Aegeline inspired synthesis of novel β3-AR agonist improves insulin sensitivity in vitro and in vivo models of insulin resistance

Article abstract of DOI:10.1016/j.metabol.2018.03.001

Background and Purpose: In our drug discovery program of natural product, earlier we have reported Aegeline that is N-acylated-1-amino-2- alcohol, which was isolated from the leaves of Aeglemarmelos showed anti-hyperlipidemic activity for which the QSAR s

Full text of DOI:10.1016/j.metabol.2018.03.001

Accepted Manuscript
Aegeline inspired synthesis of novel β3-AR agonist improves
insulin sensitivity in vitro and in vivo models of insulin resistance
Sujith Rajan, Sabbu Satish, Kripa Shankar, Sukanya Pandeti, Salil
Varshney, Ankita Srivastava, Durgesh Kumar, Abhishek Gupta,
Sanchita Gupta, Rakhi Choudhary, Vishal M. Balaramnavar,
Tadigoppula Narender, Anil N. Gaikwad
PII:
S0026-0495(18)30067-2
doi:10.1016/j.metabol.2018.03.001
YMETA 53750
DOI:
Reference:
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Accepted date:
22 August 2017
1 March 2018
Please cite this article as: Sujith Rajan, Sabbu Satish, Kripa Shankar, Sukanya Pandeti,
Salil Varshney, Ankita Srivastava, Durgesh Kumar, Abhishek Gupta, Sanchita Gupta,
Rakhi Choudhary, Vishal M. Balaramnavar, Tadigoppula Narender, Anil N. Gaikwad
,
Aegeline inspired synthesis of novel β3-AR agonist improves insulin sensitivity in
vitro and in vivo models of insulin resistance. The address for the corresponding author
was captured as affiliation for all authors. Please check if appropriate. Ymeta(2018),
doi:10.1016/j.metabol.2018.03.001
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ACCEPTED MANUSCRIPT
Aegeline inspired synthesis of novel β3-AR agonist improves insulin sensitivity in vitro and
in vivo models of insulin resistance.
1,2*
3*
1
3
1,2
Sujith Rajan , Sabbu Satish , Kripa Shankar ,Sukanya Pandeti , Salil Varshney ,
1
,2
1,2
1
1,2
Ankita Srivastava , Durgesh Kumar Abhishek Gupta ,Sanchita Gupta , Rakhi
,
4
4
2,3
1,2#
Choudhary , Vishal M. Balaramnavar ,Tadigoppula Narender , Anil N Gaikwad
.
Running title: Compound 10C revert insulin resistance by inducing browning
*
Both authors have contributed equally
1
Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow - 226031, India
Academy of Scientific and Innovative Research, CSIR-CDRI.
2
3
Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow
226031, India.
-
4
Global Institute of Pharmaceutical Education and Research, Jaspur Road, Kashipur,
Uttatarkhand, India, 244713.
#
Corresponding Authors: Anil NilkanthGaikwad, Division of Pharmacology, CSIR-Central
Drug Research Institute, Lucknow, Uttar Pradesh, India.
Tel.: +91 522 2771940x4876; fax: +91 522 2771941
E-mail:anil_gaikwad@cdri.res.in

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Graphical Abstract

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ABSTRACT
BACKGROUND AND PURPOSE
In our drug discovery program of natural product, earlier we have reported Aegeline that is N-
acylated-1-amino-2- alcohol, which was isolated from the leaves of Aeglemarmelos showed anti-
hyperlipidemic activity for which the QSAR studies predicted the compound to be the β3-AR
agonist, but the mechanism of its action was not elucidated. In our present study, we have
evaluated the β3-AR activity of novel N-acyl-1-amino-3-arylopropanol synthetic mimics of
aegeline and its beneficial effect in insulin resistance. In this study, we have proposed the novel
pharmacophore model using reported molecules for antihyperlipidemic activity. The reported
pharmacophore features were also compared with the newly developed pharmacophore model
for the observed biological activity.
EXPERIMENTAL APPROACH
Based on 3D pharmacophore modeling of known β3AR agonist, we screened 20 synthetic
derivatives of Aegeline from the literature. From these, the top scoring compound 10C was used
for further studies. The in-slico result was further validated in HEK293T cells co-trransfected
with human β3-AR and CRE-Luciferase reporter plasmid for β3-AR activity.The most active
compound was selected and β3-AR activity was further validated in white and brown adipocytes
differentiated from human mesenchymal stem cells (hMSCs). Insulin resistance model
developed in hMSC derived adipocytes was used to study the insulin sensitizing property. 8
week HFD fed C57BL6 mice was given 50mg/Kg of the selected compound and metabolic
phenotyping was done to evaluate its anti-diabetic effect.

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RESULTS
As predicted by in-silico 3D pharmacophore modeling, the compound 10C was found to be the
most active and specific β3-AR agonist with EC value of 447nM. The compound 10C activated
5
0
β3AR pathway, induced lipolysis, fatty acid oxidation and increased oxygen consumption rate
OCR) in human adipocytes. Compound 10C induced expression of brown adipocytes specific
(
markers and reverted chronic insulin induced insulin resistance in white adipocytes. The
compound 10C also improved insulin sensitivity and glucose tolerance in 8 week HFD fed
C57BL6 mice.
CONCLUSION
This study enlightens the use of in vitro insulin resistance model close to human physiology to
elucidates the insulin sensitizing activity of the compound 10C and edifies the use of β3AR
agonist as therapeutic interventions for insulin resistance and type 2 diabetes.
Key words:β3-AR agonist, Browning, hMSC derived adipocytes, Insulin resistance.

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1
. Introduction
Earlier as part of drug discovery program, we have identified Aegeline isolated from the leaves
of AegeloMarmelos as a potent antidyslipidemic agent with predicted β3-AR activity in QSAR
study[1]. We have further reportedthat synthetic derivatives of the compound Aegeline also
show LDL oxidation and anti-oxidant property[2].Based on theseprevious studies, we
hypothesize that Aegeline and its analogues may also induce browning phenomenon and
improve insulin sensitivity. Brown adipose tissue (BAT) is primarily responsible for non-
shivering thermogenesis. BAT is highly vascularized and is very sensitive to sympathetic
hormones such as norepinephrine, which triggers gene expression to proliferate and activate non-
shivering thermogenesis via UCP1[3-5]. Sympathetic nerve activity increases UCP1 mRNA
levels mainly through the effects of gene transcription. This response is mediated predominantly
by β-adrenergic receptors and cAMP and to a lesser extent by α1-AR[6-8]. Apart from cold
exposure, non-shivering thermogenesis can also be induced using β3-AR agonists such as BRL-
3
5135 and CL-316243[9, 10]. CL-316243 β3-AR agonist increases serum level of free fatty
acids and increase energy expenditure and decrease food intake despite reduced leptin level in
the animal model[11]. Unfortunately, in both humans and primates, these β3-AR agonists
demonstrated limited efficacy[12, 13]. Class I β3-AR agonist were based on rodent β3-AR,
which share only 79% similarity to humans[14]. The above difference in receptor homology
have rendered class I β3-AR less effective on humans. Clinical trials using class I β3-AR agonist
such as CL316243 and BRL37344 for obesity and diabetes were disappointing due to lack of
selectivity, potency and poor pharmacokinetics.
In this study,we have synthesized series of moleculescontaining pharmacophore predicted to be
responsible for anti-hyperlipidemic and LDL-Oxidation6A-D to 10A-Dbased on the earlier

