptor is through AdipoR1/R2 (R). Additionally, the anti-inflammatory feature of AdipoAI is also due to involvement of Adiponectin receptor APPL1(R). Investigation of AdipoAI-Adipoq signaling pathways through mRNA gene expressions of AdipoR1, AdipoR2 and pPargc1a at different doses of AdipoAI in comparison to AdipoRON in C2C12 myotubes revealed that accelerating dose of AdipoAI (0-5mM) observed significant increase in AdopoR1 and pPargc1a gene expression levels in comparison to AdipoRON (0-5uM) (Fig 1B, D). Furthermore, gene expression of AdipoR2 was also dose dependently increased in AdipoAI until 2.5mM in comparison to AdipoRON, whereas 5mM dose observed an increase in AdipoRON treated group (Fig 1C) correlating our assumption that AdipoAI is a potential adiponectin agonist.
As activity of adiponectin is through AMP-activated protein kinase (AMPK),peroxisome-activated receptor (PPAR)-a (Gesta, Tseng, & Kahn, 2007; Yamauchi, Kamon, Waki, et al., 2003) & APPL1, a downstream subunit, involved in adiponectin signaling (Mao et al., 2006), we designed the experimental plan as per our earlier findings, where AdipoAI graded doses increased the gene expression profiles of AdipoR1/R2 and Ppargc1-a. We treated C2C12 myotubes with graded doses of AdipoAI and AdipoRON for 90 mins followed by immunoblotting assay that confirmed the ability of AdipoAI to activate AMPK and was subsequently compared to that of AdipoRON (Fig 1E). As shown in Fig 1E administration of AdipoAI induced dose dependent phosphorylation of AMPK and APPL1 in comparison to AdipoRON, however PPARg produced no statistically significant results. Over all these data affirms our prediction that lower doses of AdipoAI could act as a potent activator of AMPK and APPL1.