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Chemico-Biological Interactions 268 (2017) 24e30

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Chemico-Biological Interactions

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Antidiabetic effect of SN158 through PPARa/g dual activation in ob/obmice

Yujung Jung a, 1, Yongkai Cao b, c, 1, Suresh Paudel c, Goo Yoon d, Seung Hoon Cheon c,Gyu-Un Bae e, Li Tai Jin f, Yong Kee Kim e, *, Su-Nam Kim a, **

a Natural Products Research Institute, Korea Institute of Science and Technology, Gangneung, Gangwon-do 25451, Republic of Koreab Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, Chinac College of Pharmacy and Research Institute of Drug Development, Chonnam National University, Gwangju 61186, Republic of Koread College of Pharmacy, Mokpo National University, Muan 58554, Republic of Koreae College of Pharmacy, Sookmyung Women's University, Seoul 04310, Republic of Koreaf School of Pharmaceutical Sciences, Key Laboratory of Biotechnology Pharmaceutical Engineering, Wenzhou Medical University, Wenzhou 325000, China

a r t i c l e i n f o

Article history:Received 12 September 2016Received in revised form30 January 2017Accepted 23 February 2017Available online 24 February 2017

Keywords:(E)-N-(4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl)acryloyl)phenyl)-4-tert-butylbenzamideSN158PPARa/g agonistInsulin sensitivity

* Corresponding author. College of Pharmacy, Sook100 Cheongpa-ro 47-gil, Yongsan-gu, Seoul 04610, Re** Corresponding author. Natural Products ResearchScience and Technology, 290 Daejeon-dong, GangnRepublic of Korea.

E-mail addresses: yksnbk@sookmyung.ac.kr (Y(S.-N. Kim).

1 These two authors contributed equally to this wo

http://dx.doi.org/10.1016/j.cbi.2017.02.0140009-2797/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

In this study, we aimed to demonstrate the antidiabetic potential of (E)-N-(4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl)acryloyl) phenyl)-4-tert-butylbenzamide (SN158) through peroxisome proliferator-activated receptor (PPAR)-a/g dual activation. SN158 interacted with both PPARa and PPARg, andincreased their transcriptional activities. Simultaneously, SN158 treatment led to an increase in adipo-genic differentiation of 3T3-L1 preadipocytes and fatty acid oxidation in hepatocytes. In addition, glucoseuptake in myotubes was significantly increased by SN158 treatment. Finally, SN158 significantly loweredthe plasma levels of glucose, triglycerides, and free fatty acids in ob/ob mice without severe weight gainand hepatomegaly. These results suggest that SN158 can be useful as a potential therapeutic agentagainst type 2 diabetes and related metabolic disorders by alleviating glucose and lipid abnormalities.

© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Peroxisome proliferator-activated receptors (PPARs) regulatethe various metabolic pathways involved in glucose and lipidmetabolism, and any dysregulation of these pathways can lead toabnormalities in glucose and lipid metabolism, obesity, and othercardiovascular diseases [1]. The PPAR family comprises three sub-types: PPARa, PPARb/d, and PPARg [2]. PPARa and PPARg are themost intensively studied PPARs, as they have critical roles inregulating glucose and lipid metabolism as well as in fatty acid b-oxidation [3,4]. These receptors are considered important

myung Women's University,public of Korea.Institute, Korea Institute of

eung-si, Gangwon-do 25451,

.K. Kim), snkim@kist.re.kr

rk.

pharmacological targets for the treatment of type 2 diabetes mel-litus (T2DM) or dyslipidemia. PPARa activation results in decreasedtriglyceride levels, while enhancing the high-density lipoprotein(HDL) cholesterol levels via stimulation of intracellular fatty acidoxidation, cellular fatty acid uptake, and lipoprotein metabolism[5]. In other studies, PPARa activationwas found to increase insulinsensitivity and ameliorate impaired glucose tolerance in T2DMpatients [6,7]. PPARg is the molecular target of thiazolidinediones(TZDs), which reverse insulin resistance and are used in the clinicaltreatment of T2DM [8]. Despite their beneficial antidiabetic po-tential, TZDs have several side effects. Troglitazone was withdrawnfrom clinical use because of hepatotoxicity, and it was replaced byrosiglitazone and pioglitzone. However, rosiglitazone appears tohave an increased risk of cardiovascular events; pioglitazoneshowed several adverse effects such as bone fractures, edema,weight gain, and increased incidence of heart failure [9e12]. Theseundesirable side effects of TZDs appear to be associated with thefull activation of the PPARg target genes in diverse tissues, which isrelated to the non-specificity of this class of ligands [13]. This notionis consistent with the observation that some non-TZD ligands,

