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BBA - Molecular and Cell Biology of Lipids

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Olfactory receptor 43 reduces hepatic lipid accumulation and adiposity inmice

Chunyan Wu, Trung Thanh Thach, Yeon-Ji Kim, Sung-Joon Lee⁎

Department of Biotechnology, School of Life Sciences and Biotechnology for BK21 PLUS, Korea University, Seoul 02841, Republic of Korea

A R T I C L E I N F O

Keywords:Olfr43Olfactory receptor(−)-carvoneLipid accumulationcAMP response element-binding protein

A B S T R A C T

Olfactory receptors are primarily expressed in nasal olfactory epithelium, but these receptors are also ectopicallyexpressed in diverse tissues. In this study, we investigated the biological functions of Olfr43, a mouse homolog ofhuman OR1A1, in cultured hepatocytes and mice to assess its functionality in lipid metabolism. Olfr43 wasexpressed in mouse hepatocytes, and Olfr43 activation by a known ligand, (−)-carvone, stimulated cAMP re-sponse element-binding protein (CREB) activity. In ligand-receptor binding studies using site-directed muta-genesis, (−)-carvone binding required two residues, M257 and Y258, in Olfr43. In the mouse study, oral ad-ministration of (−)-carvone for 5 weeks in high-fat diet-fed mice improved energy metabolism, includingreductions in hepatic steatosis and adiposity, and improved glucose and insulin tolerance. In mouse livers andcultured mouse hepatocytes, Olfr43 activation simulated the CREB-hairy and enhancer of split 1 (HES1)-per-oxisome proliferator-activated receptor (PPAR)-γ signaling axis, leading to a reduction in hepatic triglycerideaccumulation in the mouse liver. Thus, long-term administration of (−)-carvone reduces hepatic steatosis. Theknockdown of Olfr43 gene expression in cultured hepatocytes negated these effects of (−)-carvone. In conclu-sion, an ectopic olfactory receptor, hepatic Olfr43, regulates energy metabolism via the CREB-HES1-PPARγsignaling axis.

1. Introduction

Obesity is currently the most prevalent metabolic disease worldwideand is closely associated with nonalcoholic fatty liver disease and avariety of metabolic syndromes, including hyperlipidemia and insulinresistance [1,2]. Multiple receptor-dependent signaling pathwaysmodulate hepatic lipid metabolism, but the etiology and mechanism ofhepatic steatosis is not fully understood.

Olfactory receptors (ORs) comprise the largest multigene family ofclass A rhodopsin-like G protein couple receptors (GPCRs) [3]. ORs areprimarily expressed in the cilia of olfactory sensory neurons in the ol-factory epithelium [4]. Notably, ORs are ectopically expressed and playcritical roles in a variety of non-nasal tissues, including sperm, pan-creas, kidney, muscle, the carotid body, and adipose tissues [5–9].Recently, our research group demonstrated the biological functions ofOlfr544 in mouse liver and adipose tissues. Activation of Olfr544 byazelaic acid, an endogenous ligand, stimulated fatty acid oxidation

(FAO) and lipolysis in the liver and adipose tissues, respectively, re-wired fuel preference to fats and reduced adiposity in mice [9]. Ad-ditionally, we reported that OR1A1, a human homolog of Olfr43, isexpressed in human hepatocytes [10]. Activation of OR1A1 by itsagonist, (−)-carvone, modulates hepatic triglyceride accumulation[10].

The importance of carvone on disease prevention and therapeuticshas been preciously suggested. (−)-Carvone and (+)-carvone are thetwo enantiomers of carvone, both of which are widely used in the foodand flavor industry. Two carvone isomers are active monoterpeneconstituents of the essential oils (EOs) of spearmint (Mentha spicata),and caraway (Carum carvi) [11]. Carvone is effective against a widespectrum of human pathogenic fungi and bacteria [12], and it is alsoproven to be in a class of chemopreventive agents that may activate thedetoxifying enzyme glutathione S-transferase, which is correlated withanticarcinogenic activity [13,14]. Administration of carvone has beenreported to prevent chemically induced lung and forestomach

https://doi.org/10.1016/j.bbalip.2019.01.004Received 30 May 2018; Received in revised form 30 December 2018; Accepted 5 January 2019

