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\begin{document}

\title{Antidiabetic features of AdipoAI, a novel AdipoR agonist.}



\author[1]{Qisheng Tu}%
\author[1]{Akash Ahuja}%
\author[1]{Elissa Zboinski}%
\author[1]{Siddhartha das}%
\author[1]{Zoe Xiaofang Zhu}%
\author[1]{Qian Ma}%
\author[1]{Ying Xie}%
\author[1]{Jake Chen}%
\affil[1]{Tufts University School of Dental Medicine}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Adiponectin is an antidiabetic endogenous adipokine that have protective
role in unfavorable metabolic sequalae arising from obesity. Recent
evidence suggests a sinister link between hypoadiponectinemia and
development of insulin resistance/ type 2 diabetes (T2D). The insulin
sensitizing property of adiponectin is through specific adipoq receptors
R1 \& R2, activation of AMP-activated protein kinase (AMPK) and
peroxisome proliferator-activated receptor (PPAR)\selectlanguage{greek}α \selectlanguage{english}pathways. AdipoAI is
a novel synthetic analogue of endogenous adiponectin with possibly
similar pharmacological effects. Thus, there is a need of orally active
small molecules that activate Adipoq subunits, and its downstream
signaling could ameliorate obesity related type 2 diabetes. This study
was aimed to access the effects of AdipoAI on obesity and T2D. Through
\emph{in-vitro} and \emph{in-vivo} analyses, we investigated the
anti-diabetic potentials of AdipoAI and compared it with AdipoRON,
another orally active adiponectin receptors agonist. Our results showed
that \emph{In-vitro} treatment of AdipoAI (0-5\selectlanguage{greek}µ\selectlanguage{english}M) increased adiponectin
receptor subunits AdipoR1/R2 with increase in AMPK and APPL1 protein
expression in C2C12 myotubes. Similarly, \emph{in-vivo}, oral
administration of AdipoAI (25 mg/kg) observed similar effects as that of
AdipoRON (50 mg/kg) with improved control of blood glucose and insulin
sensitivity in diet-induced obesity (DIO) mice models. Further, AdipoAI
significantly reduced epididymal fat content with decrease in
inflammatory markers and increase in PPAR-a and AMPK levels and
exhibited hepatoprotective effects in liver. Further, AdipoAI and
AdipoRON also observed similar results in adipose tissue. Thus, our
results suggest that low doses of orally active small molecule agonist
of adiponectin AdipoAI can be a promising therapeutic target for obesity
and T2D.%
\end{abstract}%



\sloppy


Antidiabetic features of AdipoAI, a novel AdipoR agonist.

Akash Ahuja\textsuperscript{1}, Elissa Zboinski\textsuperscript{1},
Siddhartha das\textsuperscript{1}, Xiaofang Zhu\textsuperscript{1}, Qian
Ma\textsuperscript{1,3}, Ying Xie\textsuperscript{1,4}, Qisheng
Tu\textsuperscript{1},Jake Chen\textsuperscript{1,2}

\textsuperscript{1} Division of Oral Biology, Department of
Periodontology, Tufts University School of Dental Medicine, Boston,
Massachusetts, USA

\textsuperscript{2} Dept. of Developmental, Molecular and Chemical
Biology, Tufts School of Medicine; Graduate School of Biomedical
Sciences, Tufts University, Boston, Massachusetts, USA

\textsuperscript{3} Department of General Dentistry, Affiliated
Stomatological Hospital of Nanjing Medical University, Jiangsu, China

\textsuperscript{4} Department of Periodontology, Peking University
School and Hospital of Stomatology, Beijing, China

\textbf{*Correspondence: Jake Chen: jk.chen@tufts.edu}

\textbf{Qisheng Tu: qisheng.tu@tufts.edu}

\textbf{Running title} : AdipoAI improves type 2 diabetes.

Abstract

Adiponectin is an antidiabetic endogenous adipokine that have protective
role in unfavorable metabolic sequalae arising from obesity. Recent
evidence suggests a sinister link between hypoadiponectinemia and
development of insulin resistance/ type 2 diabetes (T2D). The insulin
sensitizing property of adiponectin is through specific adipoq receptors
R1 \& R2, activation of AMP-activated protein kinase (AMPK) and
peroxisome proliferator-activated receptor (PPAR)\selectlanguage{greek}α \selectlanguage{english}pathways. AdipoAI is
a novel synthetic analogue of endogenous adiponectin with possibly
similar pharmacological effects. Thus, there is a need of orally active
small molecules that activate Adipoq subunits, and its downstream
signaling could ameliorate obesity related type 2 diabetes. This study
was aimed to access the effects of AdipoAI on obesity and T2D.
Through\emph{in-vitro} and \emph{in-vivo} analyses, we investigated the
anti-diabetic potentials of AdipoAI and compared it with AdipoRON,
another orally active adiponectin receptors agonist. Our results showed
that \emph{In-vitro} treatment of AdipoAI (0-5\selectlanguage{greek}µ\selectlanguage{english}M) increased adiponectin
receptor subunits AdipoR1/R2 with increase in AMPK and APPL1 protein
expression in C2C12 myotubes. Similarly, \emph{in-vivo} , oral
administration of AdipoAI (25 mg/kg) observed similar effects as that of
AdipoRON (50 mg/kg) with improved control of blood glucose and insulin
sensitivity in diet-induced obesity (DIO) mice models. Further, AdipoAI
significantly reduced epididymal fat content with decrease in
inflammatory markers and increase in PPAR-\selectlanguage{greek}α \selectlanguage{english}and AMPK levels and
exhibited hepatoprotective effects in liver. Further, AdipoAI and
AdipoRON also observed similar results in adipose tissue. Thus, our
results suggest that low doses of orally active small molecule agonist
of adiponectin AdipoAI can be a promising therapeutic target for obesity
and T2D.