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reported SAR activity. We used 3D pharmacophore modeling to predict the β3-AR activity of
synthesized derivative of Aegeline.Among the derivatives, compound 10Cwas found to be most
active and was further studied in white and brown adipocytes differentiated from human
mesenchymal stem cells (hMSC). These cells are of human origin, thus we were able to
circumvent the problems of selectivity and receptor homology faced during the development of
class-Iβ3-AR. In the present study, we found increased expression of brown adipocytes specific
marker, β-oxidation and lipolysis in white adipocytes upon treatment with compound 10C. The
compound also increased mitochondrial biogenesis and uncoupled respiration in white
adipocytes. As β3-AR agonists are reported to increase insulin sensitivity, we tested the
compound in chronic insulin (CI) induced insulin resistance (IR) inhMSC derived adipocytes.
Compound 10C increased phosphorylation of AKT and reverted CI induced IR. The specificity
study using β1-AR and β2-AR indicated the compound to be a selective β3-AR agonist.The in
vivo study on HFD induced insulin resistant C57BL6 mice further validated the in vitro results.
2
. Materials and Methods
2
.1 Synthesis of compound 10C
To a stirred solution of phenol and K CO in dry acetone epichlorohydrin was added and the
2
3
reaction mixture then refluxed until phenol was consumed. The reaction mixture was filtered
washed with acetone and dried under reduced pressure,diluted with water,and extracted with
ethylacetate.The organic layer was dried over anhydrous sodium sulphate,solvent was evaporated
under reduced pressure to obtain crude reaction product.This was further purified by column
chromatography over silica gel(60-120 mesh) to get pure oxirane(6-10).A mixture of oxiranes(6-
0
1
0) and Aromatic nitriles in dry DCM was stirred for 15 min at 0 c , then added BF *OEt drop
3
2

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0
wise maintaining temperature 0 c. Reaction mixture was stirred for15 min and ice was removed,
reaction was allow to stir at room temperature. Completion of reaction was monitored by TLC
and quenched with saturated sodium bicarbonate. The aqueous phase was extracted with
ethylacetate(3*50 ml),and the combined organic layer were dried over Na SO ,filtered and
2
4
concentrated in vacuum. Column chromatography on silica gel(60-120 mesh) using ethylacetate
and hexane as eluent provided the desired compounds(6A-10D).Detailed experimental data and
procedure for the synthesis of all the compounds along with compound 10Care given in the
supporting information. The schematic diagram of synthesis of Compound 10C is given in figure
1
E.
2
.2 Screening of compounds in CREB-luciferase assay system
HEK293T cells were co-transfected with human β1AR/β2AR/β3-AR plasmids (β1 and β2-AR
plasmids were purchased from Addgene and β3-ARwas purchased from University of science
and technology Missouri).CREB luciferase plasmid was gifted by Dr. SabyashachiSanyal as
described in figure legends. peGFPC1 plasmidDNA3 (purchased from Addgene) was used as a
transfection control. After 48hr of transfection cells were given with compound treatment as
mentioned in figure legend and lysate was prepared. Luciferase activity was measured by
providing luciferin substrate to the lysate and reading was measured inPerkin Elmer Envision
2
100 luminometer. Luciferase readings were normalized by GFP readings.
2
.3 Common feature pharmacophore generation
The model was developed as per the protocol reported previously by Prathipati et al (1). Detailed
procedure is provided in supplementary information.

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2
.4 hMSC culture and differentiation in to white and brown adipocyte
hMSCs were cultured in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine
serum (Gibco) and antibiotics. hMSCs were differentiated into white and brown adipocytes as
mentioned previously [15].Briefly, hMSCs were treated with differentiation cocktail containing
5
00μM IBMX, 5μg/ml insulin, 1μM dexamethasone and 200μM indomethacin (All purchased
form Sigma Chemicals). Cells were maintained in differentiation cocktail containing media for 9
days and media was changed every third day. For differentiation of hMSC into brown adipocyte,
above mentioned cocktail was used except indomethacin was replaced with 0.5µm rosiglitazone
and 2nM of tri-iodothyronine.
2
.5 Insulin resistance development
Insulin resistance model was developed as described previously [15]. Briefly, fully differentiated
adipocytes were incubated with 500pM of insulin (Sigma, Cat No.I5500) for 72hrs. Medium was
changed every 24 hrs to replenish the insulin level. After 72 hrs, cells were washed extensively
using KRH buffer (121 mMNaCl, 4.9 mMKCl, 1.2 mM MgSO , 0.33 mM CaCl ) supplemented
4
2
with 5mM glucose and 0.5% BSA (Step down media) prior to acute insulin pulsing.
2
.6 Oil Red O staining
Performed as described elsewhere[16, 17].
2
.7 [3H] 2-Deoxyglucose uptake
[
3H] 2-DOG glucose uptake was performed in differentiated adipocytes as described
previously[15]. Glucose uptake was measured in triplicate and normalized with total protein

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content. Radioactivity was measured in cell lysate by liquid scintillation counter (Beckman
Coulter, LS 6500, Beckman Coulter Inc. USA) and represented as fold difference.
2
.8 Real Time PCR
Total RNA was isolated from cells using TRIZOL reagent (Invitrogen CA, USA). cDNA was
prepared using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City,
CA, USA). Quantitative real time PCR analysis of different genes was performed in Light Cycler
4
80 (Roche Diagnostics) using SYBR Green master mix (Roche diagnostics). Relative change in
gene expression was calculated using (2-∆∆Ct) method and normalized with endogenous
reference gene (18S rRNA). The primer sequences of the genes analyzed is provided in
supplementary table 1.
2
.9 JC-1 and TMRE staining
JC-1 (M34152) and TMRE (T669) dye were procured from Invitrogen Life Technologies and
experiments were performed as manufacturer’s protocol. Briefly, fully differentiated adipocytes
were given with compound 10C (1µM) treatment for 72hrsand stained with TMRE (500nm) and
JC1 (2µM) for 30mins. The cells were washed with PBS and Images were takenwithLeica
DFC450 C microscope.
2
.10 Mitochondrial respiration study
To study mitochondrial respiration activity, hMSC were seeded in 8 well flux analyzer cell plate
in at a density of 10,000 cells/well and differentiated into white adipocytes as mentioned above.
OCR of white adipocytes with/without compound treatment was determined using XFp
Extracellular Flux analyzer (Seahorse Bioscience) in presence of 1µM oligomycin, 1µM FCCP

