PPARγγγ antagonist Gleevec improves insulin...

PPARγγγγ antagonist Gleevec improves insulin sensitivity and promotes

the browning of white adipose tissue

Running title : Gleevec blocks PPARγγγγ phosphorylation at Ser273

Sunsil Choia, Eun-Sun Kim

a, Ji-Eun Jung

a, David P. Marciano

b, Ala Jo

c, Ja Young Koo

c,

Soo Youn Choia, Yong Ryoul Yang

a, Hyun-Jun Jang

a, Eung-Kyun Kim

a, Jiyoung Park

a,

Hyug Moo Kwona, In Hee Lee

d, Seung Bum Park

c, e, Kyung-Jae Myung

f, Pann-Ghill

Suha, Patrick R. Griffin

b, Jang Hyun Choi

a,*

a Department of Biological Sciences, Ulsan National Institute of Science and Technology

(UNIST), Ulsan, 689-798, Korea

b Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida,

Jupiter FL, USA

c Department of Chemistry, Seoul National University, Seoul, 151-747, Korea

d Department of Medical Chemistry, Hyundai Pharm Co., Ltd., Suwon, Gyonggi 443-270,

Korea

e Department of Biophysics and Chemical Biology/N-Bio institute, Seoul National University,

Seoul, 151-747, Korea

f Center for Genomic Integrity (CGI), Institute for Basic Science (IBS), Department of

Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan,

689-798, Korea

Page 2 of 40Diabetes

Diabetes Publish Ahead of Print, published online January 6, 2016

* Corresponding author

Prof. Jang Hyun Choi

Department of Biological Sciences, Ulsan National Institute of Science and Technology

(UNIST), Ulsan, 689-798, Korea

Phone : 82-52-217-2543

Fax : 82-52-217-5219

e-mail : [email protected]

Words: 3999; Figure: 6; Supplementary Figure: 9

Page 3 of 40 Diabetes

Abstract

Blocking phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ)

at Ser273 is one of the key mechanisms for anti-diabetic drugs to target PPARγ. Using high

throughput phosphorylation screening, we here describe that Gleevec blocks CDK5-mediated

PPARγ phosphorylation devoid of classical agonism as a PPARγ antagonist ligand. In high

fat-fed mice, Gleevec improved insulin sensitivity without causing severe side effects

associated with other PPARγ-targeting drugs. Furthermore, Gleevec reduces lipogenic and

gluconeogenic gene expression in liver and ameliorates inflammation in adipose tissues.

Interestingly, Gleevec increases browning of white adipose tissue (WAT) and energy

expenditure. Taken together, Gleevec exhibits greater beneficial effects on both glucose/lipid

metabolism and energy homeostasis by blocking PPARγ phosphorylation. These data

illustrate that Gleevec could be a novel therapeutic agent for use in insulin resistance and type

2 diabetes.


As the prevalence of obesity has exploded over the last several decades, associated

metabolic disorders, including type 2 diabetes, dyslipidemia, hypertension, and

cardiovascular diseases, have also increased dramatically. As PPARγ agonists,

thiazolidinediones (TZDs), which include pioglitazone, represent synthetic insulin-sensitizing

drugs that have been widely prescribed for the treatment of type 2 diabetes (1, 2). However,

the use of TZDs is associated with unwanted side effects, including weight gain, fluid

retention, bone fracture, cardiovascular disease, and bladder cancer (3-7). Thus, the United

States Food and Drug Administration (FDA) recently restricted the use of one TZD,

rosiglitazone, for the treatment of type 2 diabetes.

PPARγ is a master regulator of adipocyte differentiation, glucose and lipid

metabolism, and inflammation (8-10). Recently, we demonstrated that phosphorylation of

PPARγ at Ser273 (pS273) is linked to obesity and insulin resistance (11). Phosphorylation

does not globally alter its transcriptional activity, but dysregulates a specific set of genes with

roles in obesity and diabetes (11, 12). Moreover, both TZDs and SPPARMs inhibit cyclin-

dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation at Ser273 (11-13). More

specifically, non-agonist PPARγ ligands (SR1664 or UHC1) are anti-diabetic and have

reduced signals of undesirable side effects caused by TZDs (12, 13). These observations

indicate that blocking pS273 without classical agonism is an important mechanism to

consider in the development of novel anti-diabetic drugs targeting PPARγ.

In the present study, we screened a chemical library for compounds that inhibit

pS273 in vitro, and found that Gleevec, a well-known anti-cancer drug, blocked PPARγ

phosphorylation as a PPARγ ligand without classical agonism. Gleevec improved insulin

sensitivity without the commonly observed side effects of TZDs in mice fed a high-fat diet

(HFD). Furthermore, it negatively regulated pro-inflammatory responses and glucose


production in white adipose tissue (WAT) and liver, respectively. Importantly, it increased

energy expenditure by regulating a thermogenic program in subcutaneous WAT (sWAT),

resulting in anti-obesity effects. Our results demonstrate that Gleevec is a potent therapeutic

agent for both diabetes and obesity.

Research Design and Methods

Cell Culture

3T3-L1, HEK-293 and Raw264.7 cells were obtained from ATCC (VA, USA) and

cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum.

FLAG-PPARγWT

and FLAG-PPARγS273A

were subcloned into pMSCV-puro retroviral vector

(Agilent Tech., CA, USA). Adipocyte differentiation of 3T3-L1 or mouse embryonic

fibroblast (MEFs) expressing PPARγWT

or PPARγS273A

were induced differentiation as

previously described (11). Fully differentiated 3T3-L1 and MEFs or Raw264.7 cells were

preincubated with Gleevec for 24 h and treated with TNF-α (50 ng/ml) for 3 h or LPS (10

ng/ml) for 6 h, respectively. All chemicals for cell culture were obtained from Sigma (MO,

USA) unless otherwise indicated.