\textbf{Keywords:} Adiponectin, AdipoAI, Obesity, Type 2 diabetes,
Inflammation, Diet induced obesity

\textbf{Significance Statement}

Obesity is a concerning health issue, and has doubled in more than 70
countries, resulting in escalation of obesity-related disease like type
2 diabetes (T2D). The adiponectin receptors (AdipoR1 and AdipoR2) have
emerged as important targets for controlling inflammation, obesity and
T2D. To maximize the potential of adiponectin receptors in targeting the
fundamental etiopathology of obesity and T2D, we have designed,
synthesized, Adiponectin receptor agonist named AdipoAI (standing for
anti-inflammation) which in our pre-clinical trials on high fat diet
(HFD) observed reduced glucose resistance, insulin tolerance, promoted
adiponectin signaling in liver and adipose tissues of obese mice, and
resulted decrease liver inflammation. Thus, our trial has affirmed,
AdipoAI as a potent agonist of adiponectin with anti-diabetic,
hepatoprotective, anti-inflammatory features.

\textbf{Introduction}

The exponential growth in the incidence of Type 2 diabetes (T2D) is
largely due to sedentary lifestyles and changes in dietary preferences,
which adversely affects metabolism, immune system etc.(Daryabor,
Atashzar, Kabelitz, Meri, \& Kalantar, 2020; Fahed, El-Hage-Sleiman,
Farhat, \& Nemer, 2012; Leroith \& Accili, 2008). The duo viz. obesity
and T2D are known for their unfavorable consequences on adipose tissue,
muscle, liver, and pancreatic islets which further results in altered
levels of chemokines and cytokines among others, leading to insulin
resistance syndrome (Nikolajczyk, Jagannathan-Bogdan, Shin, \& Gyurko,
2011; Tsalamandris et al., 2019). Hence, inflammatory conditions such as
metabolic inflammation is linked to obesity and contributes downstream
to the pathogenesis of T2D and its clusters of characteristics
manifestations (Walker \& Colledge, 2013).

Further, adiponectin (Adipoq), an antidiabetic adipokine (Karamian,
Moossavi, \& Hemmati, 2021) has been well documented for its possible
therapeutic potentials (Choi, Doss, \& Kim, 2020; Karamian et al., 2021;
J. Y. Kim, Barua, Jeong, \& Lee, 2020). Recent understanding of
adiponectin receptors (AdipoR1 and AdipoR2) has paved the way forward in
understanding the development of insulin resistance and progressive
obesity linked diseases such as T2D (Whitehead, Richards, Hickman,
Macdonald, \& Prins, 2006). Moreover, reduction in adiponectin hormone
secretion from adipose tissue is linked to insulin resistance in T2D as
reported in animal trials where adiponectin improved insulin resistance
(Okada-Iwabu et al., 2013). Interestingly, a recent orally active small
molecule AdipoR agonist (AdipoRON) has exhibited favorable anti-diabetic
features such as ability to bind AdipoR1/R2, activation of AMPK and
PPAR-\selectlanguage{greek}α \selectlanguage{english}pathways, decreasing insulin resistance and glucose tolerance in
mice models in high fat diet (Okada-Iwabu et al., 2013).

Besides these, T2D also results in uncontrolled hepatic glucose
production, which leads to hyperglycemia as noted in diabetic
pathologies. Studies also revealed that activation of peroxisome
proliferator-activated receptors (PPARs) involves cascading effects on
various signaling pathways which results in suppressing hepatic glucose
production (Awazawa et al., 2011).Moreover, reports on animal and human
trials suggests that adiponectin can decrease hepatic gluconeogenesis
and stimulation of fatty acid oxidation in liver (Choi et al., 2020;
Howlader, Sultana, Akter, \& Hossain, 2021). Furthermore, adiponectin
signaling in liver is linked to AdipoR2 which activates peroxisome
proliferator-activated alpha (PPARa) (Achari \& Jain, 2017) suggesting
its therapeutic role in T2D. Additionally, the insulin sensitizing
properties of adiponectin is through increase in fatty-acid oxidation
via activation of AMPK (AMP-activated protein kinase) and regulation of
peroxisome proliferator-activated receptor (PPAR)-a (Tomas et al., 2002;
Yamauchi et al., 2002).

We previously reported the anti-inflammatory properties of AdipoAI (Qiu
et al., 2021; Wu et al., 2022). AdipoAI attenuated NF-\selectlanguage{greek}κ\selectlanguage{english}B (nuclear factor
kappa B), MAPK (mytogen activated protein kinase) and c-MAF (Qiu et al.,
2021) signaling pathways through activation of Adiponectin signaling
subunits AdipoR1 and APPL1 in Lipopolysaccharide (LPS)- induced
macrophages and decreased the production of cytokines. Furthermore,
AdipoAI was found to inhibit osteoclastogenesis at lower dose and
inhibited the expression of proinflammatory mediators in periodontal
tissues (Wu et al., 2022) indicating its potential candidature towards
activation of adiponectin receptor against development of T2D pathology.

In this investigation, we hypothesized that our chemically designed and
characterized AdipoAI small molecule have potent anti-diabetic
therapeutic potential similar to Adiponectin and thereby we evaluated
its \emph{in-vitro} and \emph{in-vivo} effects in comparison to a known
anti-diabetic small molecule agonist AdipoRON (Okada-Iwabu et al., 2013)
for further scientific affirmation.

\textbf{Materials and Methods}

\textbf{Animal experiment}

All the animals reported were approved by the animal care committee of
Tufts University, Boston, MA. Diet induced obese (DIO, C57BL/6) male
mice at 17 weeks of age were purchased from Jackson's laboratory and
housed control pathogen free conditions (22\selectlanguage{ngerman}°C) 12h light-dark cycle. One
week after arrival mice were divided into three groups (n=6 per group)
as follows CMC-Na (Carvoxymethylcellulose-sodium salt, thermoscientific,
A18105.36) 0.5\% (HFD group), AdipoAI 25mg/kg (HFD along with AdipoAI),
AdipoRON 50 mg/kg (HFD along with AdipoRON) were fed with high fed diet
consisting of 58\% fat, 25.6\% carbohydrates and 16.4\% protein with
access to water for 4 weeks. Small molecule AdipoAI (25 mg/kg) and
AdipoRON (50 mg/kg) were dissolved and administered in 0.5\% CMC-Na
(carboxymethyl cellulose) orally by 18G curved gavage as reported by
others (Shi et al., 2013).Oral treatment was introduced at 18 weeks and
continued for 4 weeks (2X/week) and mice weights were recorded carefully
every week as illustrated in concept map Fig 3A. At the end of
3\textsuperscript{rd} and 4\textsuperscript{th} week oral glucose and
insulin tolerance test were performed.

