Discussion
Diabetes mellitus is a chronic metabolic disease that is increasing in
prevalence globally (Cheng & Fantus, 2005), affecting more than 6% of
the US population with reports of ~ 25% in some
geriatric group, and manifesting impaired glucose tolerance (IGT)
(Inzucchi, 2002) and associated complications. Hyperglycemia i.e.
elevated glucose concentrations/glucotoxicity from Diabetes has
unfavorable foreseeable effects on multiple organs, where it results in
dysfunction in insulin secretion from pancreas and faulty insulin gene
expression through interfering various pathways that ultimately leads to
low glucose sensitization and beta-cell stress(Hasnain et al., 2014),
eventually resulting in irreversible beta-cell failure. Low-grade
inflammation, another metabolic forerunner of diabetes, is being
constantly associated with insulin resistance and obesity (Shoelson,
Herrero, & Naaz, 2007; Shoelson, Lee, & Goldfine, 2006). Therefore,
designing molecules that are efficient in targeting hyperglycemic and
inflammatory pathways could be the most important components in the
overall strategy in preventing and controlling diabetes and its
sequelae.
Latest scientific evidence indicates that adiponectin could result in
nullification of cases in obesity and metabolic diseases such as T2D
(Balasubramanian et al., 2022; Y. Kim et al., 2018). In view of all
these recent developments, adiponectin receptors (AdipoR1 and AdipoR2)
and its scaffold proteins APPL1 and APPL2 have emerged as important
target for controlling excess inflammation, a condition frequently
associated with T2D. While AdipoRON, the first-generation agonist of
adiponectin receptors, has been shown to prevent diet-induced
inflammation and insulin resistance in mice (Iwabu et al., 2010;
Okada-Iwabu et al., 2013), To maximize the potential of adiponectin
receptors in targeting the one of the cascading effects required for
pathogenesis of T2D fundamental etiopathology of T2D, we have designed,
synthesized, and characterized a synthetic and orally active small
molecule agonist of adiponectin AdipoAI and compared our results with
AdipoRON invitro and invivo . In the present study, we
demonstrated that AdipoAI could behave as an antidiabetic adipokine and
is analogous to AdipoRON (Akimoto, Maruyama, Kawabata, Tajima, &
Takenaga, 2018), supporting our notion that AdipoAI could be used as an
anti-diabetic therapeutic agent and the active properties of which were
noted to be similar and/or marginally superior to AdipoRON. Confirming
our prediction, that showed the exposure of AdipoAI initiated signaling
pathways of adiponectin and AMPK activation in C2C12 myotube cells, with
improved serum, gene expression and histopathological profiles in DIO
(diet induced obesity) mouse study models respectively in comparison to
AdipoRON.
We previously reported the anti-inflammatory properties of AdipoAI that
was found to be dependent on adiponectin receptor subunits viz. AdipoR1
and APPL1 suggesting that AdipoAI could mimic effects of adiponectin
(Qiu et al., 2021). It was also claimed that decrease in adiponectin
levels is accompanied by decrease in AMPK and PPARa activation,
resulting in increased susceptibility for diabetes through decrease in
fatty acid synthesis and increase in fatty acid oxidation (Handa et al.,
2014). AMPK and PPAR activation play an important role in cell survival
and energy production (Jager, Handschin, St-Pierre, & Spiegelman, 2007;
Yamauchi et al., 2002). Further, in skeletal muscle cells, adiponectin
pathway activation is known to be associated with AMPK, p38MAPK and
PPARa activity (Yamauchi, Kamon, Waki, et al., 2003). Additionally,
APPL1 interacts with adiponectin receptors in mammalian cells suggesting
that APPL1, AMPK and adiponectin are interconnected (Mao et al., 2006).
Our results collectively suggest that lower doses of AdipoAI
significantly increased the mRNA expression levels of peroxisome
proliferator-activated receptor Ppargc1a, AdipoR1/R1, and increase
protein expression of AMPK and APPL1 in C2C12 myotubes when compared
with AdipoRON (Yamauchi et al., 2007).