In vitro kinase assay

Active Cdk5/p35 or ERK was purchased from Millipore. In vitro kinase assay was

performed as previously described (11). Briefly, 0.5 µg of recombinant PPARγ (Cayman

Chem., MI, USA) were incubated with active CDK or ERK kinases in kinase assay buffer (25

mM Tris-HCl pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM

Na3VO4, 10 mM MgCl2) containing 10 µM ATP for 15 min at 30°C. Rb (Cell Signaling

Technology, MA, USA) was used as a positive control.


Immunoprecipitation and immunoblotting

HEK-293 cells expressing PPARγ were treated with TNF-α (50 ng/ml) and total cell

lysates were incubated with FLAG M2 agarose (Sigma-Aldrich, MO, USA) at 4°C.

Immunoprecipitates and total cell lysates were analyzed with phospho-specific antibody

against ser273 (11) or anti-PPARγ antibody (Santa Cruz, TX, USA).

Primary hepatocytes isolation and glucose production assay

Primary mouse hepatocytes were isolated by the two-step collagenase perfusion

method from male C57BL/6 after HFD as previously described (14). Primary hepatocytes

were plated and treated with Gleevec for 24 h following to treat forskolin (10 µM) for 6 h.

The glucose concentration in the media were measured by glucose assay kit (Sigma-Aldrich,

MO, USA)

Gene expression analysis

Total RNA was isolated from cells or tissues using Trizol reagents (Invitrogen, CA,

USA). The RNA was reverse-transcribed using ABI reverse transcription kit. Quantitative

PCR reactions were performed with SYBR green fluorescent dye using an ABI9300 PCR

machine. Relative mRNA expression was determined by the ∆∆-Ct method normalized to

TATA-binding protein (TBP) levels.

Reporter gene assay


HEK-293 cells were transfected with pDR-1 luciferase reporter plasmid, PPARγ,

PPARα or PPARδ, RXRα and pRL-renillia, respectively (Invitrogen, CA, USA). Reporter

gene assay was performed as previous described (11).

Animals

All animal experiments were performed according to procedures approved by Ulsan

National Institute of Science and Technology’s Institutional Animal Care and Use Committee.

5-week-old male C57BL/6J mice (DBL, Korea) were fed a high fat diet (60% kcal fat,

D12492, Research Diets Inc., NJ, USA) for 10 weeks. After 7-day-intraperitoneal (i.p.)

injection with Gleevec (25 mg/kg; the human equivalent dose would be around1500mg/daily)

or vehicle mice were injected with D-glucose (1.5 g/kg body weight) after overnight

starvation or human insulin (0.75 U/kg body weight) after 6 h starvation for glucose tolerant

tests (GTTs) or insulin tolerance tests (ITTs), respectively. To determine the energy

expenditure and inflammation, mice were injected daily 20 mg/kg of Gleevec or vehicle for 3

weeks. Oxygen consumption, carbon dioxide production, and food intake were measured by

the Comprehensive Laboratory Animal Monitoring System (CLAMS™; Columbus

Instruments, OH, USA) and body temperatures were measured rectally using digital

thermometer. Blood glucose levels were determined using tail blood and glucometer. Serum

insulin (Crystal Chem., IL, USA) and serum cholesterol, TG and FFAs were determined by

ELISA (Cayman Chem., MI, USA & Millipore, MA, USA).

Statistical analysis

Data are presented as means ± standard errors of the means (SEMs) as indicated in

the figure legends. Comparisons between two groups were made by unpaired two-tailed


Student's t tests. P values of <0.05 were considered statistically significant. Microsoft Excel

was used for statistical calculations.

Results

Gleevec blocks CDK5-mediated PPARγ phosphorylation at Ser273

To identify alternative non-agonist PPARγ ligands that block CDK5-mediated

PPARγ phosphorylation at Ser273, chemical screening was performed using an in vitro

kinase assay with 780 FDA-approved drugs. Among the positive candidates, we noted with

interest that Gleevec, a potent-anti-cancer drug (Fig. 1A), blocked pS273. Although the

beneficial effects of Gleevec on glucose metabolism have been demonstrated previously, the

molecular and cellular mechanisms remain unclear. Therefore, we focused on the molecular

mechanisms how Gleevec regulates glucose/fat metabolism. As shown in Fig. 1B, Gleevec

inhibited PPARγ phosphorylation in a dose-dependent manner, and its inhibitory effect was

similar to that of rosiglitazone at 10 µM. However, it does not inhibit the CDK5-mediated

phosphorylation of retinoblastoma (Rb), another CDK5 substrate (Fig. 1C) (11). Furthermore,

Gleevec blocked ERK-mediated PPARγ phosphorylation, indicating that Gleevec directly

targets PPARγ and inhibits pS273 regardless of the kinases (15) (Supplementary Fig. 1). In

human embryonic kidney 293 (HEK293) cells expressing PPARγ, Gleevec significantly

inhibited TNF-α-mediated PPARγ phosphorylation (Fig. 1D). Because Gleevec has been

widely used for the treatment of chronic myelogenous leukemia (CML) by specifically

targeting BCR-Abl tyrosine kinase, we tested whether other BCR-Abl tyrosine kinase

inhibitors block PPARγ phosphorylation. As shown in Fig. 1E, while nilotinib, dasatinib, and

sunitinib (Fig. 1A) inhibited pS273 in a similar manner as that of Gleevec, they also blocked


Rb phosphorylation by CDK5, indicating that only Gleevec targets PPARγ. These results

suggest that Gleevec specifically blocks pS273, independent of its BCR-Abl targeting.