Y. Jung et al. / Chemico-Biological Interactions 268 (2017) 24e30 25

which are also full agonists and exhibit antidiabetic activity,showed similar side effects [13]. Therefore, the development ofnovel PPARg agonists that partially modulate its target genes isconsidered as one of the most promising approaches for thedevelopment of antidiabetic agents [13e16]. Despite the weakactivation of receptors, partial PPARg agonists may have higherselectivity and fewer side effects [17e19].

There have been intensive research efforts to develop PPARa/gdual agonists because the activation of both PPARa and PPARgwitha single drug can simultaneously normalize glucose and lipidimpairment. Nevertheless, the dual activation of PPARa/g still offersan attractive therapeutic option, particularly if the agonists canpartially modulate the expression of their target genes [20].Recently, (E)-N-(4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl)acryloyl) phenyl)-4-tert-butylbenzamide (SN158) has been re-ported as a PTP1B inhibitor [21]. In this study, we further charac-terized the pharmacological properties of SN158 as a partial PPARa/g dual agonist and antidiabetic agent.

2. Materials and methods

2.1. Chemicals and cell culture

(E)-N-(4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl)acryloyl)phenyl)-4-tert-butylbenzamide (SN158) (Fig. 1A) was synthe-sized by Professor S. H. Cheon [21]. Troglitazone, pioglitazone,rosiglitazone, GW6471, and WY14643 were purchased fromSigma-Aldrich Co. (St Louis, MO). CV-1, 3T3-L1, and C2C12 cellswere obtained from ATCC and cultured in Dulbecco's modifiedEagle's medium (DMEM; Invitrogen, Carlsbad, CA), supple-mented with 10% fetal bovine serum (FBS; HyClone Labora-tories, Logan, UT) and 1% penicillin/streptomycin (Invitrogen).HepG2 cells were purchased from ATCC and cultured in Mini-mum Essential Mediumwith Earle's Balanced Salts (MEM/EBSS),supplemented with 10% FBS, 1% penicillin/streptomycin, 1�non-essential amino acid (WelGENE, Daegu, Korea), and 1 mMsodium pyruvate (WelGENE) at 37 �C with 5% CO2.

2.2. Ligand binding assay

The LanthaScreen™ TR-PRET PPAR competitive binding assay(Invitrogen) was performed according to the manufacturer'sguidelines [22].

2.3. 2-NBDG glucose uptake assay

C2C12 myoblasts were differentiated into myotubes, and 2-NBDG glucose uptake assay was performed according to previousreport [23].

2.4. Adipocyte differentiation

Adipocyte differentiation was induced by treating the cells witha differentiation mixture (DM) containing 10 mg/mL insulin, 1 mMdexamethasone, and 0.5 mM 3-Isobutyl-1-methylxanthine with10% FBS in DMEM for 48 h before the cells were switched to amaintenance medium with 10% FBS and 10 mg/mL insulin. Aftertreatment for additional 48 h, the medium was replaced with 10%FBS-DMEM. Thereafter, the mediumwas changed every 2 days. Thetest drugs were administered at the initiation of differentiation andwith every medium change for 8 days. The lipid accumulation inthe cells was detected by Oil Red O staining.