\textbf{Cell culture}

C2C12 mouse myoblast cell lines were obtained from ATCC, CRL-1772) and
were allowed to grow in 90\% Dulbecco's modified high glucose Eagle's
medium (DMEM) containing 10\% v/v fetal bovine serum (FBS) in 10cm cell
culture dish at 37°C in 5\% CO\textsubscript{2} incubator until 80\%
confluent. The media was replaced to myogenic differentiation medium
containing 98\% DMEM and 2\% v/v horse serum and was changed every other
day for 7 days. The differentiated myotubes were then plated in 96, 6
and 12 wells plates for RT-PCR and immunoblot analysis.

\textbf{CCK8 assay}

CCK-8 assay was performed as described previously (Qiu et al., 2021)
briefly C2C12 cells (0.05X10\textsuperscript{4} cells/well) were plated
in 96 well plate overnight (day 0), following AdipoAI and AdipoRON
treatment for 24hours. After 24hours treatment CCK-8 (cell counting kit)
(Dojindo, Rockville, MD, USA) assay was performed according to
manufacture instruction the media was discarded carefully and 10ul of
CCK-8 was added to the plates and incubated for 2h in incubator. The
absorbance was recorded at 450nmn and calculated with respect to
nontreated control groups.

\textbf{Realtime PCR}

Real time PCR was carried out according to methods previously described
(Ahuja, Kim, Sung, \& Cho, 2020; Wu et al., 2022). DNA free RNA was
extracted from the C2C12 myotubes using zymo quick RNA mini kit (R1055)
from with DNase treatment and RNA from tissues were isolated using
Trizol (Invitrogen) according to manufacture protocol. Briefly 1ug of
total RNA was reverse transcribed using first strand cDNA synthesis kit
(thermos fisher) real time PCR was performed in triplicate using Bio-Rad
icycler following manufacture protocols. The relative gene expression of
the target genes was calculated by using 2\selectlanguage{english}\textsuperscript{-[?][?]ct} method
(Qiu et al., 2021), the values were normalized to the housekeeping gene
GPADH. Primer sequence of the respected primers are mentioned in table
no1.

\textbf{Western blot analysis}

C2C12 myotubes (1X10\textsuperscript{6}) were incubated at different
concentrations of AdipoAI and AdipoRON for 90 minutes. The cells were
then washed with ice-cold PBS. The pellet was lysed using ice-cold 1X
NUPAGE LDS lysis buffer (Invitrogen) approximately 200ul of the sample
buffer were added to each sample the cells were vortex briefly and
centrifuge at 8,000rpm for 5 mins, the supernatant was collected and
heated at 70degC for 10min. The proteins were than loaded in 4.12\%
bis-tris gel using MES running buffer (Invitrogen) at 100V for 2 hours
(Qiu et al., 2021; Wu et al., 2022), proteins were then transferred to
0.2 um polyvinylidene difluoride membrane using transfer buffer
(Tris-base, glycine,10\% SDS, methanol). Membrane was blocked using 3\%
bovine serum albumin (BSA) in TBST for 1 hour followed by incubation
with specific antibody (p-AMPK, APPL1, PGAC-y) overnight. After primary
antibody the membrane were than washed 3 times with TBST for 10minutes
and incubated with HRB labeled secondary antibody containing 3\% BSA for
1h at room temperature, after incubation the membrane were washed 3
times with TBST and were visualized using ECL reagent (Ahuja et al.,
2020).

\textbf{Oral glucose and Insulin tolerance test}

DIO Mice were starved overnight (16h), followed by oral administration
of glucose (1mg/kg) were given to each group (n=6). Blood glucose levels
were evaluated prior to dosing, and carefully monitored every 15 minutes
time interval for a period of 120 mins, following oral administration.
Insulin tolerance test (0.75\selectlanguage{english} IU insulin per kg body weight) was
performed in treated DIO mice models after 6 hours of fasting. Blood
glucose was monitored at specific time interval (0 -120 mins) by using a
sensitive glucometer.

\textbf{Histology and Immunostaining of liver tissues}

Histology staining was performed as described previously with few
modifications (Qiu et al., 2021). Briefly, Liver tissues from mouse were
collected from all the treated groups after postmortem and fixed in ice
cold 4\% paraformaldehyde (PFA) overnight at 4degC. The following day
samples the sections were paraffin embedded and were cut in 4um
thickness following deparaffinization, the sections were stained with
hematoxylin for (5-15 mins) washed and counter stained with eosin. The
slides were quickly dehydrated and sealed using mineral oil. The images
were documented at 20X magnification on Olympus BX53.

For immunostaining 4-um liver sections were cut and mounted on slides
from all the groups the sections were deparaffinized with xylene and
dehydrated in ethanol. The procedure was performed according to
manufacture instructions (ab209101, Rabbit specific IHC polymer
detection kit). The sections were immersed in 0.3\% H2O2/methanol was
used for 30 mins to reduce nonspecific staining to remove exogenous
peroxidase avtivity, next the sections were incubated in primary
antibody Phospho-AMPKa (Thr 172) (Rabbit mAb\# 2535, cell signaling)
overnight at 4degC in 3\% BSA in PBS, followed by PBS washing, the slides
were stained with anti-rabbit IgG. DAB was used for color rendering, and
counter stained with hepatotoxicant, dehydrated, and sealed. Images were
documented using Olympus BX53 at 20X magnification.

\textbf{Statistical analysis}

All the data are presented at mean +- SD, the analysis was performed by
using GraphPad version 9.4.1 software. Statistical analysis was
performed using one-way ANOVA with multiple comparison between groups,
the values of p\textless{}0.05 were considered as statistically
significant.