Gleevec is a PPARγ ligand that lacks classical agonism

Next, a LanthaScreen TR-FRET competitive binding assay was performed to assess

whether Gleevec directly binds PPARγ. The half-maximum inhibitory concentration (IC50) of

Gleevec (1-3 µM) was higher than that of rosiglitazone (0.1µM) (Fig. 2A). Gleevec did not

induce transcriptional activity of PPARγ at any concentration tested (Fig. 2B). A coactivator

recruitment assay showed that Gleevec impaired recruitment of the CBP coactivator to

PPARγ, which was recruited to PPARγ in the presence of rosiglitazone (Fig. 2D).

Furthermore, Gleevec did not transcriptionally activate either PPARα or PPARδ

(Supplementary Fig. 2).

It has been reported that full agonist ligands form a hydrogen bond with tyrosine 473

on PPARγ H12; this interaction stabilizes the agonist conformation and allows H12 to serve

as the coactivator binding site (AF-2 surface) (16). Therefore, the stability of the AF2 surface

is an important determinant for the transactivation of PPARγ. In hydrogen/deuterium

exchange (HDX) analyses of the PPARG LBD, Gleevec binding induced no change in H12

conformational dynamics, in contrast to rosiglitazone that strongly stabilized the same region

(Fig. 2C and Supplementary Fig. 3). This suggests that the conformational stability of H12,

unaffected by Gleevec is consistent with the observed absence of transcriptional activity (Fig.

2B) and coactivator recruitment (Fig. 2D). Next, we compared the in silico docking

simulations of Gleevec and SR1664 (Supplementary Fig. 4). The docking score for the LBD

of PPARγ revealed that Gleevec fits in a manner similar to that of SR1664 in the proper

binding mode.


To further determine whether Gleevec lacks classical agonism, we tested its effects on

adipocyte differentiation (8, 9). As shown in Fig. 2E, rosiglitazone dramatically stimulated

adipocyte differentiation of fibroblasts (pre-adipocytes), whereas Gleevec did not increase

lipid accumulation. Moreover, the expression of adipogenic markers was also increased by

rosiglitazone, but not by Gleevec (Fig. 2F). Then, we tested the ability of Gleevec to regulate

gene expression in fully differentiated adipocytes (Fig. 2G). As shown in Fig. 2G, Gleevec

upregulated the expression of 11/17 (64%) diabetic genes dysregulated by pS273 in fully

differentiated adipocytes (Fig. 2G) (11). These data suggest that Gleevec is not a classical

transcriptional agonist of PPARγ, but specifically regulates pS273.

Gleevec improves insulin sensitivity in HFD-induced obese mice

Next, we determined whether Gleevec exerts anti-diabetic properties in vivo. After

wild type C57BL/6 mice were fed an HFD for 10 weeks, glucose tolerance tests (GTTs) and

insulin tolerance tests (ITTs) were performed following treatment with 25 mg/kg/day

Gleevec for 7 days. Both GTTs and ITTs were markedly improved without affecting body

weight (Fig. 3A, B, E). Treatment with Gleevec in HFD-fed mice significantly decreased

pS273 (Fig. 3C). Furthermore, the potency of phosphorylation inhibition was positively

correlated with improved glucose tolerance (Fig. 3D). Control mice that received vehicle only

remained hyperinsulinemic, but Gleevec substantially reduced insulin levels in these mice

(Fig. 3F), and the level of fed blood glucose was significantly reduced (Supplementary Fig.

5A). Insulin resistance, as computed by HOMA-IR, showed a clear improvement following

treatment with Gleevec (Fig. 3G). In addition, serum triglyceride (TG) and free fatty acid

(FFA) levels significantly decreased in Gleevec-treated mice (Supplementary Fig. 5B-C).

Treatment with Gleevec also altered the expression of 9/17 (52.9%) diabetic genes

dysregulated by pS273, all in the direction predicted for inhibition of pS273 (Fig. 3H) (11).


The expression of adiponectin seemed to be unaffected by treatment with Gleevec for 1 week,

but we observed the expressions of both adiponectin and adipsin were significantly increased

by treatment with Gleevec for 3 weeks in WAT (data not shown). This difference could be

caused by other environmental factors such as the communications with immune cells

because other diabetic genes dysregulated by phosphorylation of PPARγ did not exactly

matched in between adipocytes and adipose tissue after treating with Gleevec. Taken together,

these results indicate that Gleevec has a potent insulin-sensitizing effect with preferential

regulation of diabetic genes sensitive to pS273.

Gleevec results in significantly reduced common TZDs’ side effects

TZDs such as rosiglitazone can cause weight gain and fluid retention, all of which

contribute to increased cardiac dysfunction (3). They also increase the risk for bone fracture

by reducing bone formation and bone mineral density (4). As shown in Fig. 3I, treatment

human mesenchymal stem cells (hMSCs) with rosiglitazone reduced the expression of genes

involved in bone formation, including bone sialoprotein (Bsp), osteocalcin (Ocn), and osterix

(Osx). Importantly, Gleevec did not affect the expression of these gene sets (Fig. 3I). An

increase in body fat was also observed following treatment with rosiglitazone, but Gleevec

treatment did not cause any changes in either body fat or lean mass percentage (Fig. 3K, L).

As indicated in Fig. 3J, Gleevec had no detectable effect on hemodilution compared to either

vehicle or rosiglitazone treatment.

