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.