\textbf{Results}

\emph{In-vitro} \textbf{evaluation of Adipo-AI on adiponectin
signaling.}

To screen the optimal concentration and to calculate the
IC\textsubscript{50} 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 IC\textsubscript{50} values of AdipoAI and AdipoRON
obtained from CCK8 assay was 4.7mM and 6.22mM respectively (Fig.1A).
Therefore, the approximate IC\textsubscript{50} 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.

\textbf{AdipoAI administration improved insulin resistance and glucose
tolerance in DIO mice models}

The DIO mouse models were subjected to glucose and insulin tolerance
test in accordance with our perceived plan of interventional strategy
for developing T2D pathology (Fig 2A, B). OGTT showed time dependent
decrease in serum glucose profile for AdipoAI and AdipoRON groups when
compared to those of CMC (Carboxymethylcellulose) groups (Fig 2A).
Further, insulin tolerance test resulted in gradual reduction in the
levels of serum glucose in AdipoAI and AdipoRON groups when compared to
that of CMC group suggesting that AdipoAI have comparable beneficial
features as that of AdipoRON (Fig 2B).

\textbf{Fat distribution and body weights of mouse models exposed to
AdipoAI}

Further, comparative monitoring, from a period of 4 weeks, in DIO mice
models, revealed that the AdipoAI group had decrease weight at 20 and 21
weeks compared to that of AdipoRON and CMC groups (Fig 3B). However,
these observations were statistically insignificant. Interestingly, the
weights of iWAT (inguinal white adipose tissue) (Fig 3C) and eWAT
(epididymal white adipose tissue) (Fig 3D) were decreased for both
AdipoAI and AdipoRON group but AdipoAI mouse demonstrated the most
weight reduction for eWAT among all the groups significantly (Fig 3D).
The weight of liver also correlated to our assumption (Fig 3E).

\textbf{AdipoAI and AdipoRON activates different adiponectin subunits in
different tissues.}

Reports indicates that obesity may be related with liver dysfunction,
(Marchesini, Moscatiello, Di Domizio, \& Forlani, 2008) further,
literature also suggests that metabolic or physiological status of liver
are altered in obesity and in T2D pathology (De Silva et al., 2019).
Therefore, considering all these factors the hepatic screening of the
DIO mice models was performed which interestingly revealed that the
AdipoAI group increased gene expression profiles for Ppargc1a, AdipoR2
and Ppara (Fig 4 A, B \& C) with simultaneously decreased the expression
of IL-6, INF-b, TNF-a (Fig F, G \& H) a potent inflammatory biomarker.
We further tested weather AdipoAI could result in phosphorylation of
AMPK by performing immunoblotting and IHC staining of liver tissue
treated with AdipoAI, AdipoRON and CMC (Fig 4 D \& E). Immunostaining
and protein expression against AMPK known to improves insulin
sensitivity and glucose homeostasis (Coughlan, Valentine, Ruderman, \&
Saha, 2014) we observed increased and prominent Phosphorylation of AMPK
in both AdipoAI and AdipoRON liver tissue (Fig 4 D\&E). Further,
H\&E-stained images of AdipoAI group showed evenly populated hepatocytes
with mostly normal cytoplasmic contents and architecture (Fig 4I). The
CMC and AdipoRON group exhibited sporadic infiltration with disarranged
hepatocytes (arrowhead) and diffuse fatty changes. The overall results
suggest that AdipoAI can results in phosphorylation of AMPK and
activation of adiponectin subunits with decrease in cytokine levels in
liver.

\textbf{AdipoAI increases AdipoR1-PGC1-a expression in adipose tissues.}

As PGC1a (pPARGC1a) regulation is involved in lipid metabolism in muscle
adipocytes (Bogacka, Xie, Bray, \& Smith, 2005; Liang \& Ward, 2006; Lin
et al., 2002; Patti et al., 2003). We assume that AdipoAI and AdipoRON
oral administration regulates adiponectin and PGC1a expression levels in
white adipose tissue (WAT). In WAT AdipoAI and AdipoRON both increase
AdipoR1, pPARGC1a, PPARA gene expression levels in comparison to DIO
mice (Fig 5A, C \& D). Higher gene expression of all the three tested
gene was observed in AdipoAI treated group, suggesting that AdipoAI
could activate adiponectin signaling pathways through AdipoR1 and lower
doses of AdipoAI may influence the lipid metabolism in WAT which could
be a potential target.

\textbf{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 \textasciitilde{} 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 \emph{invitro} and \emph{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 PPAR\selectlanguage{greek}α
\selectlanguage{english}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.

\textbf{Conflict of Interest}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

\textbf{Data Availability Statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

\textbf{Author Contributions}

AA and EZ contributed equally to the manuscript by planning and
performing the experiments. AA did the data acquisition, analysis, and
interpretation. AA, XZ, QT, JK contributed to design and critically
revised the manuscript, AA, SD, QT, XZ and JK drafted and critically
revised the manuscript All authors critically discussed, corrected, and
reviewed the manuscript.

\textbf{Funding}

This work was supported by National Institutes of Health (NIH) grants
R01DE25681, R01DE26507, R01DE30074 and R01DK131444 to Jake Chen; NIH
R01DE030129 and Tufts Collaborates Award D740207 to Qisheng Tu

\textbf{Reference}

Achari, A. E., \& Jain, S. K. (2017). Adiponectin, a Therapeutic Target
for Obesity, Diabetes, and Endothelial Dysfunction. \emph{Int J Mol Sci,
18} (6). doi:10.3390/ijms18061321

Ahuja, A., Kim, E., Sung, G. H., \& Cho, J. Y. (2020). STAT3
Differentially Regulates TLR4-Mediated Inflammatory Responses in Early
or Late Phases. \emph{Int J Mol Sci, 21} (20). doi:10.3390/ijms21207675

Akimoto, M., Maruyama, R., Kawabata, Y., Tajima, Y., \& Takenaga, K.
(2018). Antidiabetic adiponectin receptor agonist AdipoRon suppresses
tumour growth of pancreatic cancer by inducing RIPK1/ERK-dependent
necroptosis. \emph{Cell Death Dis, 9} (8), 804.
doi:10.1038/s41419-018-0851-z