Next, we analyzed the expression of cardiac genes associated with heart failure or

hypertrophy. Cardiac hypertrophy is characterized by increased protein synthesis and

enlarged cardiomyocytes, leading to increased cardiac muscle mass (17). As shown in Fig.

3N, the expression of natriuretic peptide type B (Bnp), myosin heavy chain β (β-Mhc), and

nandrolone phenylpropionate (Npp) were increased in rosiglitazone-treated mice. However,


Gleevec did not alter their expression. Consistent with these results, HFD-fed mice treated

with rosiglitazone, but not Gleevec, showed increased heart weight (Fig. 3M). These results

strongly suggest that Gleevec greatly improves insulin sensitivity without the associated

adverse effects observed following treatment with most TZDs in vivo.

Gleevec ameliorates hepatic steatosis and reduces hepatic glucose production.

Obesity-induced insulin resistance is associated with fatty liver disease, and

dysregulation of hepatic glucose output greatly contributes to hyperglycemia in both humans

and mice. Histological observations revealed that Gleevec remarkably reduced hepatic

steatosis in HFD-induced obese mice (Fig. 4A). Consistent with hematoxylin and eosin (H&E)

staining, it also decreased the expression of hepatic lipogenic genes, including acetyl-CoA

carboxylase 1 (Acc-1), fatty acid synthase (Fasn), and stearoyl-CoA desaturase-1 (Scd-1) (Fig.

4B). Furthermore, Gleevec reduced the expression of gluconeogenic genes, such as glucose-

6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (Pepck) in the livers of

HFD-fed mice (Fig. 4B, C).

Next, we examined whether the reduced expression of gluconeogenic genes by

Gleevec in vivo is a direct cell-autonomous effect. As shown in Fig. 4D and E, Gleevec

directly decreased the expression of G6pase and Pepck (Fig. 4D) and glucose production (Fig.

4E) in primary hepatocytes. We also determined whether pS273 plays a role hepatic

gluconeogenesis. When wild-type PPARγ (PPARγWT

) or a phosphorylation-deficient PPARγ

mutant (PPARγS273A

) was expressed in primary hepatocytes, PPARγS273A

suppressed the

expression of G6pase and Pepck genes more efficiently than PPARγWT

without altering the

expression of PPARγ itself (Fig. 4F). These results strongly suggest that Gleevec improves

hepatic steatosis and directly regulates hepatic glucose production in a pS273-dependent

manner.


Gleevec ameliorates adipose tissue inflammation.

Many studies have reported that obesity is associated with a state of chronic low-

grade inflammation that facilitates the development of insulin resistance (18, 19). Therefore,

suppression of inflammation in adipose tissue may have therapeutic potential for the

treatment of enhanced inflammatory responses in obesity. Therefore, we determined whether

Gleevec suppresses chronic inflammation in the obese state. As shown in Fig. 5A,

histological sections of WAT in HFD-fed mice treated with Gleevec for 21 days had smaller

adipocytes than those of vehicle-treated mice; they also had fewer crown-like structures

formed by aggregated macrophages in adipose tissue (Fig. 5A) (20). To further investigate the

effects of Gleevec on inflammation in WAT, we assessed the expression of pro-inflammatory

and macrophage marker genes. As shown in Fig. 5B and C, pro-inflammatory genes such as

interleukin-6 (Il-6), monocyte-chemoattractant protein-1 (Mcp-1), and tumor necrosis factor-

α (Tnf-α) were significantly reduced in WAT following treatment with Gleevec whereas IL-

1β was not changed. Moreover, the expression of macrophage marker genes (F4/80, cd68,

and cd11b) and the M1 macrophage marker gene, cd11c, were also downregulated by

Gleevec. Interestingly, Gleevec-treated HFD-fed mice showed significantly increased

expression of M2 macrophage markers, including arginase (Arg) and mannose receptor (Mrc-

1) in WAT (Fig. 5D).

In previous study, we have reported that pS273 of PPARγ directly effects on

suppressing M1 macrophage activation (13). Thus we next investigated whether pS273 of

PPARγ is directly involved in anti-inflammatory activity in adipocytes and M2 polarization

in macrophages. Overexpression of PPARγS273A

in PPARγ-deficient mouse embryonic

fibroblast (MEF) cells significantly blocked pro-inflammatory gene expression compared to

that of PPARγWT

(Fig. 5E). In macrophages, overexpression of PPARγS273A

promoted


interleukin 4 (IL-4)-mediated M2 macrophage activation (Fig. 5F). Consistent with these

results, Gleevec significantly suppressed TNF-α-induced pro-inflammatory gene expression

in both 3T3-L1 adipocytes and primary macrophages (Supplementary Fig. 6A, B).

Furthermore, in vitro transwell chemotaxis assay showed that macrophages had lower

chemotactic capacity toward conditioned media (CM) from Gleevec-treated 3T3-L1 cells

compared to vehicle-treated CM (Supplementary Fig. 6C). Taken together, these data indicate

that blocking pSer273 by Gleevec causes decreased macrophage inflammation and

infiltration to WAT by substantially suppressing the pro-inflammatory the pro-inflammatory

responses in adipocytes, thus ameliorating adipose inflammation.

Gleevec increases energy expenditure and adaptive thermogenesis.

Recent studies have shown that PPARγ ligands affect energy balance by promoting

browning of WAT (21). Therefore, we investigated whether Gleevec regulates energy

expenditure using comprehensive lab animal monitoring system (CLAMS). As shown in Fig.