Abbreviations: CRE, cyclic adenosine monophosphate response element; CREB, cyclic adenosine monophosphate response element-binding protein; DGAT, dia-cylglycerol acyltransferase; EC50, half maximal effective concentration; EOs, essential oils; FAO, fatty acid oxidation; GPCRs, G protein couple receptors; GPAM,mitochondrial glycerol-3-phosphate acyltransferase; HFD, high-fat diet; HES1, hairy and enhancer of split 1; HOMA-IR, homeostatic model assessment - insulinresistance; L32, ribosomal protein L32; PKA, protein kinase A; ORs, olfactory receptors; siRNA, small interfering RNA; WT, wild-type

⁎ Corresponding author.E-mail address: junelee@korea.ac.kr (S.-J. Lee).

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carcinoma development [15], increase the number of leucocytes inBalb/c mice [16], and reduce adiposity and improve insulin resistancein mice fed a high-fat diet (HFD) [17]. Additionally, (−)-carvoneshowed an antiproliferative effect for the growth of MCF-7 cells (breastadenocarcinoma) and HT-29 (colon adenocarcinoma) [18]. It is pos-sible that the effects may be carried out in part by (−)-carvone andOR1A1/Olfr43. Thus, more in depth in vivo research of (−)-carvonemay lead to more effective treatments.

(−)-Carvone is a strong ligand for OR1A1 [19,20], with a halfmaximal effective concentration (EC50) value of< 1 μM [21]. Activa-tion of OR1A1, a member of the OR family 1 subfamily A, mediated by(−)-carvone modulates hepatic triglyceride metabolism in culturedhuman hepatocytes through the cAMP-protein kinase A (PKA)-cAMPresponse element-binding protein (CREB) signaling pathway [10]. Theacceptable daily intake of (−)-carvone is 1.87mg/kg body weight foradults [22]. The source of (−)-carvone in blood and tissue is diet, and(−)-carvone is thought to activate mouse liver receptors including ec-topic ORs. In this paper, we note that Olfr43, a murine homolog ofOR1A1, is expressed in the mouse liver, and its activation is induced by(−)-carvone. To investigate the interaction between Olfr43 and(−)-carvone, we derived the three-dimensional structure of the Olfr43:(−)-carvone complex using homology modeling and docking and per-formed mutation analysis. We further investigated the effects of Olfr43activation by (−)-carvone on hepatic lipid metabolism in HFD-fedmice.

2. Materials and methods

2.1. Cell culture and reagents

Hana3A cells were a gift from Dr. Hiroaki Matsunami at DukeUniversity. Hana3A cells were maintained in Eagle's minimal essentialmedium with Earle's balanced salts solution (HyClone, Logan, UT, USA)supplemented with 10% fetal bovine serum (HyClone) and 1% peni-cillin/streptomycin (Welgene Inc., Daegu, Korea) in a 37 °C incubatorwith a 5% CO2 atmosphere. Mouse Hepa1c1c-7 hepatocytes (KoreanCell Line Bank, Seoul, Korea) were cultured in Eagle's alpha modifica-tion minimum essential medium (HyClone) containing 10% fetal bo-vine serum and 1% penicillin/streptomycin. The (−)-carvone and for-skolin (adenylyl cyclase activator) were obtained from Sigma (St. Louis,MO, USA), and the A-23187 was purchased from Cayman (Ann Arbor,MI, USA). The dual-luciferase reporter assay system was purchasedfrom Promega (Fitchburg, WI, USA). Anti-CREB (1:500), anti-p-CREB(Ser133; 1:500), anti-α-tubulin (1:1000), anti-β-actin (1:1000), anti-PPARγ (1:1000), and anti-DGAT2 (1:2000) antibodies were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-HES1 an-tibody was obtained from Abcam (CA, USA), and anti-flag antibody waspurchased from Origene (MD, USA). Anti-mouse, anti-rabbit IgG-HRPsecondary antibodies (1:5000), and Alexa Fluor 488-tagged anti-IgGsecondary antibody (1:1000) were obtained from Invitrogen (Carlsbad,CA, USA).