Awazawa, M., Ueki, K., Inabe, K., Yamauchi, T., Kubota, N., Kaneko, K.,
. . . Kadowaki, T. (2011). Adiponectin enhances insulin sensitivity by
increasing hepatic IRS-2 expression via a macrophage-derived
IL-6-dependent pathway. \emph{Cell Metab, 13} (4), 401-412.
doi:S1550-4131(11)00083-0 {[}pii{]}

10.1016/j.cmet.2011.02.010

Balasubramanian, P., Schaar, A. E., Gustafson, G. E., Smith, A. B.,
Howell, P. R., Greenman, A., . . . Anderson, R. M. (2022). Adiponectin
receptor agonist AdipoRon improves skeletal muscle function in aged
mice. \emph{Elife, 11} . doi:10.7554/eLife.71282

Bogacka, I., Xie, H., Bray, G. A., \& Smith, S. R. (2005). Pioglitazone
induces mitochondrial biogenesis in human subcutaneous adipose tissue in
vivo. \emph{Diabetes, 54} (5), 1392-1399. doi:10.2337/diabetes.54.5.1392

Browning, J. D., Baker, J. A., Rogers, T., Davis, J., Satapati, S., \&
Burgess, S. C. (2011). Short-term weight loss and hepatic triglyceride
reduction: evidence of a metabolic advantage with dietary carbohydrate
restriction. \emph{Am J Clin Nutr, 93} (5), 1048-1052.
doi:10.3945/ajcn.110.007674

Cheng, A. Y., \& Fantus, I. G. (2005). Oral antihyperglycemic therapy
for type 2 diabetes mellitus. \emph{CMAJ, 172} (2), 213-226.
doi:10.1503/cmaj.1031414

Choi, H. M., Doss, H. M., \& Kim, K. S. (2020). Multifaceted
Physiological Roles of Adiponectin in Inflammation and
Diseases.\emph{Int J Mol Sci, 21} (4). doi:10.3390/ijms21041219

Coughlan, K. A., Valentine, R. J., Ruderman, N. B., \& Saha, A. K.
(2014). AMPK activation: a therapeutic target for type 2
diabetes?\emph{Diabetes Metab Syndr Obes, 7} , 241-253.
doi:10.2147/DMSO.S43731

Daryabor, G., Atashzar, M. R., Kabelitz, D., Meri, S., \& Kalantar, K.
(2020). The Effects of Type 2 Diabetes Mellitus on Organ Metabolism and
the Immune System. \emph{Front Immunol, 11} , 1582.
doi:10.3389/fimmu.2020.01582

David, Z., Boyer, T. D., Wright, T. L., \& Manns, M. P.
(2006).\emph{Zakim and Boyer's hepatology : a textbook of liver disease}
(5th ed.). Philadelphia, PA: Saunders Elsevier.

De Silva, N. M. G., Borges, M. C., Hingorani, A. D., Engmann, J., Shah,
T., Zhang, X., . . . Lawlor, D. A. (2019). Liver Function and Risk of
Type 2 Diabetes: Bidirectional Mendelian Randomization
Study.\emph{Diabetes, 68} (8), 1681-1691. doi:10.2337/db18-1048

Fahed, A. C., El-Hage-Sleiman, A. K., Farhat, T. I., \& Nemer, G. M.
(2012). Diet, genetics, and disease: a focus on the middle East and
north Africa region. \emph{J Nutr Metab, 2012} , 109037.
doi:10.1155/2012/109037

Gesta, S., Tseng, Y. H., \& Kahn, C. R. (2007). Developmental origin of
fat: tracking obesity to its source. \emph{Cell, 131} (2), 242-256.
doi:10.1016/j.cell.2007.10.004

Gwaltney-Brant, S. M. (2021). Chapter 8 - Nutraceuticals in hepatic
diseases. In R. C. Gupta, R. Lall, \& A. Srivastava
(Eds.),\emph{Nutraceuticals (Second Edition)} (pp. 117-129): Academic
Press.

Handa, P., Maliken, B. D., Nelson, J. E., Morgan-Stevenson, V., Messner,
D. J., Dhillon, B. K., . . . Kowdley, K. V. (2014). Reduced adiponectin
signaling due to weight gain results in nonalcoholic steatohepatitis
through impaired mitochondrial biogenesis. \emph{Hepatology, 60} (1),
133-145. doi:10.1002/hep.26946

Hasnain, S. Z., Borg, D. J., Harcourt, B. E., Tong, H., Sheng, Y. H.,
Ng, C. P., . . . McGuckin, M. A. (2014). Glycemic control in diabetes is
restored by therapeutic manipulation of cytokines that regulate beta
cell stress. \emph{Nat Med, 20} (12), 1417-1426. doi:10.1038/nm.3705

Howlader, M., Sultana, M. I., Akter, F., \& Hossain, M. M. (2021).
Adiponectin gene polymorphisms associated with diabetes mellitus: A
descriptive review. \emph{Heliyon, 7} (8), e07851.
doi:10.1016/j.heliyon.2021.e07851

Inzucchi, S. E. (2002). Oral antihyperglycemic therapy for type 2
diabetes: scientific review. \emph{JAMA, 287} (3), 360-372.
doi:10.1001/jama.287.3.360

Iwabu, M., Yamauchi, T., Okada-Iwabu, M., Sato, K., Nakagawa, T.,
Funata, M., . . . Kadowaki, T. (2010). Adiponectin and AdipoR1 regulate
PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1. \emph{Nature, 464}
(7293), 1313-1319. doi:10.1038/nature08991

Jager, S., Handschin, C., St-Pierre, J., \& Spiegelman, B. M. (2007).
AMP-activated protein kinase (AMPK) action in skeletal muscle via direct
phosphorylation of PGC-1alpha. \emph{Proc Natl Acad Sci U S A, 104}
(29), 12017-12022. doi:10.1073/pnas.0705070104

Karamian, M., Moossavi, M., \& Hemmati, M. (2021). From diabetes to
renal aging: the therapeutic potential of adiponectin. \emph{J Physiol
Biochem, 77} (2), 205-214. doi:10.1007/s13105-021-00790-4

Kasper, D. L. (2015). \emph{Harrison's principles of internal
medicine}(19th edition / editors, Dennis L. Kasper, Anthony S. Fauci,
Stephen L. Hauser, Dan L. Longo, J. Larry Jameson, Joseph Loscalzo.
ed.). New York: McGraw Hill Education Medical.