2.5. Gene expression analysis

Total RNA was isolated from 3T3-L1 adipocytes using the TRIzolreagent (Invitrogen) according to the manufacturer's instructions.cDNA synthesis and quantitative real-time PCR (Q-PCR) analyseswere performed according to previous report [23]. The primer setsfor mC/EBPa were 50-AGGTGCTGGAGTTGACCAGT-30 and 50-CAGCCTAGAGATCCAGCGAC-30 (NM_00768); mAdiponectin, 50-AGCCTGGAGAAGCCGCTTAT-30 and 50-TTGCAGTA-GAACTTGCCAGTGC-30 (NM_009605); mGAPDH, 50-TTGTTGCCAT-CAACGACCCC-30 and 50-GCCGTTGAATTTGCCGTGAG-30

(NM_008084); hCPT1a, 50- AGACGGTGGAACAGAGGCTGAAG-30 and50-TGAGACCAAACAAAGTGATGATGTCAG-30 (NM_001876.1);hACOX2, 50- GCGGACATGGCTACTCAAAGC-30 and 50-GCAGTG-CACCTTAGCAGCCTG-30 (NM_003500.1); and hGAPDH, 50- ACCA-CAGTCCATGCCATCAC-30 and 50-TCCACCACCCTGTTGCTGTA-30

(NM_002046).

2.6. Cell-based transactivation assay

CV-1 cells were seeded into 24-well plates and cultured for 24 h.The reporter gene activities for PPARs were measured using theDual-Luciferase® Reporter Assay System (Promega) according toprevious report [19].

2.7. Animal studies

All the experiments were performed according to the proced-ures approved by the KIST's Institutional Animal Care and UseCommittee (IRB code No. AP201335). Six-week-old male C57BL/6JHamSlc-ob/ob mice were purchased from Shizuoka LaboratoryAnimal Center (Japan). The mice were housed under conditions ofcontrolled temperature (23 ± 2 �C), humidity (55± 5%), and stan-dard light cycles (12 h light/dark). The mice were orally adminis-tered 30 mg/kg pioglitazone or 30, 100 mg/kg SN158 daily for 3weeks for the analysis of biomarkers in blood. For glucose-tolerance tests, 6-week-old mice were orally administered 30 mg/kg pioglitazone or 30,100mg/kg SN158 daily for 3weeks and fastedovernight before the oral administration of 2 g/kg D-glucose. Theglucose levels were measured in tail vein blood at the indicatedtime intervals using an Accu-Chek glucometer (Roche Diagnostics,Indianapolis, IN). At the end of the experimental period, the liver/body weight ratios were measured, and blood samples were ob-tained from the abdominal aorta to determine the plasmabiomarker concentrations. The triglyceride and free fatty acid (FFA)levels were measured using SICDIA L TG reagent (Eiken ChemicalCo., Japan) and NEFAZYME-S (Eiken Chemical Co.), respectively.

2.8. Statistics

The data are expressed as the mean ± standard deviation (S.D.)of three independent experiments. The differences between themean values in the two groups were analyzed using one-wayanalysis of variance (ANOVA). P < 0.05 was considered statisti-cally significant.

3. Results

3.1. SN158 is a partial PPARa/g dual agonist

To investigate the pharmacological properties of SN158 (Fig.1A),we examined the effects of SN158 on the transcriptional activity ofPPARs. SN158 treatment led to an increase in the transcriptionalactivities of both PPARa and PPARg in a dose-dependent manner;however, their efficacies were much weaker than those of the

Fig. 1. SN158 binds to and partially activates both PPARa and PPARg. (A) Structure of (E)-N-(4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl)acryloyl) phenyl)-4-tert-butylbenzamide(SN158). (B) Binding affinities of SN158 to PPARa and PPARg were analyzed using the LanthaScreen™ TR-FRET competitive binding assay, as described in Materials and methods.Transcriptional activation of PPARa (C) and PPARg (D) by SN158 was analyzed using a reporter gene assay in CV-1 cells. The cells were treated with 10 mM WY14643 (WY), 10 mMpioglitazone (PIO), or various concentrations of SN158 (1, 3, 10, or 30 mM) for 24 h. The cells were harvested and analyzed by luciferase assay as described in Materials and methods.Each bar represents the mean ± S.D. of three independent experiments. *P < 0.05 vs. control (CON). (E) The CV-1 cells transfected with PPARg and PPRE-Luc plasmids were treatedwith 30 mM SN158 in the absence or presence of TZDs (10 mM pioglitazone (PIO), 10 mM troglitazone (TRO), or 1 mM rosiglitazone (ROSI)) for 24 h. The luciferase assay was performedas described in Materials and methods. Each bar represents the mean ± S.D. of three independent experiments.