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and 0.5 µM Rotenone/Antimycin A mixture. To study metabolic pathway, we used inhibitors of
enzyme mitochondrial pyruvate carrier (UK509, 2µM), Glutaminase (BPTES, 3µM) and
carnitinepalmitoyltransferase (Etomoxir, 4µM). Readings were normalized with total protein
concentration.
2
.11 cAMP Assay
2
5µg of protein was taken for measurement of cAMP level in adipocytes with or without
compound 10C (1µM) treatment for 24hrs according to manufacturer’s instruction manual (Cell
Signaling Technologies).
2
.12 Glycerol release Assay
Glycerol release assay was done using RANDOX kit (GLY 105) purchased from Randox
Laboratories, Antrim, UK. Briefly, fully differentiated adipocytes were kept in serum free high
glucose media for 2hrs before incubating with varying concentration of compound 10C for
another 2hrs as given in figure legend (Fig. 3C). The supernatant was collected and glycerol was
measured as per manufacturer’s instruction manual.
2
.13 Glut4 translocation Assay
3
T3-L1 mature adipocytes were transfected with HA-Glut4-GFP using Gene Pulser electro-
poration system (Biorad) and plated onto a coverslip placed in 6 well plate. After 24hrs of
electroporation, the media was changed and CI treatment was given with/without 1µM
compound 10C as mentioned in the figure legend. Cells were incubated in high glucose media
for 2hrs and 1nM insulin stimulation was given for 20 min. Cells were fixed and incubated with
anti HA primary antibody overnight at 4ºC. Cells were washed 3 to 4 times with PBS and

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incubated with anti-mouse secondary antibody conjugated with Alexafluor-594 for 30mins.
Images were taken on confocal microscope.
2
.14 Western immunoblotting
Western blotting was performed as described previously (27340034). Antibodies pAKTSer 473,
pAS160 Thr 642 (#8881), AKT protein (#4691), AS160 protein (#2670), pHSLSer 660 (#4126),
pHSLSer563 (#4139), HSL protein (#18381), PGC1α (#2178), UCP1 (14670) were purchased
from Cell Signaling Technology (Beverly, MA),PRDM16 (106410) from abcam, (Cambridge,
USA). Anti-β-actinPKA-Cα (sc-903), and PKA-Cβ (sc-904) were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Image J software (NIH) was used for densitometric
quantification of protein bands.
2
.15 Cell Viability Assay
4
MTT assay: 3T3-L1 cells were seeded in 96 well plate at a density of 1×10 cells per well. Cells
were allowed to grow in complete media for 24hrs. Cells were then treated with various
concentrations (1 µM to 1000 µM) of compound 10C. After 24hrs media containing compound
1
0C was removed and cells were incubated for 3hrs with media containing MTT at a
concentration of 0.5µg/ml. The supernatant was aspirated and DMSO was added to each well.
Absorbance was taken at 570 nm using multi-well plate reader. The result given is the
representative of three independent experiments. Error bars in the graph represent mean ± SD.
2
.16 Animal care and treatment
The study was conducted in C57BL6 male mice at the animal facility of CSIR- Central Drug
Research Institute, Lucknow. All experimental procedures were approved by the institutional

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animal ethics committee and were conducted in accordance with the guidelines of the Committee
for the purpose of Control and Supervision of Experiments on Animals (CPCSEAs), India. The
micewere housed at temperature of 23±2 °C; humidity 50–60%; light 300 Lux at floor level with
regular 12h light cycle. 24 male mice were housed (six mice/cage) and given water and normal
chow diet ad libitum. Compound 10C (50mg/kg) was given orally to 8 week HFD fed mice. n= 6
mice/group were used in the study. Body weight and food intake of the mice were recorded
weekly.
2
.17 Organ collection
After 12 weeks of treatment as given in experimental design (Figure 6A) mice were fasted for
hrs and euthanized using anesthetic ether. Epididymalwhite adipose tissue (eWAT) was excised
6
from mice after acute pulsing with insulin (0.75U/kg). Tissues were processed as per the
0
subjected techniques and/or stored at -80 C till use.
2
.18 Intraperitoneal Glucose tolerance test (IPGTT)
IPGTT was performed as described previously [15]. Briefly, mice were fasted for 8hrs. Basal
th
fasting glucose (0 minutes) was measured with the help of Accu-Check Active glucometer by
tail cut method. For glucose tolerance test, glucose (2g/kg) was administered intra-peritoneal,
and blood glucose was measured at 15, 30, 60, 90, and 120 minutes.
2
.19 Insulin tolerance test (ITT)
ITT was performed in 5hrs fasted mice. Regular Insulin (0.75U/kg, Sigma Aldrich, USA) was
injected intra-peritoneal, and glucose was measured at 0, 15, 30, 60 and 90 minutes from tail cut
method.

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2
.20 Statistical analysis
Results are expressed as mean ± SD of at least three to five separate experiments. Densitometry
of western blots is expressed as mean ± SD of three independent experiments. Statistical
significance was determined by Student’s t-test and one way ANOVA for real time
results.EC was calculated by non-linear regression curve, where actual concentrations are
5
0
converted to logarithmic value and plotted in log agonist verse response curve(GraphPad Prism
version 5). p values < 0.05 were considered significant.
3
. Results
3
.1 Chemistry:
The synthesis of N-acylated 1- amino 3- aryloxy 2- propanols by known protocol i.e.Ritter
reaction. The oxirane were prepared with the substituted phenol with epichlorohydrin in acetone
in presence of base followed by ring opening with different benzonitriles. The ring opening
reaction of 2-(phenoxymethyl) oxiranes(6-10) in benzonitriles in presence of BF *OEt ,led to
3
2
synthesis of N-(2 hydroxy-3-phenoxypropyl)benzamides (6A-6D; 7A-7D; 8A-8D; 9A-9D and
1
3
0A-10D) in good yield.
.2 Pharmacophore modeling: β3-AR 3D pharmacophore generation
In this work we redeveloped a Pharmacophore model reported previously for β3-AR agonist
using the reported dataset as well as parameters. The developed Pharmacophore model has good
corroboration between the activity reported and predicted for a set of molecules under study. The
redeveloped Pharmacophore model was used as query for predicting the test set of compounds
including the series under study for β3-AR. previously. The model was used to predict the