Kawano, J., \& Arora, R. (2009). The role of adiponectin in obesity,
diabetes, and cardiovascular disease. \emph{J Cardiometab Syndr, 4} (1),
44-49. doi:CMS030 {[}pii{]}

10.1111/j.1559-4572.2008.00030.x

Kim, J. Y., Barua, S., Jeong, Y. J., \& Lee, J. E. (2020). Adiponectin:
The Potential Regulator and Therapeutic Target of Obesity and
Alzheimer's Disease. \emph{Int J Mol Sci, 21} (17).
doi:10.3390/ijms21176419

Kim, Y., Lim, J. H., Kim, M. Y., Kim, E. N., Yoon, H. E., Shin, S. J., .
. . Park, C. W. (2018). The Adiponectin Receptor Agonist AdipoRon
Ameliorates Diabetic Nephropathy in a Model of Type 2 Diabetes. \emph{J
Am Soc Nephrol, 29} (4), 1108-1127. doi:10.1681/ASN.2017060627

Leroith, D., \& Accili, D. (2008). Mechanisms of disease: using
genetically altered mice to study concepts of type 2 diabetes. \emph{Nat
Clin Pract Endocrinol Metab, 4} (3), 164-172. doi:10.1038/ncpendmet0729

Liang, H., \& Ward, W. F. (2006). PGC-1alpha: a key regulator of energy
metabolism. \emph{Adv Physiol Educ, 30} (4), 145-151.
doi:10.1152/advan.00052.2006

Lin, J., Wu, H., Tarr, P. T., Zhang, C. Y., Wu, Z., Boss, O., . . .
Spiegelman, B. M. (2002). Transcriptional co-activator PGC-1 alpha
drives the formation of slow-twitch muscle fibres. \emph{Nature, 418}
(6899), 797-801. doi:10.1038/nature00904

Liu, Y., Trnka, M. J., Guan, S., Kwon, D., Kim, D. H., Chen, J. J., . .
. Correia, M. A. (2020). A Novel Mechanism for NF-kappaB-activation via
IkappaB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced
Inflammation. \emph{Mol Cell Proteomics, 19} (12), 1968-1986.
doi:10.1074/mcp.RA120.002316

Mao, X., Kikani, C. K., Riojas, R. A., Langlais, P., Wang, L., Ramos, F.
J., . . . Dong, L. Q. (2006). APPL1 binds to adiponectin receptors and
mediates adiponectin signalling and function. \emph{Nat Cell Biol, 8}
(5), 516-523. doi:10.1038/ncb1404

Marchesini, G., Moscatiello, S., Di Domizio, S., \& Forlani, G. (2008).
Obesity-Associated Liver Disease. \emph{The Journal of Clinical
Endocrinology \& Metabolism, 93} (11\_supplement\_1), s74-s80.
doi:10.1210/jc.2008-1399

Nigro, E., Scudiero, O., Monaco, M. L., Palmieri, A., Mazzarella, G.,
Costagliola, C., . . . Daniele, A. (2014). New insight into adiponectin
role in obesity and obesity-related diseases. \emph{Biomed Res Int,
2014} , 658913. doi:10.1155/2014/658913

Nikolajczyk, B. S., Jagannathan-Bogdan, M., Shin, H., \& Gyurko, R.
(2011). State of the union between metabolism and the immune system in
type 2 diabetes. \emph{Genes Immun, 12} (4), 239-250.
doi:10.1038/gene.2011.14

Okada-Iwabu, M., Yamauchi, T., Iwabu, M., Honma, T., Hamagami, K.,
Matsuda, K., . . . Kadowaki, T. (2013). A small-molecule AdipoR agonist
for type 2 diabetes and short life in obesity. \emph{Nature, 503}
(7477), 493-499. doi:10.1038/nature12656

Patti, M. E., Butte, A. J., Crunkhorn, S., Cusi, K., Berria, R.,
Kashyap, S., . . . Mandarino, L. J. (2003). Coordinated reduction of
genes of oxidative metabolism in humans with insulin resistance and
diabetes: Potential role of PGC1 and NRF1. \emph{Proc Natl Acad Sci U S
A, 100} (14), 8466-8471. doi:10.1073/pnas.1032913100

Qiu, W., Wu, H., Hu, Z., Wu, X., Tu, M., Fang, F., . . . Chen, J.
(2021). Identification and characterization of a novel adiponectin
receptor agonist adipo anti-inflammation agonist and its
anti-inflammatory effects in vitro and in vivo. \emph{Br J Pharmacol,
178} (2), 280-297. doi:10.1111/bph.15277

Rasmussen, M. S., Lihn, A. S., Pedersen, S. B., Bruun, J. M., Rasmussen,
M., \& Richelsen, B. (2006). Adiponectin receptors in human adipose
tissue: effects of obesity, weight loss, and fat depots. \emph{Obesity
(Silver Spring), 14} (1), 28-35. doi:10.1038/oby.2006.5

Ruan, H., \& Dong, L. Q. (2016). Adiponectin signaling and function in
insulin target tissues. \emph{J Mol Cell Biol, 8} (2), 101-109.
doi:10.1093/jmcb/mjw014

Ruderman, N. B., Carling, D., Prentki, M., \& Cacicedo, J. M. (2013).
AMPK, insulin resistance, and the metabolic syndrome. \emph{J Clin
Invest, 123} (7), 2764-2772. doi:10.1172/JCI67227

Santamarina, A. B., Oliveira, J. L., Silva, F. P., Carnier, J.,
Mennitti, L. V., Santana, A. A., . . . Oyama, L. M. (2015). Green Tea
Extract Rich in Epigallocatechin-3-Gallate Prevents Fatty Liver by AMPK
Activation via LKB1 in Mice Fed a High-Fat Diet. \emph{PLoS One, 10}
(11), e0141227. doi:10.1371/journal.pone.0141227