Latest scientific evidence indicates that adiponectin could result in
nullification of cases in obesity and metabolic diseases such as T2D
(Kawano & Arora, 2009; Nigro et al., 2014). Advantageously,
contribution of adiponectin in decreasing triglyceride, suppressed
inflammation, decrease glucose intolerance and insulin resistance
through activation of AMPK are acknowledged (Tomas et al., 2002) are
beneficial in T2D cases (Kasper, 2015). We observed the similar pattern
in our DIO mice models where biweekly administration of AdipoAI
(25mg/kg) decreased inguinal and epididymal fat content. We noted the
mRNA levels of isolated WAT that were found to be higher in AdipoR1 and
compliment similar studies with increased expression of PGC-1a & Ppara
genes (Rasmussen et al., 2006). There could be several possible
mechanisms for the reduced fat accumulation in response to AdipoAI
exposure. One of the possibilities might be AdipoAI induced increased
fat oxidation (Weyer et al., 2000) with another possibility of reduction
in triglyceride uptake and its subsequent storage in adipose tissue
(Browning et al., 2011). Further, to the discussion PGC-1a is a key
molecule in mitochondrial biogenesis and is associated with insulin
resistance and obesity (Shen et al., 2022). Specific overexpression of
PGC-1 a expression in adipocyte improved metabolic disfunction in HFD
mice and improved insulin sensitivity (Shen et al., 2022).
Further AdipoAI decreased blood glucose levels during oral glucose and
insulin tolerance test, which was similar to AdipoRON groups, suggesting
that lower AdipoAI (25mg/kg) administration was potent enough in
exerting similar effects to that of mean doses of AdipoRON (50 mg/kg).
The interpretation of the obtained results could be extrapolated from
similar trail with another adiponectin agonist where the assessment of
whole-body insulin sensitivity of DIO mice models reveled the
involvement of hepatic fibroblast growth factor 21 (FGF21), that is
linked to short-term adiponectin agonist-induced hepatic PPARα
activation (Wang et al., 2022).
The pathological features/manifestations of T2D and thereby we proceeded
with the screening of respective parameters for specific
feature/manifestations in DIO mouse study models. We planned liver
evaluation as adiponectin and its mechanism of action, which acts mainly
through AdipoR2 (Yamauchi, Kamon, Ito, et al., 2003), are
correspondingly found to be concentrated in the aforementioned organ
(Ruan & Dong, 2016). As noted in our study favorable effects were
exhibited by AdipoAI on livers of DIO mouse models which increase in
gene expression of adiponectin subunit AdipoR2 in AdipoAI and AdipoRON,
interestingly AdipoAI increased pPargc1a, pPara mRNA expression levels
with reduced pro-inflammatory markers (TNF-a, IL1-b, IL-6) in liver
tissues these overall results suggest hepato-protective features AdipoAI
in lower doses compared to AdipoRON. These data were further
corroborated by immunoblotting and immunohistochemistry staining which
revealed increased expression of phosphorylation of AMPK in both the
treated groups, suggesting that lower dose of AdipoAI could
phosphorylate AMPK which in turn inhibits inflammation, ER and oxidative
stress, (Ruderman, Carling, Prentki, & Cacicedo, 2013).
Apart from its sporadic or zonal steatosis pathology, that have strong
association with diabetes, insulin resistance, and obesity, all
contributing towards further lipid accumulation in hepatic parenchymal
cells leading to hepatic steatosis, one of the most common liver
diseases reported globally. As speculated, the H&E staining of DIO
mouse liver tissue exposed to AdipoAI group showed relatively normal
hepatic architecture organized mostly in loose rows and separated by
sinusoids. However, CMC and AdipoRON group showed disarranged
hepatocytes with appearance of diffused fat vacuoles with frank
Mallory-Denk bodies (Liu et al., 2020). indicating towards development
of microvesicular steatosis (David, Boyer, Wright, & Manns, 2006). The
histologic features resembling steatohepatitis found in control groups
could be most probably be due to insulin resistance associated with
obesity (Gwaltney-Brant, 2021). Our data suggest that AdipoAI likely
have an influence on the increase in adiponectin secretion, which
subsequently lead to one mechanism of AMPK activation through LKB1 as
reported in a similar trial (Santamarina et al., 2015).
In conclusion, our trial has affirmed the anti-diabetic features viz.
hepatoprotective, normoglycemic, anti-inflammatory and weight reduction
effects of AdipoAI in DIO mice models at significantly lower doses when
compared to AdipoRON. The results will contribute towards the
interpretation and prediction of possible clinically relevant effects in
future studies and devising interventional strategies of T2D and its
characteristics manifestations.