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compound 10C which was predicted as active in the reproduced model. The compound 10C was
used as a ligand for the Pharmacophore query of β3-AR. The mapping resulted in the predicted
activity of 312.411 nM. These results are very good in terms of activity so we preceded our
compound for further biological evaluation starting from invitro analysis.
3
.3 Screening of human β3-AR agonist using CREB luciferase assay system
HEK 293T cells transfected with human β3-AR and CREB-Luciferase (cyclic AMP response
element binding protein) plasmids,were treated with series of 20 compounds (500nM) containing
common class II β3-ARpharmacophore for 6hrs. Three compounds 6B, 8D and 10C showed a
significant increase in luciferase activity, among which compound 10C had highest luciferase
activity, which is represented as fold difference (Figure 2A). The EC value of Aegelinewas
5
0
found to be 1.298e-005 (Figure 2B). The compound 10C treatment for 6 and 12 hrs increased
luciferase activity in concentration-dependent manner(Figure 2C and D).Treatment of compound
1
0C on HEK 293T cells transfected with human β3-AR increased intracellular cAMP level in
concentration dependent manner (Figure 2E). The EC value of the compound 10C was found
50
to be 447nM ± 8.00445e-07 and the activity got saturated above 1µm concentration (Figure 2B
and C). The compound 10C showed better human β3-ARactivity compared to class I β3-
ARagonist, BRL37344 (Figure 2F). The compound 10C showed no cytotoxicity on white
adipocytes differentiated from hMSCs even at the concentration as high as 100μM (Figure 2G).
To check the activity of compound 10C on other isoforms of β-AR, HEK 293T cells co-
transfected with human β1-AR, β2-AR and β3-AR along with CREB luciferasewere treated
witheither isoproterenol (10µM) or compound 10C(1µM) for 6hrs and luciferase assay was
performed. Isoproterenol was used as a positive control as it is a non-selective β-AR agonist.

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Compound 10C showed β3-AR specific activity (Figure 2H) which was further validated at
various concentrations (Figure 2I).
3
.4 Compound 10Cinduces lipolysis and activates β-oxidation.
Expression and phosphorylation of proteins involved in theβ3-AR pathway were found to be
increased 15mins onwards incompound 10Ctreated white adipocytes.In order to have clear
window for biological alterations, we have used 1µM of compound 10C for treatment in general
unless otherwise specified. The EC value was found to be 447nM and its activity saturated at
5
0
concentration above 1µM as observed in figure (1B and 1C). We observed increased expression
of protein kinase A (PKA) catalytic α and β subunit on treatment with compound 10Cinwhite
adipocytes. Phosphorylation of HSL at both Ser 563 and 660 was increased on treatment with
compound 10C. UCP1 expression was also found to be increasedon treatment with compound
1
0C (Figure 3A& Supplementary Figure 2A). We found the concentration dependent increasein
the expression and phosphorylation of the above mentioned proteins involved in theβ3-AR
pathway on treatment with compound 10C in white adipocytes differentiated from hMSCs.
Concentration dependent activation of the β3-AR pathway on treatment with the compound 10C
was more prominent in brown adipocytes compare to white adipocytes (Figure
3
B&Supplementary Figure 2B). This might be due to higher expression of β3-AR in brown
adipocytes compared to white adipocytes[18]. The compound 10C showed concentration
dependent increase in lipolysis as evident from the glycerol release assay (Figure 3C). To
confirm whether the compound 10C induces lipolysis, white adipocytes were treated with
increasing concentration of compound 10C (100nm to 100μM) for 72hrs. Oil red O staining of
white adipocytes treated with the compound 10C indicate decreased lipid content with increasing
concentration of compound 10C, as evident from the image (Figure 3D) and Oil red O

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absorbance (Figure 3E). White adipocyte on treatment with the compound 10C for 20mins
(acute) showed increased maximal respiration and spare respiratory capacity in presence of
proton gradient uncoupler FCCP (Figure 3F). Oxygen consumption rate was measured in the
compound 10C treated white adipocytes in presence of different metabolic pathway inhibitors
such as UK509 (Mitochondrial pyruvate carrier inhibitor) for the glycolytic pathway, BPTES
(Glutaminase inhibitor) for the Glutamate pathway andEtoxomir (Carnitinepalmitoyltransferase
1
A inhibitor) for the β-oxidation (Figure3G). The compound 10C treated white adipocytes
showed significant reduction in OCR in presence of CPT1A inhibitor, which in turn indicates
that these cells utilize more fatty acid as fuel for β oxidation compared to untreated adipocytes.
The above results thusconfirm that compound 10C increases lipolysis and β oxidation.
3
.5 Chronic treatment of compound 10C induces transdifferentiation from white to brown
adipocyte:
Above experimental results of mitochondrial respiration study and mechanistic study of the
compound 10C showed induction of brown adipocyte properties. β3-ARagonist are known to
induce non-shivering thermogenesis and browning phenomenon. As anticipated white adipocytes
treated with compound 10C for 72hrs time period showed increased expression of transcription
factors associated with brown adipocytes such as PRDM16, PGC1α, NRF1, DIO2, CIDEA, and
EVA1 (Figure4A). The compound 10C treatment also led to the increased expression of genes
related to mitochondria such as UCP1, COX, CytC, ATP5G1 and fatty acid oxidation related
genes such as CPT1A, MCAD FATP1 (Figure 4B and C). One of the strikingobservation is
increased expression of Carnitinepalmitoyltransferase 1A (CPT1A) enzyme in compound10C
treated white adipocytes. Compound 10C increased expression of brown adipocyte specific
markers such as PRDM16, UCP1 and PGC1α expression at protein level in both cases a) hMSC