Y. Jung et al. / Chemico-Biological Interactions 268 (2017) 24e3026

positive controls (Fig. 1C and D). In addition, SN158 had minimaleffect on PPARd transactivation and no detectable effect on retinoidX receptor alpha (RXRa) activity (data not shown). We next exam-ined the binding affinities of SN158 toPPARa andPPARgbyusing theLanthaScreen competitive binding assay. SN158 could bind to bothPPARa and PPARg (Fig. 1B). The binding affinity of SN158 to PPARawasmuchweaker than that of the positive control GW6471,which isbecause of theweak ability of SN158 to activate PPARa transcriptionin comparison to that of the positive control (Fig. 1B). However,despite the weak PPARg transactivation potential of SN158, itsbinding affinity to PPARg (IC50 ¼ 70 nM) was much stronger thanthat of pioglitazone (IC50 ¼ 1629 nM), indicating that SN158 func-tions as apartial agonistof PPARg receptor. To confirmthat SN158actas a partial agonist, we examined the effect of SN158 on the PPARg-reporter gene activity in the presence of PPARg-full agonists. Thereporter gene activation by PPARg-full agonists was significantlysuppressed bySN158 treatment (Fig.1E), supporting that SN158 actsas a partial agonist. These findings indicate that SN158 acts as apartial PPARa/g dual agonist by binding to PPARa and PPARg.

3.2. SN158 induces both adipocyte differentiation and fatty acidoxidation

It has been demonstrated that PPARg agonists enhance thedifferentiation of various preadipocytes and stem cells into matureadipocytes, indicating that PPARg plays a key role in adipocytedifferentiation. The adipogenic potential of SN158 was examinedusing a 3T3-L1 preadipocyte system. Treatment with the PPARgagonist pioglitazone led to a dramatic increase in the adipogenicdifferentiation of 3T3-L1 cells as evidenced by Oil Red O staining(Fig. 2A and B). This observation was in accordance with the resultthat SN158 causes lipid droplet accumulation in adipocytes(Fig. 2A). Simultaneously, adipogenic differentiation was stronglyenhanced by SN158 treatment, which was comparable to thatobserved with pioglitazone (Fig. 2A and B). We next examined theeffect of SN158 on the expression of adipogenic transcription factorCCAAT/enhancer binding protein a (C/EBPa) and the marker geneadiponectin in differentiated adipocytes. After the induction ofadipocyte differentiation by SN158, the mRNA levels of C/EBPa and

Fig. 2. SN158 upregulates the expression of genes associated with adipocyte differentiation or fatty acid oxidation. (A) Lipid droplets in differentiated 3T3-L1 cells were stained with OilRed O. PIO: pioglitazone (10 mM), SN158 (10 mM), DM: differentiation media. (B) The accumulated lipid was quantified by measuring the OD values at 520 nm. (C) The expressionlevels of adipogenic marker genes were analyzed by Q-PCR. Each bar represents the mean ± S.D. of three independent experiments. *P < 0.05 vs. control (CON). (D) HepG2 cellstransfected with human PPARawere incubated with WY14643 (WY; 10 mM) or SN158 (10 mM) for 48 h. The relative gene expressionwas analyzed by Q-PCR. Each bar represents themean ± S.D. of three independent experiments. *P < 0.05 vs. control (CON).

Y. Jung et al. / Chemico-Biological Interactions 268 (2017) 24e30 27

adiponectin were measured using Q-PCR. The mRNA levels of C/EBPa and adiponectin increased in the SN158-treated adipocyteswhen compared to those in the control group (Fig. 2C). The increasein C/EBPa levels by SN158 was quite similar to that of the piogli-tazone -treated group; however, the adiponectin levels were muchlower, indicating that SN158 partially modulates PPARg target geneexpression. To confirm the PPARa agonistic effect of SN158, we nextdetermined its effect on fatty acid b-oxidation. The expressionlevels of PPARa target genes involved in b-oxidationweremeasuredin PPARa-transfected HepG2 cells. The expression levels ofcarnitine-palmitoyl transferase 1a (CPT1a) and acyl-CoA oxidase 2(ACOX2) were significantly increased by SN158 treatment (Fig. 2D).These results strongly support the hypothesis that SN158 enhancesfatty acid b-oxidation via PPARa activation.