6A and B, we observed a highly significant increase in energy expenditure in Gleevec-treated

mice compared to vehicle-treated controls. Importantly, there was no change in respiratory

exchange ratio (RER), indicating that Gleevec did not stimulate any substantial shift from

carbohydrate to fat-based fuels (Supplementary Fig. 7). Importantly, these changes in energy

expenditure in Gleevec-treated mice were not dependent on food intake or physical activity

(Fig. 6B). At the molecular level, Gleevec produced a significant increase in broad

brown/beige fat thermogenic and mitochondrial genes, including Ucp-1, Pgc-1α, and cox-5b

in WAT and interscapular brown adipose tissue (BAT) (Fig. 6C, Supplementary Fig. 8A). In

addition, Gleevec stimulated the expression of beige adipocyte marker (Cd137 and Tmem26)

and β-oxidation genes (carnitine palmitoyltransferase 1b, Cpt1b) (Fig. 6C).


To further examine the effects of Gleevec on cold-induced thermogenesis in vivo, mice

were challenged with cold exposure at 4°C. Acute exposure (9 h) to cold significantly

dropped rectal temperatures (Fig. 6D). As expected, Gleevec-treated mice showed a more

resistant phenotype (decreased rectal temperature) against cold (Fig. 6D). WAT morphology

was also analyzed under these conditions by H&E staining. As shown in Fig. 6E, multilocular

adipocytes were observed in the sWAT of Gleevec-treated mice. Furthermore, the expression

of thermogenic and mitochondrial genes, including Ucp-1, Cidea, Cox5b, Atp5b and Cpt1b

were significantly increased in scWAT and BAT of Gleevec-treated mice following cold

exposure (Fig. 6F and Supplementary Fig. 8B).

To determine whether the browning effect of Gleevec is cell-autonomous, Gleevec was

administered to the stromal vascular fraction (SVF) of inguinal WAT during differentiation in

vitro. There were no detectable differences in the expression of Ucp-1 or Pgc-1α in fully

differentiated adipocytes (Supplementary Fig. 9), indicating that Gleevec may induce adipose

tissue thermogenesis in vivo independent of its direct action on adipocytes.

Recent studies have demonstrated that IL-4 activates M2 macrophage polarization and

alternatively activated macrophage produces catecholamine to be important for induction of

thermogesis in WAT and BAT (22). As Gleevec promoted the expression of M2 marker

genes, including Arg and Mrc-1 in adipose tissue (Fig. 5C), we next examined the expression

of IL-4, IL-13, and thyrosine hydroxylase (Th), the rate-limiting enzyme to synthesize

catecholamines in WAT. As shown in Fig. 6G and H, treatment with Gleevec resulted in

significantly increased expression of IL-4, IL-13 and Th either during cold exposure (Fig. 6G)

or in normal housing temperature (22°C) (Fig. 6H). Taken together, these results indicate that

Gleevec increases energy expenditure by upregulating beige/brown fat thermogenic genes in

vivo. And these effects were induced by a phenotypic switch in adipose tissue macrophages,


along with increase expression of Th possibly via inducing expression of the IL-4 and IL-13.

Discussion

Full agonist PPARγ ligands, including TZDs, have been widely used to treat Type 2

diabetes (8, 9, 2). However, patients treated with these drugs have exhibited higher incidence

of serious adverse effects, such as weight gain, bone fracture, and congestive heart failure

compared with other oral hypoglycemic agent. Recent studies have shown that the insulin-

sensitizing effects of PPARγ ligands are not dependent on classical agonism, but rather are a

consequence of ligand-dependent inhibition of pS273 by CDK5/ERK kinases (11, 12, 15).

More specifically, non-agonist PPARγ ligands, such as SR1664 and UHC1, have illustrated

similar glucose-lowering effects as TZDs while lacking the commonly observed side effects.

These studies have allowed us the opportunity to find a potential compound for the treatment

of type 2 diabetes. Thus, we aimed to discover compounds that block pS273 and lack

classical agonism with high binding affinity to PPARγ. Drug repositioning is the application

of known drugs and compounds to new indications, which can save time and costs because

they have already been tested in humans and detailed information is available on their

pharmacology, formulation, and potential toxicity (23). Thus, we screened PPARγ ligands

that block pS273 with an FDA-approved drug library and Gleevec was determined to fit these

criteria.

Gleevec, a specific BCR-Abl kinase inhibitor, is a well-known anti-cancer drug that

exhibits dramatic effects for the treatment of CML and gastrointestinal stromal tumors

(GISTs) (24, 25). In addition to its anti-neoplastic activity, several studies have reported that

Gleevec has a blood glucose-lowering effect in patients suffering from both CML and type 2


diabetes (26, 27) or GISTs (28). Restoration of insulin sensitivity or amelioration of

hyperlipidemia has also been noted in non-diabetic CML patients who show significant

insulin resistance at diagnosis (29, 30). Although the beneficial effects of Gleevec on glucose

and lipid metabolism have been demonstrated previously, the molecular and cellular

mechanisms remain unclear. Previous studies have demonstrated that Gleevec controls

hyperglycemia by preserving pancreatic β-cell mass or reducing endoplasmic reticulum (ER)

stress in diet-induced obese rats or diabetic db/db mice (31-34), while the present study

clearly demonstrates that the potent insulin-sensitizing action of Gleevec is derived from a

distinct molecular mechanism. Gleevec acts as a PPARγ antagonist ligand that directly blocks

pS273 while lacking classical agonism (Fig. 2 and Supplementary Fig. 1-4). Several lines of

evidence have shown improved insulin sensitivity through blocking pS273 by Gleevec:

Gleevec ameliorates hepatic steatosis and glucose production in liver (Fig. 4), attenuates pro-

inflammatory responses in WAT (Fig. 5), and promotes browning and energy expenditure in

WAT (Fig. 6), all of which contribute to increased insulin sensitivity (Fig. 3).