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differentiated to white adipocytes in presence of compound 10C(1μM) . b) Fully mature white
adipocytes given 72hr treatment of compound 10C (1μM) (Figure 4D). The above experiment
suggests that the compound 10Cnot only induces browning phenomenon in white adipocytes but
is also capable of differentiating hMSC to brown-like adipocytes in absence of browning
inducing agent like troglitazone. White adipocytes on 72hrs (chronic) treatment with compound
1
0C showed increased basal OCR, maximal respiration as well as increased spare respiratory
capacity (Figure4E). This indicates compound 10C increase mitochondrial respiration either by
increasing expression of the UCP-1 protein (Fig 4 C &D) or by increasing mitochondrial
biogenesis. We wanted to assess whether the increase in basal OCR incompound 10C treated
white adipocytes is due to increase in mitochondrial biogenesis or not. For the same, we
performedTMRE and JC1 staining of white adipocytes treated with 1μM of compound 10C for
7
2hrs. We observed increased mitochondrial biogenesis and membrane potential in compound
1
0C treated adipocytes (Figure 4F and G). This further confirmsahigher number of functionally
active mitochondria,a hallmark feature of brown adipocyte in white adipocytes treated with
compound 10C. These studies confirm white to brown trans-differentiation potential of the
compound10C.
3
.6 Compound 10C reverses chronic insulin induced insulin resistance in white adipocytes
differentiated from hMSC.
Chronic insulin induced IR adipocytes were treated withcompound 10C(1µM) for 24hrs and
glucose uptake was measured after stimulation with 10nm ofinsulin for 20mins as given in
(Figure 5A). Compound 10C treated CI induced IR adipocyte showed increased glucose uptake
upon insulin stimulation compared to CI induced IR adipocytes. The glucose uptake in
compound 10C treated CI induced IR adipocytes upon insulin stimulation were similar to control

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adipocytes (Figure 5A). We also observedasignificant increase in phosphorylation of AKT and
AS160 at residue Ser473 and Thr642 respectively, upon insulin stimulation in 24hrscompound
1
0C treated IR adipocytes compared to that of untreatedIR adipocytes. The phosphorylation
levels were similar to control adipocytes (Figure 5B). We also performed Glut4 translocation
assay in the CI induced IR by transfecting with HA-GLUT4-GFP. The CI induced IR adipocytes
were given 1μM compound 10C treatment for 2hrs before stimulation with 10nM insulin and
fixed for Glut4 translocation assay.Anti-HA tagged with Alexa flour-594 was used to detect
membrane bound Glut4, as the Glut4 binds to membrane the HA tag is exposed to outer surface
which binds to anti-HA tagged with Alexa-594. Glut4 translocation assay further confirmed that
on 0.5nM insulin stimulation compound 10C treated CI induced IR adipocytes showed increased
Glut4 translocation compared to IR adipocytes, which was evident on microscopic observation
as well as quantification of red/green intensity provided by the image (Figure 5C & D).
3
.7 Compound 10Ctreatment improved lipid profile and reversed HFD induced insulin
resistance in C57BL/6 mice
A schematic representation of experiment design indicating compound treatment in 8 weeks
HFD fed mice (Figure 6A). The compound 10C treatment to 8 weeks HFD fed mice did not alter
food intake but reduced whole body weight (Figure 6B and C).The compound 10C treated mice
had decreased eWAT weight compared to untreated HFD fed mice (Figure 6D). Morphological
analysis by HE staining of eWAT indicated decreased adipocytes hypertrophy in compound 10C
treated mice (Figure 6E-G). Compound 10C ameliorated HFD induced dyslipidemia in terms of
significantly decreased serum triglyceride and increased serum HDL level (Figure 6H & K). We
also found decrease pattern but not significant in serum total cholesterol and LDL after

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compound 10C treatment (Figure 6I & J). Compound 10C treatment led to increased glucose
tolerance and insulin sensitivity as evident by ITT and IPGTT (Figure 6L-O).
We did serum analysis of recommended hormones. In this cohort, compound 10C attenuated
HFD induced hyperinsulinemia and hyperlpetinemia(Supplementary Figure 3C). Results were
also matched with reduced adipocyte hypertrophy as observed in H&E staining. To check the
molecular basis of increased insulin sensitivity, we probed proteins involved in insulin signaling.
We found increased phosphorylation of AKT and AS160 in eWAT of the compound 10C treated
mice compared to untreated mice (Figure 6P).In line with in vitro findings, we checked brown
adipocyte specific markers in the compound 10C treated mice. We found increased expression of
Cpt1A, Ucp1, Pgc1α, which are known to induce browning characteristics (Figure 6Q).Similar to
in vitro results, the compound 10C activated β3-AR pathway as evident by increased
phosphorylation of HSL at Ser 563 and increased expression PKA-Cα in eWAT of HFD fed
mice (Figure 6R). From the above results, it can be concluded that the compound 10C increases
insulin sensitivity and improve lipid profile by inducing browning phenomenon in eWAT of
HFD fed mice.
4
. Discussion
A larger percentage of therapeutic drugs available today are inspired from natural products and
their synthetic moieties. In an endeavor to identify novel natural products or its analogues
capable of trans-differentiating white adipocyte to brown, we screened synthetic analogues of
Aegeline previously reported for antidyslipidemic activity and predicted to be β3-AR agonist[1].
The compound 10C identified in screening, induced browning of white adipocyte in vitro,
activated β3-AR pathways leading to increased lipolysis and β-oxidation and increased OCR by

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increasing mitochondrial biogenesis and functionality. Compound10Calso improved lipid profile
and insulin sensitivity in HFD fed mice.
Since the discovery of brown adipose tissue in adult humans in late 1990[19], the scientific
community has considered activating brown adipose tissue as a mean to utilize excess stored
fat[20]. Cloning ofhuman β3-AR receptor has further revitalized above idea as the chronic
treatment of β3-AR agonist can improve the BAT content in adult humans[21]. The anti-obesity
effect of β3-AR agonist corresponds to the activation of thermogenesis in BAT by UCP1 and
activation of lipolysis in WAT[22, 23]. Chronic stimulation of β3-AR in obese animals reduced
adiposity and increased expression of the UCP1 protein in adipose tissue[24]Given the
background, chronic treatment of compound 10C increased lipolysis assessed in terms of
glycerol release and increased UCP1 expression. As anticipatedthe compound increased fatty
acid oxidation as evident by increased expression of genes involved in fatty acid oxidation such
as CPT1A, FATP1 and mCAD. CPT1A is the main carrier of fatty acid from cytosol to
mitochondria where fatty acid is oxidized[25]. By measuring the decrease in OCR in presence of
CPT1A inhibitor on compound treated adipocytes, we were able to calculate the fatty acid
oxidation rate on compound treatment.
In the past decade, many novel β3-AR agonists have been developed. SWR-03425A, a novel
human β3-AR agonist significantly decreased blood glucose and serum insulin level in KK-Ay
mice[26]. Similarly, Mirabegronanother human β3-AR agonist currently approved by FDA to
treat overactive bladder syndrome has shown to activate brown adipose tissue in human and
increase resting metabolic rate[27]. Chronic treatment of Mirabegronalso improved glucose
disposal rate and insulin sensitivity in HFD fed mice[27]. The above studies prove beyond any
doubt that β3-ARagonists are capable of reversing insulin resistance and increase insulin