2.2. CRE-luciferase reporter assay

Hana3A cells were seeded overnight in 24-well plates. A FLAG-tagged Olfr43 expression vector or a mock vector (lacking the Olfr43coding region), CRE luciferase reporter, and Renilla expression vectorwere co-transiently transfected into the cells using Lipofectamine 2000(Invitrogen) following the manufacturer's instructions. At 18 h post-transfection, the cells were stimulated with the test compounds for 18 h.Luciferase activity was assayed using a dual-glo luciferase assay kit(Promega). The luminescence of the samples was quantified using aVictor X2 (PerkinElmer, Santa Clara, CA, USA). The firefly lumines-cence signal was normalized to that of Renilla luciferase driven by aconstitutively active SV40 promoter (pRL-SV40; Promega).

2.3. Single amino-acid substitution of Olfr43

Olfr43 plasmid was obtained from Origene (Rockville, MD, USA)with a Myc-tag at the C-terminus (MR213679). The Olfr43 plasmid wasamplified by transformation into E. coli. To induce single amino-acidsubstitutions, the Olfr43 construct was mutated by PCR using site-di-rected mutagenesis primers based on the protocol of the QuickChangekit. A tyrosine (Y) residue was mutated to phenylalanine (F), which hasa similar structure to tyrosine but lacks an eOH group, and a methio-nine (M) residue was mutated to alanine (A). The Olfr43 mutant cloneswere confirmed by sequence analysis.

2.4. Homologous modeling of Olfr43 and Olfr43:(−)-carvone docking

We modeled the structure of Olfr43 (residues 20–313) using threehigh-resolution X-ray structures of the GPCR:agonist complex as tem-plates—human kappa opioid receptor in complex with JDTic (PDB ID,4DJH), A2A adenosine receptor bound to ZM241385 (PDB ID, 3EML),and M2 muscarinic acetylcholine receptor bound to an antagonist (PDBID, 3UON). Initially, five apo models of Olfr43 were derived based onthe sequence alignment using Modeller 11.1 [23] with optimal molPDFand DOPE values of 13,907 and −36,847, respectively, compared tothe average template DOPE value of −34,460. These models were re-fined and subjected to energy minimization using Galaxy [24] andMolprobility [25]. To gain insight into their interaction, we docked(−)-carvone into Olfr43 using SwissDock [26]. A representative com-plex structure is the nearest model to the largest cluster center withminimization of free energy of the ligand-receptor complex. Three-di-mensional graphics were prepared using PyMol [27].

2.5. Mouse experiments

Healthy 8-week-old male C57BL/6 J wild-type (WT) mice wereobtained from Samtako (Gyeonggi-do, Korea). All the animal experi-ments were conducted according to protocols approved by the AnimalExperiment Committee of Korea University (Protocol No. KUIACUC-2016-97). Mice were maintained under a 12 h photoperiod, at a tem-perature of 21–25 °C and a relative humidity of 50–60%. The mice werefed a 60% HFD for 4 weeks and subsequently randomly assigned intothe test or vehicle group. Mice in the test group were fed a 60% HFDand orally administered (−)-carvone (20mg/kg body weight) for5 weeks. Mice in the vehicle group were administered ddH2O. The bodyweight and food intake of the mice were measured weekly. Mice werefasted overnight before being euthanized. Blood was collected inethylenediaminetetraacetic acid tubes (BD Vacutainer) retro-orbitallyor by cardiac puncture, centrifuged at 300×g for 20min at 4 °C toseparate plasma, and stored at −80 °C. Liver and epididymis adiposetissues were collected, frozen in liquid nitrogen, and stored at −80 °Cuntil analysis.