In our previous study, we demonstrated that blocking pS273 inhibits

lipopolysaccharide (LPS)-induced inflammation in macrophages (13). Furthermore, Gleevec

reportedly suppresses LPS- or TNF-α-induced inflammatory cytokine production in

macrophages by blocking IκB phosphorylation and subsequent DNA binding of nuclear

factor kappa B (NF-κB), thus inhibiting NF-κB activation (36). Thus, we speculate that

inhibition of pS273 by Gleevec regulates adipose tissue inflammation in the obese state.

Indeed, we observed that Gleevec redirects ATMs from an M1 to an M2 polarization state

(Fig. 5), suggesting that Gleevec reduces macrophage infiltration by suppressing the pro-

inflammatory response in adipocytes, as well as regulating M1/M2 polarization in

macrophages, thus ameliorating adipose tissue inflammation.


In the present results, we observed Gleevec induces adipose tissue thermogenesis in

vivo independent of its direct action on adipocytes. How does Gleevec promote browning of

WAT? According to recent evidence, an increase in M2 macrophages produces

catecholamine in the induction of thermogenic and β-oxidation gene expression in WAT and

BAT in cold-exposed mice (22). Furthermore, this effect is accompanied by increased

eosinophil-derived IL-4 in WAT (37). Accordingly, genetic deletion of Il4ra or Th in

myeloid cells significantly impairs the development of thermogenic beige fat in mice (37, 38).

Recently, Lee et al. reported that IL-4, derived from type 2 innate lymphoid (ILC2s),

stimulated eosinophils and directly promoted the proliferation and commitment of platelet-

derived growth factor receptor α+ (Pdgfrα

+) adipocyte precursors (APs), which are

bipotential cells that differentiate into either beige or white adipocytes within sWAT and

enhances differentiation into beige adipocytes (39). Interestingly, we have shown that

Gleevec treatment increased the proportion of M2 macrophages (Fig. 5) and the expression of

beige maker genes in sWAT (Fig. 6). Furthermore, we observed that PPARγS273A

mutant

increased IL-4-induced M2 polarization of macrophages (Fig. 5). Together, induction of IL-

4/IL-13 expression and alternative macrophage activation by inhibition of PPARγ

phosphorylation might promote regulation of expression of genes associated with

brown/beige adipose thermogenesis.

In conclusion, we found that Gleevec has potent beneficial effects on insulin

sensitivity to relieve metabolic disorders by regulating glucose and lipid metabolism without

causing several adverse effects, including body weight gain, fatty liver, fluid retention, bone

fracture, and cardiac hypertrophy, which have been observed following treatment with most

TZDs. Notably, Gleevec regulates energy expenditure by promoting the development of

beige fat in WAT. These results establish an important role for Gleevec in regulating adipose


tissue thermogenesis and white adipose plasticity towards BAT, and shed the light on the

development of novel therapeutic drugs for the treatment of obesity and type 2 diabetes.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant

funded by the Korea government (MSIP) (No. 2014M3A9D8034456)(S.S.C.), the Korean

Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea.

(HI14C2518)(J.H.C), the National Research Foundation of Korea (NRF-

2013R1A1A2060283, NRF-2011-0020163)(J.H.C), and the Institute for Basic Science (IBS-

R022-D1-2015)(J.H.C, K.J.M).

E.S.K., J.E.J., D.P.M., A.J., J.Y.K., S.Y.C., Y.R.Y., H.J.J., E.K.K., J.P., H.M.K.,

I.H.L., S.B.P., K.J.M., P.G.S., P.R.G. researched data and assisted with data interpretation.

S.S.C. and J.H.C. concived the hypothesis, designed and researched data, and wrote the

manuscript. J.H.C. is the guarantor of this work and, as such, has full access to all the data in

the study and takes responsibility for the integrity of the data and the accuracy of the data

analysis.

No potential conflicts of interest relevant to this article were reported.

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Figure Legends

Fig. 1. Gleevec blocks CDK5-mediated PPARγ phosphorylation. (A) Chemical structure

of compounds. (B) In vitro CDK5 assay on full-length PPARγ incubated with rosiglitazone or

Gleevec. (C) Phosphorylation of Rb after treating with rosiglitazone or Gleevec. (D) TNF-α-

induced phosphorylation of PPARγ in HEK-293 cells expressing PPARγ treated with

rosiglitazone or Gleevec. (E) In vitro CDK5 assay on full-length PPARγ or Rb incubated

with rosiglitazone, Gleevec, Nilotinib, Dasatinib, and Sunitinib. NT, not treated

Fig. 2. Gleevec is a PPARγ ligand that lacks classical agonism. (A) Binding affinity of

Gleevec or rosiglitazone to the LBD of PPARγ by LanthaScreen assay. (B) Transcriptional

activity of a PPAR-derived reporter gene in HEK-293 cells following treatment with

rosiglitazone, MRL24, SR1664 or Gleevec. (C) Differential HDX results are mapped onto the

PPARγ crystal structure (3PRG) comparing the LBD of PPARγ in the presence of Gleevec

(left) and rosiglitazone (right). (D) Recruitment of coactivator to PPARγ in the presence of

rosiglitazone or Gleevec by LanthaScreen assay. (E) Lipid accumulation in differentiated

3T3-L1 adipocytes treated with rosiglitazone or Gleevec following Oil-red O staining.