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sensitivity. The lipolytic action of compound 10C is partly mediated by adenylatecyclase
activity, which in turn increases intracellular cAMP level and partly by activation of HSL by
activated PKA[28]. We further used this opportunity to test the compound 10C in CI induced
insulin resistance model developed by us in white adipocytes differentiated from hMSCs [15].
The compound 10C reverted CI induced insulin resistance as evident from increased
phosphorylation of protein involved in insulin signaling, glucose uptake and GLUT4
translocation. Our in vivo results validates that compound 10Cincreases insulin sensitivity by
activating β3-AR pathway and induces browning like characteristics in eWAT as evident from
increased expression of UCP1, PGC1α, PKA-Cα and increased phosphorylation of HSL at Ser
5
63 residues.Although, we have already observed elevated energy expenditure related genes in
eWAT of compound 10C treated mice (Figure 6Q). Therefore, to address this query, we did
relative gene expression analysis in subcutaneous white adipose tissue (SAT) also. SAT is
predominantly reported for browning and thermogenesis over visceral adipose tissue (e.g.
eWAT)[29, 30]. In SAT of compound 10C treated mice, we found significantly elevated
expression of Pgc1α, Ucp1, Elvol3, and cpt1 genes as compared to untreated HFD fed
group(Supplementary Figure 3A). Morphological analysis by H&E staining showed beige
phenotype in the SAT of compound 10C treated mice(Supplementary Figure 3B). Taken
together, these results suggested that compound 10C promoted energy expenditure and browning
in SAT and therefore, exerted anti-obesity effects. Given the account, compound 10C
demonstrated overall benefit in altered metabolic status in HFD fed C57BL/6 mice in terms of
improved dyslipidemia and insulin sensitivity.
In summary, Aegeline inspired synthesis led to identification of chemical compound10C capable
of specifically activatingβ3-AR in nanomolarEC range. Compound 10Cis capable of inducing
5
0

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browning in white adipocyte and has trans-differentiating potential. Given the bioactivity and
mechanistic profile of the compound, compound 10C has an overall beneficial effect in
metabolic disorders in terms of increased lipolysis, increase thermogenesis, decreased
dyslipidemia and betterment in glucose and insulin tolerance in HFD fed animal models.
Although more insights and further developments are warranted, nevertheless, Aegeline inspired
synthetic compound 10C can be lead compound for trans-differentiating white to brown
adipocytes.
5
. Acknowledgement
We are grateful to Dr. SabyasachiSanyal for gifting CREB-Luciferase reporter plasmid for
development of β3-AR agonist screening platform. We are also thankful to staff of sophisticated
analytical instrumentation facility (SAIF) for providing access to intravital microscopy and
spectral data. We acknowledge the help provided by Prof. Nuzhat Husain (Director) Dr. Ram
ManoharLohia Institute of Medical Sciencefor approval from stem cell ethics committee. We
also acknowledge the help and support provided by staff of Sushrut Institute of Plastic Surgery
(SIPS) Hospital for collection of liposuction sample.
6
. Authors Contribution
Sujith Rajan: Wrote the manuscript and performed in-vitro experiments. SabbuSatish:
Synthesized the compounds and wrote the manuscript. Kripa Shankar: Performed in vivo
studies and checked the manuscript. Sukanya Pandeti: Synthesized compound and checked the
manuscript. Salil Varshney: Checked the manuscript and performed statistical analysis.Ankita
Srivastava: Performed experiment and check the manuscript. Durgesh Kumar: Performed in
vivo studies and checked the manuscript. Abhishek Gupta: Checked and formatted the

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manuscript.Sanchita Gupta:Performed experiment and checked the manuscript. Rakhi
Choudhary:Performed in-silico screening and developed model.Vishal M. Balaramnavar:
Performed in-silico analysis and checked the manuscript. Tadigoppula Narender:
Conceptualized the idea and checked the manuscript. Dr. Anil N Gaikwad: Conceptualized the
idea and corrected the manuscript.
7
. Funding
This research work is supported by department of biotechnology (DBT) government of India
project GAP1079 and CSIR-CDRI Network project: "Towards holistic understanding of
complex diseases: Unraveling the threads of complex disease (THUNDER) Project No:
BSC0102.

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References
[
1] Narender T, Shweta S, Tiwari P, Papi Reddy K, Khaliq T, Prathipati P, et al. Antihyperglycemic and
antidyslipidemic agent from Aegle marmelos. Bioorganic & medicinal chemistry letters. 2007;17:1808-
1.
2] Sarkar S, Sonkar R, Bhatia G, Tadigoppula N. Synthesis of new N-acryl-1-amino-2-phenylethanol
1
[
and N-acyl-1-amino-3-aryloxypropanols and evaluation of their antihyperlipidemic, LDL-oxidation and
antioxidant activity. European journal of medicinal chemistry. 2014;80:135-44.
[
3] van der Lans AA, Hoeks J, Brans B, Vijgen GH, Visser MG, Vosselman MJ, et al. Cold acclimation
recruits human brown fat and increases nonshivering thermogenesis. J Clin Invest. 2013;123:3395-
03.
4] Scarpace PJ, Matheny M, Borst S, Tumer N. Thermoregulation with age: role of thermogenesis and
uncoupling protein expression in brown adipose tissue. Proc Soc Exp Biol Med. 1994;205:154-61.
5] van Marken Lichtenbelt W. Brown adipose tissue and the regulation of nonshivering
thermogenesis. Curr Opin Clin Nutr Metab Care. 2012;15:547-52.
6] Yasuda T, Masaki T, Sakata T, Yoshimatsu H. Hypothalamic neuronal histamine regulates
4
[
[
[
sympathetic nerve activity and expression of uncoupling protein 1 mRNA in brown adipose tissue in
rats. Neuroscience. 2004;125:535-40.
[
7] Zhao J, Cannon B, Nedergaard J. alpha1-Adrenergic stimulation potentiates the thermogenic action
of beta3-adrenoreceptor-generated cAMP in brown fat cells. J Biol Chem. 1997;272:32847-56.
8] Fain JN, Mohell N, Wallace MA, Mills I. Metabolic effects of beta, alpha 1, and alpha 2
[
adrenoceptor activation on brown adipocytes isolated from the perirenal adipose tissue of fetal
lambs. Metabolism: clinical and experimental. 1984;33:289-94.
[
9] Shih MF, Taberner PV. Selective activation of brown adipocyte hormone-sensitive lipase and cAMP
production in the mouse by beta 3-adrenoceptor agonists. Biochem Pharmacol. 1995;50:601-8.
10] Klaus S, Seivert A, Boeuf S. Effect of the beta(3)-adrenergic agonist Cl316,243 on functional
differentiation of white and brown adipocytes in primary cell culture. Biochimica et biophysica acta.
001;1539:85-92.
11] Himms-Hagen J, Cui J, Danforth E, Jr., Taatjes DJ, Lang SS, Waters BL, et al. Effect of CL-316,243, a
[
2
[
thermogenic beta 3-agonist, on energy balance and brown and white adipose tissues in rats. Am J
Physiol. 1994;266:R1371-82.
[
12] Barbatelli G, Murano I, Madsen L, Hao Q, Jimenez M, Kristiansen K, et al. The emergence of cold-
induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown
adipocyte transdifferentiation. Am J Physiol Endocrinol Metab. 2010;298:E1244-53.
[
13] de Jesus LA, Carvalho SD, Ribeiro MO, Schneider M, Kim SW, Harney JW, et al. The type 2
iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue. J Clin
Invest. 2001;108:1379-85.
[
14] Igawa Y, Aizawa N, Homma Y. Beta3-adrenoceptor agonists: possible role in the treatment of
overactive bladder. Korean journal of urology. 2010;51:811-8.
15] Rajan S, Shankar K, Beg M, Varshney S, Gupta A, Srivastava A, et al. Chronic hyperinsulinemia
reduces insulin sensitivity and metabolic functions of brown adipocyte. J Endocrinol. 2016;230:275-90.
16] Beg M, Shankar K, Varshney S, Rajan S, Singh SP, Jagdale P, et al. A clerodane diterpene inhibit
[
[
adipogenesis by cell cycle arrest and ameliorate obesity in C57BL/6 mice. Molecular and cellular
endocrinology. 2015;399:373-85.
[
17] Rajan S, Puri S, Kumar D, Babu MH, Shankar K, Varshney S, et al. Novel indole and triazole based
hybrid molecules exhibit potent anti-adipogenic and antidyslipidemic activity by activating
Wnt3a/beta-catenin pathway. European journal of medicinal chemistry. 2018;143:1345-60.