2.6. Oral glucose tolerance test and insulin tolerance test

For oral glucose tolerance tests, after an overnight fast, blood wascollected from the tail vein of the mice. The mice were orally ad-ministered glucose (1.5 g/kg body weight) or the vehicle (ddH2O,100 μL). For insulin tolerance tests, mice were fasted for 4 h and in-traperitoneally injected with insulin (0.35 unit/kg body weight) or thevehicle (phosphate-buffered saline, 100 μL). Blood glucose levels weremeasured at 0, 15, 30, 60, 90, and 120min after glucose feeding orinsulin injection using a portable glucometer (Accu-Check Go; Roche,Basel, Switzerland).

2.7. Total RNA extraction and quantitative polymerase chain reaction

Total RNA was isolated from liver tissue using RNAiso Plus reagent(TaKaRa Bio Inc., Otsu, Japan), and cDNA was synthesized using the

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Rever Trace® RT Master Mix Kit (Toyobo, Osaka, Japan) according tothe manufacturer's instructions. The reverse transcriptase-polymerasechain reaction (RT-PCR) products were analyzed by agarose gel elec-trophoresis (4%) using a ChemiDoc touch imaging system with ImageLab 5.2 software (Bio-Rad, Redmond, WA, USA). A mock indicates aRT-PCR reaction with RNA samples without reverse transcription re-action. This is to test potential genomic DNA contamination in RNAsamples. Quantitative PCR (qPCR) was performed with theThunderbird™ SYBR® qPCR Mix reagent (TaKaRa Bio Inc.), an iQ5Cycler System (Bio-Rad, Hercules, CA, USA), and the primers listed inTable S1. Expression levels were normalized to that of ribosomal proteinL32.

2.8. Immunoblot analysis

Protein levels were measured by immunoblotting as previously de-scribed [9]. In brief, total proteins were extracted from mouse tissues orcultured hepatocytes using a radioimmunoprecipitation assay buffer,and protein concentrations were calculated using a bicinchoninic acidprotein assay kit (Pierce Biotechnology, Rockford, IL, USA). The de-natured proteins were separated on 10% sodium dodecyl sulfate poly-acrylamide gels, transferred to the nitrocellulose membranes (Daeillab,Seoul, Korea), and then incubated with specific primary and secondaryantibodies. Immunoblot images were obtained using the ChemiDoc™touch imaging system, and band intensities were quantified with ImageLab 5.2 software (Bio-Rad, Philadelphia, PA, USA). Protein levels werenormalized to that of α-tubulin or β-actin.

2.9. Quantification of hepatic lipids and plasma profiling

Hepatic triglyceride concentrations were quantified as previouslydescribed [9]. Plasma triglyceride, cholesterol, glucose, aspartate ami-notransferase, and alanine aminotransferase levels were quantifiedusing an automated clinical chemistry analyzer (Cobas111; Roche) withenzymatic methods according to the manufacturer's instructions. Theplasma insulin kit was obtained from Millipore (Bedford, MA, USA).The homeostatic model assessment - insulin resistance (HOMA-IR)index was calculated according to this formula:

− =×HOMA IR Glucose Insulin

405 (Glucose in mass unit of mg/dL). The in-sulin-sensitive index was derived using the inverse of the sum of thelogarithms of the fasting insulin and fasting glucose following thisformula: 1/(log(fasting insulin μU/mL)+ log(fasting glucose mg/dL)).

For intracellular triglyceride levels, Hepa1c1c-7 cells were seededovernight in 24-well plates, then lipid-loaded with free fatty acids(400 μM of palmitic acid and 400 μM of oleic acid) with 0.5% bovineserum albumin (GenDEPOT, TX, USA) for 24 h. Lipid-loaded cells werethen stimulated with (−)-carvone (100 μM) for 24 h. The cellular lipidswere extracted with 1mL hexane/isopropanol (2:1) for 30min at roomtemperature. In addition, the extracts were desiccated using aCentrifugal Vacuum Concentrator (Labocene, Korea) and then dissolvedin 95% ethanol. Cellular triglyceride levels were analyzed using anautomated clinical chemistry analyzer (Cobas111; Roche).