3.3. SN158 enhances glucose uptake in C2C12 myotubes

To ascertain the beneficial effects of SN158 on insulin sensitivityfurther, we examined the glucose uptake in C2C12 myotubes. Un-der normal conditions, the glucose uptake in C2C12 myotubes wasdramatically enhanced by insulin treatment (Fig. 3A). SN158treatment significantly enhanced glucose uptake in the absence orpresence of insulin (Fig. 3A). Next, we investigated the effects ofSN158 on glucose uptake under palmitate-induced insulin-resis-tant conditions. Glucose uptake in C2C12 myotubes under insulin-resistant conditions was dramatically enhanced by SN158 treat-ment (Fig. 3B). SN158-mediated increases in glucose uptake arequite similar with pioglitazone's effects in both normal and insulin-resistant conditions. All these results support that SN158 improvesinsulin sensitivity and enhances glucose uptake in myotubes.

3.4. SN158 alleviates glucose and lipid impairment in ob/ob mice

To evaluate the antidiabetic effects of SN158 in vivo, weadministered SN158 to obese and diabetic ob/ob mice and moni-tored the plasma glucose levels. SN158 treatment led to a signifi-cant decrease in plasma glucose levels (Fig. 4A). The glucoselowering effect at high dose (100 mg/kg) group was comparable tothat observed in pioglitazone treated group (Fig. 4A). However,unlike pioglitazone that causes significant body weight gain, SN158treatment did not promote considerable body-weight gain (Fig. 4C).Notably, we did not observe increase in liver weight in the SN158-treated group; however, the liver weight in pioglitazone-treatedanimals was significantly increased (Fig. 4D). In addition, glucosetolerance was significantly alleviated by SN158 treatment (Fig. 4B),even though the effect was weaker than that of pioglitazone. Takentogether, these results strongly suggest that SN158 can efficientlyalleviate glucose impairment without the side effects of significantweight gain and hepatomegaly. Next, we examined whether SN158is capable of alleviating lipid abnormalities. The plasma levels oftriglycerides and FFAs significantly decreased in SN158-treatedmice, which was comparable to the effects observed followingpioglitazone treatment (Fig. 4E).

4. Discussion

In this study, we demonstrated the antidiabetic potential ofSN158 as a novel partial PPARa/g dual agonist, which alleviatesglucose and lipid impairment in ob/ob mice. The essential role ofPPARs in various metabolic diseases has beenwell demonstrated byseveral functional studies: TZDs drugs improve insulin resistanceby PPARg activation, and PPARa agonists decrease the circulating

Fig. 3. SN158 enhances insulin sensitivity. Glucose uptake in C2C12 myotubes treated with pioglitazone (PIO) or SN158 was measured by 2-NBDG glucose uptake assay under normal(A) and palmitate-induced insulin-resistant conditions (B). Each bar represents the mean ± S.D. of three independent experiments. *p < 0.05 vs. control (CON), #p < 0.05 vs. insulin-treated control.

Fig. 4. Antidiabetic effects of SN158 in ob/ob mice. (A) Non-fasting blood glucose levels in pioglitazone (PIO; 30 mg/kg)-, SN158 (30 or 100 mg/kg)-treated diabetic ob/ob mice (n ¼ 7for each group). *P < 0.05 vs. control (CON). (B) Oral glucose tolerance test in ob/ob mice. The mice were fasted for 18 h and treated with glucose (2 g/kg), and the glucose levels inthe blood were measured at the baseline (t ¼ 0 min) and in the blood samples drawn at the indicated time points (n ¼ 7). The area under the curve (AUC) of glucose tolerance testwas calculated. *P < 0.05 vs. control (CON). (C) Whole-body weight changes over 3 weeks following drug administration. *P < 0.05 vs. control (CON). (D) Average liver weights ofPIO-, or SN158-treated ob/ob mice. Each bar represents the mean ± S.D. *P < 0.05 vs. control (CON). (E) The blood triglyceride and free fatty acids levels were measured as describedin Materials and Methods. Each bar represents the means ± S.D. *P < 0.05 vs. control (CON).