ACCEPTED MANUSCRIPT
[
18] Kajimura S, Saito M. A new era in brown adipose tissue biology: molecular control of brown fat
development and energy homeostasis. Annual review of physiology. 2014;76:225-49.
19] Rajan S, Gupta A, Beg M, Shankar K, Srivastava A, Varshney S, et al. Adipocyte
transdifferentiation and its molecular targets. Differentiation; research in biological diversity.
014;87:183-92.
20] Aldiss P, Betts J, Sale C, Pope M, Symonds ME. Exercise-induced 'browning' of adipose tissues.
Metabolism: clinical and experimental. 2017.
21] Ghorbani M, Himms-Hagen J. Appearance of brown adipocytes in white adipose tissue during CL
[
2
[
[
3
1
16,243-induced reversal of obesity and diabetes in Zucker fa/fa rats. Int J Obes Relat Metab Disord.
997;21:465-75.
[
22] Takakura Y, Yoshida T. [Beta 3-adrenergic receptor agonists--past, present and future]. Nihon
Yakurigaku Zasshi. 2001;118:315-20.
23] Jeong MY, Park J, Youn DH, Jung Y, Kang J, Lim S, et al. Albiflorin ameliorates obesity by inducing
[
thermogenic genes via AMPK and PI3K/AKT in vivo and in vitro. Metabolism: clinical and
experimental. 2017;73:85-99.
[
24] Nakamura Y, Nagase I, Asano A, Sasaki N, Yoshida T, Umekawa T, et al. Beta 3-adrenergic agonist
up-regulates uncoupling proteins 2 and 3 in skeletal muscle of the mouse. J Vet Med Sci. 2001;63:309-
4.
25] Lee K, Kerner J, Hoppel CL. Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an
outer membrane fatty acid transfer complex. The Journal of biological chemistry. 2011;286:25655-62.
26] Kiso T, Namikawa T, Tokunaga T, Sawada K, Kakita T, Shogaki T, et al. Anti-obesity and anti-
1
[
[
diabetic activities of a new beta3 adrenergic receptor agonist, (S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-
phenoxypropyl)]amino]ethyl]-1-propenyl] phenoxy] acetic acid ethanedioic acid (SWR-0342SA), in KK-
Ay mice. Biol Pharm Bull. 1999;22:1073-8.
[
27] Cypess AM, Weiner LS, Roberts-Toler C, Franquet Elia E, Kessler SH, Kahn PA, et al. Activation of
human brown adipose tissue by a beta3-adrenergic receptor agonist. Cell metabolism. 2015;21:33-8.
28] Duncan RE, Ahmadian M, Jaworski K, Sarkadi-Nagy E, Sul HS. Regulation of lipolysis in adipocytes.
Annual review of nutrition. 2007;27:79-101.
29] Ding H, Zheng S, Garcia-Ruiz D, Hou D, Wei Z, Liao Z, et al. Fasting induces a subcutaneous-to-
[
[
visceral fat switch mediated by microRNA-149-3p and suppression of PRDM16. Nature
communications. 2016;7:11533.
[
30] Sidossis LS, Porter C, Saraf MK, Borsheim E, Radhakrishnan RS, Chao T, et al. Browning of
Subcutaneous White Adipose Tissue in Humans after Severe Adrenergic Stress. Cell metabolism.
015;22:219-27.
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Figure Legend
1
. Structure of compounds
The structure of compounds, Aegeline (A), CL316243 (B), BRL37344 (C) and Compound 10C
D). Synthesis of Compound 10 C (E).x
(
2
. Screening of human β3-AR agonist using CREB luciferase assay system
HEK 293T cells transfected with human β3-AR and CREB- luciferase were treated with 500nM
of each compound for 6 hrsand luciferase activity was measured using Perkin Elmer Envision
2
100 luminometer All Luciferase activity was normalized with GFP.The increase in luciferase
activity is represented as fold over control. n=3, error bars represent SD, *P<0.05, **p<0.01,
*
**P<0.001 as compared by paired Student t test (A). Dose response curve of Aegeline: HEK
93T cells transfected with β3-AR and CREB luciferase were treated with log concentration
2
(
1nm, 3nm, 10nm, 30nm, 100nm, 300nm, 1µM, 3µM and 10µM) of compound Aegeline for 6hr
time interval.EC was calculated using Graphpad non-linear regression curve. n=3, error bars
50
represent SD (B). Dose response curve of compound 10C: HEK 293T cells transfected with β3-
AR and CREB luciferase were treated with log concentration (1nm, 3nm, 10nm, 30nm, 100nm,
3
00nm, 1µM, 3µM and 10µM) of compound 10C for 6 and 12hr time interval. EC50 was
calculated using Graphpad non-linear regression curve.n=3, error bars represent SD (C and D).
HEK 293T cells were transfected with β3-AR and treated with log concentration of compound
1
0C for 6hrs. The cells were lysed and intracellular cAMP level was measured using kit. n=3(E).
HEK 293T cells transfected with β3-AR and CREB- luciferase were treated with 1µM of
compound BRL37344 or10C at for 6hrs. The luciferase activity was normalized with GFP and is
represented as fold over control. n=3, error bars represent SD. *p<0.05, **p<0.01,***p<0.001 as