Expression of adipogenic marker genes (F) and gene sets regulated by PPARγ

phosphorylation (G) in these cells. All of error bars represented are S.E.M. (n=3). *p<0.05,

**p<0.01, ***p<0.001 compared with vehicle.


Fig. 3. Gleevec has potent insulin-sensitizing effects in high fat-induced obese mice

without severe side effects that TZDs have. Intraperitoneal glucose tolerant test (IPGTT) (A)

and intraperitoneal insulin tolerance test (IPITT) (B) after 7 days of treatment with vehicle or

Gleevec (25 mg/kg) in HFD-fed mice treated (n=5). Inlet, AUC. (C) Phosphorylation of

PPARγ in WAT. Quantification of PPARγ phosphorylation compared to total PPARγ was

performed (n=5). (D) Correlation between the levels of PPARγ phosphorylation normalized

to the total PPARγ protein and the changes of AUC by GTT. Pearson correlation coefficient

and p value are shown. Fasting body weight (E), fasting insulin (F), HOMA-IR (G) were

determined in these mice (n=5). (H) Expression of gene sets regulated by PPARγ

phosphorylation in WAT (n=5). (I) hMSCs were differentiated with 10 mM β-

glycerophosphate, 50 µM ascorbate-2-phosphate, 100 nM DEX treated with Gleevec or

rosiglitazone for 2 weeks. The expression of osteoblast marker genes were determined by

qPCR (n=3). Packed cell volume (PVC) in whole blood (J), the percent of body fat mass (K),

the percent of lean mass (L), heart weight (M) were measured and the expression of marker

genes for heart failure and cardiac hypertrophy in heart (N) was determined in HFD-fed mice

treated with rosiglitazone or Gleevec for 14 days (n=6). All of represented error bars are

S.E.M. (n=5). *p<0.05, **p<0.01, ***p<0.001 compared with vehicle.

Fig. 4. Gleevec reduces hepatic steatosis and glucose production in liver. (A) Histological

analysis by hematolxylin-eosin (H&E) staining of liver in HFD-fed mice treated with

Gleevec for 7 days. Expression of lipogenic (B) and gluconeogenic (C) genes in liver (n=5).

(D) Expression of gluconeogenic genes in primary hepatocytes isolated from HFD-fed mice

for 10 weeks following treatment with Gleevec (n=3). (E) Glucose concentration in the

primary hepatocyte media (n=3). (F) Expression of gluconeogenic genes and PPARγ in the


primary hepatocyes expressing PPARγWT

or PPARγS273A

(n=3). All of represented error bars

are S.E.M. *p<0.05, **p<0.01, ***p<0.001 compared with vector; ##

p<0.01 compared

between PPARγWT

and PPARγS273A

Fig. 5. Gleevec ameliorates adipose tissue inflammation. (A) Histological analysis by H&E

staining of WAT in HFD-fed mice treated with Gleevec for 21 days. Adipocyte size was

calculated on histological sections of WAT (n=6). Expression of marker genes for M1

macrophage (B), total macrophage (C) and M2 macrophage (D) (n=6). All of represented

error bars are S.E.M. *p<0.05, **p<0.01, ***p<0.001 compared with vehicle. (E) Expression

of pro-inflammatory genes in mouse embryonic fibroblast (MEFs) expressing PPARγWT

or

PPARγS273A

treated with TNF-α (50 ng/ml) (n=3). All of represented error bars are S.E.M.

*p<0.05, **p<0.01, ***p<0.001 compared with PPARγWT

; #p<0.05,

##p<0.01,

###p<0.001

compared with TNF-α treated cells; n.s, not significant. (F) Expression of pro-imflammatory

genes in Raw264.7 macrophages expressing PPARγWT

or PPARγS273A

treated with LPS (10

ng/ml) (n=3). All of represented error bars are S.E.M. **p<0.01 compared with vector and

##p<0.01 compared with PPARγ

WT ; n.s, not significant

Fig. 6. Gleevec increases energy expenditure and adaptive thermogenesis. (A-B) O2

consumption rate (VO2), CO2 production rate (VCO2), food intake, and locomotors activity

(XTOT) were measured by CLAMS in HFD-fed mice treated with Gleevec for 21 days (n=4).

(C) Expression of thermogenic, β-oxidation and beige marker genes in sWAT (n=6). (D)

Rectal temperature were measured at the indicated time points for mice placed in a cold room

(n=4). (E) H&E staining of sWAT in these mice. (F) Expression of genes for thermogenesis

and β-oxidation (n=4). Expression of Il-4, Il-13 and Th in subcutaneous WAT were


determined by qPCR with (G) or without cold exposure (H) (n=4-6). All of represented error

bars are S.E.M. *p<0.05, **p<0.01, ***p<0.001 compared with vehicle.


pPPARg

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F4/80 cd11b cd11c cd68

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Figure. 5

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Pparg

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Figure. 6

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Cidea PGC-1a PRDM16 UCP-1 cox5b ATP5B CPT-1bRe

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vehicleGleevec 20 mg/kg

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Supplementary Figure 1 Gleevec blocks ERK-mediated PPARγ phosphorylation.

In vitro ERK kinase assay on full-length PPARγ incubated with rosiglitazone or Gleevec.

pPPARg

PPARg

NT

Gle

eve

c (

10

μM

)

ATP

NT

Ro

sig

lita

zo

ne

(1

0 μ

M)

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eve

c (

1μ

M)

Gle

eve

c (

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μM

)

Gle

eve

c (

0.0

1 μ

M)


Supplymentary Figure. 2 Gleevec does not activate PPARa and PPARd.

Transcriptional activity of a PPAR-derived reporter gene in HEK-293 cells following treatment with GW7647, L165041 or Gleevec.

