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.