2.10. Histological analysis

After administration of (−)-carvone for 5 weeks, mice were eu-thanized after an overnight fast. Liver tissues were fixed in 4% paraf-ormaldehyde and stained with hematoxylin and eosin at theHistopathology Department of Anam Korea University Hospital (Seoul,Korea). Images were obtained using the Nikon Eclipse Ti-S microscope(Nikon Inc., Tokyo, Japan) and NIS Elements F software (Nikon Inc.).

2.11. Intracellular Ca2+ measurement

Hepa1c1c-7 cells were seeded overnight in 96-well plates at a

density of 4×104/well. An equal volume of 2× Fluo-4 Direct™ Ca2+

reagent loading solution (Invitrogen, CA, USA) was added to the wells,and plates were incubated at 37 °C and room temperature for 30min.The cells were then treated with 100 μM of (−)-carvone or 10 μM of A-23187 (a calcium-ionophore, positive control), and Ca2+ release wasmeasured according to the manufacturer's protocol (Invitrogen, CA,USA).

2.12. Olfr43 gene knockdown

Hepa1c1c-7 cells were seeded in six-well plates at a density of2× 105/well. At 50–60% confluence, cells were transfected with 8 μLof either scramble or Olfr43 siRNA duplex (Santa Cruz, CA, USA) usingLipofectamine 2000 (Invitrogen) following the manufacturer's instruc-tions. Cells were transfected again with scramble or Olfr43 siRNA at thesame concentration after 6 h of initial transfection. After 5 h of doubletransfection, 1 mL of fresh medium containing 20% fetal bovine serumwas added, and cells were incubated for 18 h before compound treat-ment.

2.13. β-Hydroxybutyrate analysis

Hepatic β-hydroxybutyrate levels were qualified by using a β-hy-droxybutyrate (Ketone Body) Colorimetric Assay Kit (Cayman, USA),according to the manufacturer's instructions. Briefly, mouse livers werehomogenized in the assay buffer (100mM Tris-HCl, pH 8.5) with pro-tease inhibitors, and then centrifuged at 4 °C to obtain the supernatants.The reactions were initiated by adding developer solutions to the testsamples, which is the mixture of lyophilized enzyme and colorimetricdetector, and then incubated at 25 °C in the dark for 30min. The ab-sorbance was qualified at 455 nm.

2.14. FAO

Mouse liver FAO was quantified as previously described [9]. Therate of palmitoyl-CoA oxidation was directly related to the rate of fattyacid oxidation in the HFD-fed mouse livers.

The FAO in cultured mouse hepatocytes Hepa1c1c-7 cells were as-sessed using [1− 14C] palmitate as previously described [28]. Briefly,Hepa1c1c-7 cells were seeded at a density of 104/well and in 24-wellplates for 24 h, then lipid-loaded with free fatty acids (400 μM of pal-mitic acid and 400 μM of oleic acid) with 0.5% bovine serum albumin(GenDEPOT, TX, USA) for 24 h after scramble or Olfr43 siRNA trans-fection. The cells were then stimulated with (−)-carvone (100 μM) for24 h, and then incubated with 1.75mM [1− 14C] palmitate (57mCi/mM; Perkin Elmer) at 37 °C for 1 h. The 14CO2 was qualified and theprotein concentrations of the resulting lysates were determined fornormalization.

2.15. Immunocytochemistry

Immunocytochemistry experiments were performed to check theexpression levels of Olfr43 on cell plasma membrane as previouslydescribed [9]. Briefly, Hana3A cells were seeded overnight and trans-fected with a FLAG-tagged Olfr43 vector (WT or mutant) using Lipo-fectamine 2000 (Invitrogen) for 18 h. The cells were then washed withPBS and fixed for 10min using 4% paraformaldehyde. Fixed cells wereincubated with anti-Flag antibody (1:1000; Origene) overnight at 4 °Cand Alexa Fluor 488-tagged anti-IgG secondary antibody (1:1000; In-vitrogen) for 1 h in the dark. The nuclei were stained with DAPI (In-vitrogen, CA, USA) for 5min. Then, the coverslips were embedded inanti-fade mounting solution (Thermo Fisher Scientific). Image analysiswas performed using a LSM700 confocal microscope (Carl Zeiss, Jena,Germany). Flag-Olfr43 was stained in green and nuclei were stained inblue.