A B P

PA

Ra

tra

ns

cri

pti

on

al a

cti

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(μM) (μM)

PP

AR

d t

ran

sc

rip

tio

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0.01 0.1 1 10

GW7647

Gleevec

L16540

Gleevec

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GW7647

Gleevec


Apo

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Rosiglitazone

Helix 3

% D

eu

teri

um

Time (s)

Helix 11

% D

eu

teri

um

Time (s)

Helix 12

% D

eu

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um

Time (s)

Supplementary Figure.3 Gleevec Induced Perturbation in conformational dynamics of PPARg detected by HDX-MS.

Differential HDX results are mapped onto the PPARg crystal structure (3PRG) comparing the apo ligand binding domain of PPARg in the

presence of either Gleevec or rosiglitazone. 10 µM of HIS-PPARγ LBD protein was preincubated with 1:2 molar excess of compounds. 5 μl of

protein solution was mixed with 20 μl of D2O-containing HDX buffer (20 mM KPO4 pH 7.4, 50 mM KCl) and incubated at 4 °C for 10s, 30s, 60s,

900s and 3,600s. On-exchange was quenched and the protein denatured with 25 μl of quench solution (0.1 % v/v TFA in 3 M urea). Samples were

then passed through an immobilized pepsin column and the resulting peptides were trapped on a C8 trap column. The bound peptides were

gradient-eluted (5-50 % CH3CN w/v and 0.3 % w/v formic acid) across a C18 HPLC column for 5 min at 4 °C. The eluted peptides were

subjected to electrospray ionization directly coupled to a high resolution Orbitrap mass spectrometer (Thermo Fisher, MA, USA). Each HDX

experiment was carried out in triplicate and the intensity weighted average m/z value (centroid) of each peptide isotopic envelope was calculated

with in-house HDX Workbench software.


DockScore: 77.670

DockScore: 65.905

Supplementary Figure 4 Binding mode of SR1664 or Gleevec to PPARγ LBD.

Docking simulation was performed with crystal structure of PPARγ LBD (PDB ID: 2hfp) and Discovery Studio® 1.7 (Accelrys). The binding

pose was predicted by docking simulation using the Discovery Studio 1.7® program. PPARγ ligand-binding pockets were defined from receptor

cavities, and the LigandFit module implemented in the Receptor-Ligand Interaction protocol was used for detailed calculations. X-ray crystal

structure of PPARγ ligand binding domain (2hfp) was used in the docking simulation and the subsequent structural analysis with the Discovery

Studio Visualizer 3.0® program (Accelrys Software Inc., CA, USA).


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Supplementary Figure 5 The effect of Gleevec on blood glucose level and plasma level of triglyceride and free fatty acid in HFD-fed mice.

Fed blood glucose level (A) and serum level of triglyceride (B) and FFAs (C) in HFD mice treated with Gleevec for 7 days or 21 days (same as in

Fig. 3C). Error bars are S.E.M. ***p<0.001 compared with vehicle

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Vehicle/LPS

Supplementary Figure.6 The effect of Gleevec on inflammation in adipocytes and macrophages.

Fully differentiated 3T3-L1 adipocytes (A) and primary macrophages isolated from bone-marrow (B) were treated with Gleevec for 24 h and

stimulated with TNF-a (50 ng/ml) for 3 h or LPS (10 ng/ml) for 6 h, respectively. Expression of pro-inflammatory cytokines was determined by

qPCR (n=3). (C) Migratory capacity of Raw264.7 macrophages were measured using an in vitro transwell chemotaxis assay (n=5). Mature 3T3-

L1 adipocytes were used for preparation of conditioned media (CM). 3T3-L1 adipocytes were stimulated by TNF-a (10 ng/ml) for 24 h after pre-

treatment with the indicated concentration of Gleevec for 24 h in serum-free DMEM. For the migration, 100,000 Raw264.7 cells were placed in

the upper chamber of a 5 μm polycarbonate filter (24-transwell format; Corning, Lowell, MA, USA), whereas adipocyte conditioned medium or

MCP-1 (10 ng/ml) treatment was placed in the lower chamber. After 3 h of migration, cells were fixed in formalin and stained with 4’, 6-

diamidino-2-phenylindole and observed.All of represented error bars are S.E.M. *p<0.05, **p<0.01, ***p<0.001 compared with vehicle.


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Supplementary Figure.7 The effect of Gleevec on respiratory exchange ratio (RER) in HFD-fed mice.

The RER for 5 days in HFD-fed mice treated with Gleevec for 21 days (A) and the average quantification for 12 h light and 12 h dark period in A.

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Supplementary Figure 8 The effect of gleevec on thermogenic program in brown adipose tissue (BAT) of HFD-fed mice.

Expression of thermogenic and b-oxidation genes was determined by qPCR in interscapular brown adipose tissue (BAT) of gleevec-treated HFD-

fed mice for 21 days without (A) or with 9 h cold challenge (B). Error bars are S.E.M. (n=7).*p<0.05, **p<0.01 and ***p<0.001 compared with

vehicle.


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(-) (+)

Forskolin

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(-) (+)

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n.s

Supplementary Figure.9 The effect of Gleevec on Ucp-1 and Pgc-1a in isolated stromal vascular fraction (SVF) of sWAT.

Fully differentiated sWAT SVF cells were treated with Gleevec for 3 days. Expression of Ucp-1 and Pgc-1a were determied by qPCR following

treatment of forskolin (10 mM) for 4 h. Error bars are S.E.M. (n=3). ***p<0.001 compared with forskolin-untreated vehicle. n.s, not significant


PPARγγγ antagonist Gleevec improves insulin...

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Transcript of PPARγγγ antagonist Gleevec improves insulin...