Y. Jung et al. / Chemico-Biological Interactions 268 (2017) 24e3028

lipid levels. Although many dual PPAR agonists have been synthe-sized and studied, to date there are no dual PPAR agonists availablefor clinical use because of their serious side effects. However,balanced dual PPARa/g agonists could potentially benefit patients

with diabetes and metabolic disorders [24]. In addition, whencompared to full agonists, some partial agonists may selectivelymodulate PPARg activity through a different binding mode in theligand binding pocket and by the selective recruitment of

Y. Jung et al. / Chemico-Biological Interactions 268 (2017) 24e30 29

transcriptional coactivators [25]. Notably, partial or weak agonistshave been shown to have similar efficacy as full agonist TZDs, butthey exhibit an improved side effect profile.

A few partial agonists have entered clinical development [26].Here, we showed that SN158 has dual agonistic activities for bothPPARa and PPARg; however, the binding affinity of SN158 to PPARa(980 nM) is weaker than that of the positive control GW6471(232.7 nM); its binding affinity (70 nM) to PPARg is quite strongerthan that of pioglitazone (1629 nM) (Fig. 1). Unexpectedly, despitethe higher binding affinity of SN158 to PPARg, its effect on PPARgtranscriptional activity is much weaker than that of pioglitazone,indicating that SN158 acts as a partial agonist. This assumptionwasfurther strengthened by the observation that SN158 treatmentsignificantly suppressed the PPARg-reporter gene activation byTZDs (Fig. 1E). In addition, our data show that the adipogenic po-tential of SN158 is quite similar to pioglitazone (Fig. 2A). Further-more, our observation that SN158 efficiently stimulates PPARaactivity supports the hypothesis that SN158 may primarily governlipid metabolism by fatty acid oxidation (Fig. 2D). In addition,SN158 exhibits partial agonistic function to PPARa and its targetgenes, suggesting the enhancement of lipid catabolism. Treatmentwith SN158 led to an improvement in insulin sensitivity, resultingin enhanced glucose uptake inmyotubes (Fig. 3). So far, we have notbeen able to describe the precise mechanism by which a weakpartial agonist SN158 enhances adipogenic differentiation to asimilar extent as that of the full agonist pioglitazone. Recent reportsshowed that the phosphorylation of the nuclear receptor PPARg, adominant regulator of adipogenesis and fat cell gene expression,leads to the dysregulation of a large number of genes, includingadinopectin [17,27]. In addition, PPARg phosphorylation is highlyassociated with glucose tolerance and insulin sensitivity [28].Therefore, it is possible that SN158 has an effect on the phos-phorylation of PPARg. To confirm this hypothesis, we tested PPARgphosphorylation status with phospho-specific antibody (phosho-S273 and phosho-S112, LifeSpan BioSciences, Seattle, WA), butcould not ascertain this right now due to the technical limitations.

PPARg agonists such as rosiglitazone and pioglitazone showsevere side effects including hepatomegaly, body-weight gain, andfluid retention, in both humans and mice [29e31]. Increased bodyand liver weights were observed in mice treated with pioglitazonefor 3 weeks (Fig. 4). However, our data showed that SN158 signif-icantly improved glucose impairment without hepatomegaly andbody-weight gain in ob/ob mice. The lipolysis of adipose tissueleads to the hydrolysis of triglycerides and release of FFAs [32]. Thecirculating levels of FFAs are usually elevated in obesity and type 2diabetes [33]. The PPAR partial agonist SN158 may have a distinctadvantage for its pharmacological application, because a number ofstudies have shown that PPARg partial agonists including selectivePPAR modulators have decreased side effect profiles whencompared with those of full agonists [34e36].

5. Conclusion

In summary, it was revealed that SN158 has beneficial effects inthe improvement of glucose and lipid metabolism disorders byselectively activating both PPARa and PPARg. SN158 treatment isnot associated with severe adverse reactions such as weight gainand hepatomegaly that have been observed for other PPAR ago-nists. Therefore, SN158 has a strong potential for further develop-ment as antimetabolic agents to correct glucose and lipidabnormalities as well as insulin resistance.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Acknowledgments

This study was supported by grants from the Korea Institute ofScience and Technology, Republic of Korea (no. 2Z04850) and theSookmyung Women's University Research Grants (1-1403-0042).

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.cbi.2017.02.014.

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