Shen, S. H., Singh, S. P., Raffaele, M., Waldman, M., Hochhauser, E.,
Ospino, J., . . . Peterson, S. J. (2022). Adipocyte-Specific Expression
of PGC1alpha Promotes Adipocyte Browning and Alleviates Obesity-Induced
Metabolic Dysfunction in an HO-1-Dependent Fashion. \emph{Antioxidants
(Basel), 11} (6). doi:10.3390/antiox11061147

Shi, J., Cao, B., Zha, W. B., Wu, X. L., Liu, L. S., Xiao, W. J., . . .
Liu, C. X. (2013). Pharmacokinetic interactions between
20(S)-ginsenoside Rh2 and the HIV protease inhibitor ritonavir in vitro
and in vivo. \emph{Acta Pharmacol Sin, 34} (10), 1349-1358.
doi:10.1038/aps.2013.69

Shoelson, S. E., Herrero, L., \& Naaz, A. (2007). Obesity, inflammation,
and insulin resistance. \emph{Gastroenterology, 132} (6), 2169-2180.
doi:10.1053/j.gastro.2007.03.059

Shoelson, S. E., Lee, J., \& Goldfine, A. B. (2006). Inflammation and
insulin resistance. \emph{J Clin Invest, 116} (7), 1793-1801.
doi:10.1172/JCI29069

Tomas, E., Tsao, T. S., Saha, A. K., Murrey, H. E., Zhang Cc, C., Itani,
S. I., . . . Ruderman, N. B. (2002). Enhanced muscle fat oxidation and
glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase
inhibition and AMP-activated protein kinase activation. \emph{Proc Natl
Acad Sci U S A, 99} (25), 16309-16313. doi:10.1073/pnas.222657499

Tsalamandris, S., Antonopoulos, A. S., Oikonomou, E., Papamikroulis, G.
A., Vogiatzi, G., Papaioannou, S., . . . Tousoulis, D. (2019). The Role
of Inflammation in Diabetes: Current Concepts and Future
Perspectives.\emph{Eur Cardiol, 14} (1), 50-59.
doi:10.15420/ecr.2018.33.1

Walker, B. R., \& Colledge, N. R. (2013). \emph{Davidson's principles
and practice of medicine e-book} : Elsevier Health Sciences.

Wang, Y., Liu, H., Zhang, R., Xiang, Y., Lu, J., Xia, B., . . . Wu, J.
(2022). AdipoRon exerts opposing effects on insulin sensitivity via
fibroblast growth factor 21-mediated time-dependent mechanisms. \emph{J
Biol Chem, 298} (3), 101641. doi:10.1016/j.jbc.2022.101641

Weyer, C., Pratley, R. E., Salbe, A. D., Bogardus, C., Ravussin, E., \&
Tataranni, P. A. (2000). Energy expenditure, fat oxidation, and body
weight regulation: a study of metabolic adaptation to long-term weight
change. \emph{J Clin Endocrinol Metab, 85} (3), 1087-1094.
doi:10.1210/jcem.85.3.6447

Whitehead, J. P., Richards, A. A., Hickman, I. J., Macdonald, G. A., \&
Prins, J. B. (2006). Adiponectin--a key adipokine in the metabolic
syndrome. \emph{Diabetes Obes Metab, 8} (3), 264-280.
doi:10.1111/j.1463-1326.2005.00510.x

Wu, X., Sun, Y., Cui, R., Qiu, W., Zhang, J., Hu, Z., . . . Chen, J.
(2022). A novel adiponectin receptor agonist (AdipoAI) ameliorates type
2 diabetes-associated periodontitis by enhancing autophagy in
osteoclasts. \emph{J Periodontal Res, 57} (2), 381-391.
doi:10.1111/jre.12969

Yamauchi, T., Kamon, J., Ito, Y., Tsuchida, A., Yokomizo, T., Kita, S.,
. . . Kadowaki, T. (2003). Cloning of adiponectin receptors that mediate
antidiabetic metabolic effects. \emph{Nature, 423} (6941), 762-769.
doi:10.1038/nature01705

nature01705 {[}pii{]}

Yamauchi, T., Kamon, J., Minokoshi, Y., Ito, Y., Waki, H., Uchida, S., .
. . Kadowaki, T. (2002). Adiponectin stimulates glucose utilization and
fatty-acid oxidation by activating AMP-activated protein
kinase.\emph{Nat Med, 8} (11), 1288-1295. doi:10.1038/nm788

nm788 {[}pii{]}

Yamauchi, T., Kamon, J., Waki, H., Imai, Y., Shimozawa, N., Hioki, K., .
. . Kadowaki, T. (2003). Globular adiponectin protected ob/ob mice from
diabetes and ApoE-deficient mice from atherosclerosis. \emph{J Biol
Chem, 278} (4), 2461-2468. doi:10.1074/jbc.M209033200

Yamauchi, T., Nio, Y., Maki, T., Kobayashi, M., Takazawa, T., Iwabu, M.,
. . . Kadowaki, T. (2007). Targeted disruption of AdipoR1 and AdipoR2
causes abrogation of adiponectin binding and metabolic actions.\emph{Nat
Med, 13} (3), 332-339. doi:10.1038/nm1557\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figures}