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tested by Student t test (F). White adipocytes differentiated fromhMSC was treated with log
concentration of compound 10C for 24hrs and MTT activity was performed Absorbance was
measured at 540nM. n=3, error bars represent SD (G). HEK 293T cells transfected with β1, β2
and β3-AR along with CREB luciferase were given compound 10C (1µM) or isoproterenol
(10µM) treatment for 6hr as shown in figure G. n=3, error bars represent SD. *p<0.05,
*
*p<0.01,***p<0.001 as tested by Student t test (H). Similar to above experiments, human β1-
AR, β2-AR and β3-AR along with CREB luciferase reporter transduced HEK cells were given
compound 10C treatment(Log concentration) for 6hr interval and luciferase reading was taken.
(I). All luciferase readings were normalized with GFP. n=3, error bars represent SD.
3
. Compound 10C induces lipolysis and activates β-oxidation.
White adipocytes differentiated from hMSC were treated with 1μM compound 10C. Protein was
isolated at different time intervals as given in figure and subjected to western blot analysis for
following proteins, PKA-Cα, PKA-Cβ, pHSL (Ser 563 and 660 residue), HSL protein, UCP-1
and actin (A). White and brown adipocytes differentiated from hMSC were treated with
increasing concentration of compound 10C for 30mins as given in figureB. Protein was isolated
and subjected to western blot analysis for following proteins PKA-Cα, PKA-Cβ, pHSL (Ser 563
and 660 residue), HSL protein, UCP-1 and actin (B). Glycerol release assay: white adipocytes
were treated with varying concentration of compound 10Cfor 24hrsas shown in figure and
glycerol release was measured as per manufactures protocol. n=3, error bars represent SD (C).
Fully mature white adipocytes were treated with varying concentrations of compound 10C for
7
2hrs. White adipocytes were stained with ORO and images were acquired on Leica DFC450 C
microscope at 10 and 40X (upper left corner) magnification (D). Accumulated ORO was
extracted and absorbance was measured at 490 nm (E). n=3, error bar indicate SD, ***P<0.001,

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*
*P<0.01 and *P<0.05 as tested by Student t test. Mitochondrial respiration study: white
adipocytes were treated with 1μM compound 10C for 20mins before measuring OCR in presence
of ATP synthase inhibitor (1µM oligomycin), proton uncoupler (1µM FCCP) and electron
transport chain inhibitors (0.5µMRotenone/Antimycin mix) was measured with Seahorse
Bioscience XFp Extracellular Flux analyzer, n=3, error bars represent SD, **P<0.005,
*
**P<0.001 as tested by Student t test (F). OCR of white adipocytes with and without 10C
treatment for 72hrs were measured in presence of UK509 (2µM) (Mitochondrial pyruvate carrier
inhibitor), BPTES (3µM) (Glutaminase inhibitor), Etoxomir (4µM)
Carnitinepalmitoyltransferase 1A inhibitor) (G). The below graph shows OCR calculated in
(
presence of above mentioned inhibitors with/without compound treatment. The decrease in OCR
in presence of various inhibitors as shown in graph corresponds to metabolic pathways
glycolysis, glutamate pathway and β-oxidation pathway. n=3, error bars represent SD,
*
**P<0.001 as compared by paired Student t test (G).
4
. Chronic treatment of compound10C induces browning of white adipocytes.
Expression profile of transcription factors in white adipocytes treated with compound 10C at
μM concentration for 72hrs. n=3, error bars represent SD, ***P<0.001 as tested one way
1
ANOVA and Bonferroni’s post-test analysis (A). Real-Time PCR analysis of genes related to
mitochondrial biogenesis and fatty acid oxidation in white adipocytes treated with compound
1
0C at 1μM concentration for 72hrs. n=3, error bars represent SD, ***P<0.001 as tested two
way ANOVA and Bonferroni’s post-test analysis (B and C). Western blot analysis of PRDM16,
PGC1α and UCP1 in white and brown adipocytes treated with compound 10C at 1μM
concentration for 72hrs and hMSC differentiated to white adipocytes in presence of compound
1
0C (1μM) which is denoted by symbol * in the figure. Densitometry of representative blots

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normalized with actin. n=3, error bar indicate SD, ***P<0.001, **P<0.01 and *P<0.05 as tested
by Student t test (D).White adipocytes were treated with compound 10C for 72hrs and OCR was
measured at basal and in the presence of ATP synthase inhibitor (1µM oligomycin), proton
uncoupler (1µM FCCP) and electron transport chain inhibitors (0.5µMRotenone/Antimycin mix)
was measured with Seahorse Bioscience XFp Extracellular Flux analyser, n=3, error bars
represent SD, ***P<0.001 as tested by Student t test (E).White adipocytes were treated with
1
μM 10C for72hrs and stained with TMRE dye. Cells were excited at 405nM and image was
taken at 20X magnification using Leica DFC450 C microscope. The graph besides the image
shows the relative quantification of mean red intensity normalized with DAPI. n=3, error bars
represent SD, ***P<0.001 as tested by Student t test (F). Similar to above experiment white
adipocytes were treated with 1μM 10C for72hrs prior staining with JC-1 dye. Cells were excited
with 405 or 488nM and Image was taken at 20X magnification using Leica DFC450 C
microscope. Images were analyzed using image J software. The red fluorescence show high
membrane potential mitochondria (J aggregates) and green florescence show low membrane
potential mitochondria (L monomers). The graph on the right hand side is the relative
quantification of red to green ratio normalized with nuclear stain DAPI. n=3, the error bars
denote SD, **P<0.001 as tested by Student t test (G).
5
.Compound 10C increases AKT phosphorylation and reverses chronic insulin induced
insulin resistance in white adipocytes differentiated from hMSC.
Control and CI treated adipocytes were incubated with or without 1μM compound 10C for last
2
4hrs of CI treatment. 10nM insulin stimulation was given as indicated in the figure. Glucose
uptake reading was normalized with total protein concentration. n=3, the error bars indicate SD,
**P<0.001 as tested by one way ANOVA and Bonferroni post-test analysis (A). Similar to
*