Figure 1: Comparative \emph{in vitro} studies of AdipoAI and AdipoRON to
determine cellular toxicity and mRNA gene expression levels: (A)
Cellular toxicity and behavioral patterns of C2C12 myotubes treated with
AdipoAI and AdipoRON (0-10 mM) after 24 hrs incubation by using CCK8
assay; determined at 450 nm absorbance spectra. Cell cytotoxicity test
reveals IC\textsubscript{50} values of AdipoAI and AdipoRON.
Quantitative gene expression analysis of (B) AdipoR1 (C) AdipoR2 (D)
Ppargc1a in C2C12 myotubes treated with AdipoAI and AdipoRON (0-5 uM)
after 1.5 hours of treatment. (E) Adiponectin signaling in
differentiated C2C12 myotubes treated with different concentrations of
AdipoAI and AdipoRON for 1.5 hours with respect to controls (untreated).
. Statistical significance was calculated based on ordinary one-way
analysis of variance (ANOVA) with multiple comparison using GraphPad
Prism 9.4.1 software and all the values are presented as mean \selectlanguage{ngerman}± SD with
three independent experiments sets with *p\textless{}0.05;
**p\textless{}0.001.Figure 2. Oral Glucose tolerance (OGTT) and Insulin
tolerance test (ITT) test at 20 and 21 weeks in diet induced obesity
(DIO) mice models for accessing AdipoAI efficacy. (A) Oral glucose
tolerance test tested at 20 weeks in DIO mice treated with CMC (0.5\%),
AdipoAI (25 mg/kg) and AdipoRON (50 mg/kg). (B) Insulin tolerance test
tested at 21 weeks in DIO mice treated with CMC (0.5\%), AdipoAI (25
mg/kg) and AdipoRON (50 mg/kg). Data are represented as mean ± SD of 6
mice per group.; *p\textless{}0.05, **p\textless{}0.001 compared to CMC
vehicle.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2.} Body and adipocyte weight of DIO mice treated with
CMC, AdipoAI and AdipoRON for 4 weeks (A) Concept strategy of \emph{in
vivo} trial (B) Comparative Body weights measured from 18-21 weeks in
DIO mice treated with CMC (0.5\%), AdipoAI (25 mg/kg) and AdipoRON (50
mg/kg) (C \& D) changes in fat Inguinal and epididymal fat contents for
gauging comparative adipose tissue biology in DIO mice models (E) liver
weights of DIO mice with respective treated groups recorded at 21 weeks.
Data were analyzed by using ordinary 1-way followed by \selectlanguage{polish}Š\selectlanguage{english}id\selectlanguage{ngerman}ák's post hoc
test. all the values are presented as mean ± SD with three independent
experiments sets with *p\textless{}0.05; **p\textless{}0.01.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Oral Glucose tolerance (OGTT) and Insulin tolerance
test (ITT) test at 20 and 21 weeks in diet induced obesity (DIO) mice
models for accessing AdipoAI efficacy (A) Mice were starved overnight,
and oral administration of glucose (1mg/kg) were given blood glucose
levels were evaluated prior to dosing (0-15 min), and carefully
monitored every 15 minutes time interval for a period of 120 mins,
following oral administration. The AdipoAI (25 mg/kg) exposed mice
models revealed a decreased in blood glucose levels and have similar
profile of OGTT with respect to another established bioactive molecule
such as AdipoRon (50 mg/kg). (B) Insulin tolerance test (0.75 IU insulin
per kg body weight) was performed in AdipoAI and AdipoRON treated DIO
mice models after 6 hours of fasting. Blood glucose was monitored at
specific time interval (0 -120 mins) by using a sensitive glucometer
depicting comparable curves of AdipoAI and AdipoRON. Data are
represented as mean \selectlanguage{ngerman}± SD of 6 mice per group.; *p\textless{}0.05,
**p\textless{}0.001 compared to CMC vehicle.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Effect of AdipoAI treatment on adiponectin subunits
expression and hepatic microarchitecture in liver tissues. (A, B \& C)
mRNA Gene expression profiles of Ppargc1a, AdipoR2 in liver of 21 weeks
old DIO mice treated with CMC (0.5\%), AdipoAI (25 mg/kg) and AdipoRON
(50 mg/kg) administered biweekly respectively (D) Western blot analysis
phosphorylation of AMPK in liver samples treated with indicated amount
of AdipoAI and AdipoRON (E) Immunohistochemistry staining profiles of
p-AMPK levels in liver tissues from mice study models showing increase
AMPK localization in AdipoAI treated groups (E, F and G) mRNA expression
of inflammatory cytokines IL-6, TNF-a, IL-1B in liver tissues treated
with CMC, AdipoAI, AdipoRON biweekly for four weeks (H) Hematoxylin and
eosin staining depict improved hepatic details compared to control. Data
were analyzed by using ordinary 1-way followed by multiple comparison
between groups. all the values are presented as mean \selectlanguage{ngerman}± SD with three
independent experiments sets with *p\textless{}0.05; **p\textless{}0.01,
***p\textless{}0.001.

.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5.} AdipoAI activates adiponectin receptor subunit
AdipoR1. (A, B, C \& D) A tissue distribution of AdipoR1, AdipoR2,
Ppargc1a, Ppara was assessed by mRNA expression in WAT adipose tissue of
DIO mice treated with CMC (0.5\%), AdipoAI (25mg/kg) and AdipoRON (50
mg/kg). Data were analyzed by using ordinary 1-way followed by multiple
comparison between groups. all the values are presented as mean \selectlanguage{ngerman}± SD
with three independent experiments sets with *p\textless{}0.05;
**p\textless{}0.01, ***p\textless{}0.001.

Table 1: RT-PCR primers sequence\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Gene & Primer sequence\tabularnewline
\midrule
\endhead
AdipoR1 & TCAGGGATTGCTCTACTGATTATG\tabularnewline
& AGACGATGGAGAGGTAGATGAG\tabularnewline
AdipoR2 & AGGCTGGCTAATGCTTATGG\tabularnewline
& GATGTGGAAGAGCTGATGAGAG\tabularnewline
pPARGC1a & CGACAGCTATGAAGCCTATGAG\tabularnewline
& CTTCTGCCTCTCTCTCTGTTTG\tabularnewline
IL6 F & GTTGCCTTCTTCTTGGGACTGATG\tabularnewline
& CTGGCTTTGTCTTTCTTGTTATC\tabularnewline
TNF & GGTGCCTATGTCTCAGCCTCTT\tabularnewline
& GCCATAGAACTGATGAGAGGGAG\tabularnewline
IL1b & ACATCAGCACCTCACAAGCA\tabularnewline
& TTAGAAACAGTCCAGCCCCATG\tabularnewline
pPARA & ACGATGCTGTCCTCCTTGATG\tabularnewline
& GCGTCTGACTCGGTCTTCTTG\tabularnewline
GAPDH & AGGTCGGTGTGAACGGATTTG\tabularnewline
& TGTAGACCATGTAGTTGAGGTCA\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