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2.16. Statistical analysis

All data were expressed as the mean ± SEM, and statistical com-parisons of two or more groups were performed using Student's t-test orone-way analysis of variance (ANOVA) followed by Tukey's honestlysignificant difference (HSD) test. A value of P < 0.05 was consideredsignificant.

3. Results

3.1. Olfr43 activates the PKA-CREB pathway in mouse hepatocytes

RT-PCR (Fig. 1A) and qPCR (Fig. S1) analysis indicated that Olfr43was expressed in the mouse livers. We performed qPCR to confirm theectopic expression profile of Olfr43 in various mouse tissues (Fig. S1).Olfr43 showed the highest expression in nose, followed by liver (Fig.S1). Olfr43 was also expressed in mouse kidney, heart, spleen, colon,small intestine and muscle (Fig. S1). (−)-Carvone is an agonist ofOR1A1 [20,21,29,30], a human homolog of Olfr43, the activation ofwhich increases intracellular cAMP levels and activates the PKA-CREBsignaling pathway [10]. To determine whether (−)-carvone binds toand activates Olfr43, we performed CRE-reporter luciferase assays inHana3A cells. Hana 3A cells stably express the OR-trafficking cofactorsGαolf, RPT1, RPT2, and REEP, which enhance cell surface expression ofOR proteins [31]. A FLAG-tagged Olfr43 expression vector or mockvector (lacking the Olfr43 coding sequence) was transiently transfectedinto Hana3A cells. We found that (−)-carvone induced a dose-depen-dent expression of the CRE-luciferase reporter in Olfr43-overexpressingHana3A cells, with an EC50 value of 46.4 ± 2 μM (Fig. 1B). Therefore,Olfr43 is expressed in mouse hepatocytes and (−)-carvone is a ligand ofOlfr43.

3.2. Binding of (−)-carvone to Olfr43 requires M237 and Y258

To obtain mechanistic insight into (−)-carvone recognition byOlfr43, we derived a molecular docking model for Olfr43:(−)-carvoneby homology modeling and performing a mutation analysis of the re-sidues that participate in the interaction. Olfr43 possesses seventransmembrane helices and a cytoplasmic C-terminal domain (Fig. 1C).In our Olfr43 model, the disulfide bond was located between C97 in EL2and C179 at the N-terminus of TM3, which is conserved among rho-dopsin receptors. A Ramachandran plot showed that 97% (283/292) ofall the residues were in the allowed region, which indicates that ourOlfr43 receptor structure was reliable [32]. Importantly, (−)-carvonewas docked in a hydrophobic binding pocket formed by amino acidsfrom transmembrane domains III (M104, A108), V (M198, L201, V205),and VI (M257, Y258), and ECL2 (I181) (Fig. 1B). The complex modelalso indicated a hydrogen bond interaction between the carbonyloxygen of the ligand (O7) and the hydroxyl group (-OH) of Y258(Fig. 1C). To verify our model and determine whether the interactionbetween Y258 and (−)-carvone plays an important role in receptoractivation, we mutated the tyrosine (Y) residue to phenylalanine (F),which lacks an –OH group. The Y251 residue reportedly forms a hy-drogen bond with (S)-(−)-citronellal, which is important for receptoractivation [33]. Thus, we also assessed a Y251F mutant. The proteinsreferenced above were expressed in Hana3A cells and the expressionlevels of Olfr43 (WT and mutants) in cell membrane were comparable(Fig. S2). Based on the EC50 value of (−)-carvone for Olfr43 activation,we used 100 μM of (−)-carvone to induce Olfr43 activation (Fig. 1E).The CRE-luciferase results indicated that (−)-carvone significantly in-creased CREB activity in Olfr43 WT-transfected cells. Y251F Olfr43showed similar activity to that of the WT in (−)-carvone-stimulatedcells, whereas the activity of Y258F Olfr43 was significantly reduced(Fig. 1E). When stimulated with (−)-carvone, the activity of Y258FOlfr43 did not significantly induce CREB activity but its effect on CREBactivation was slightly higher than that of of the vehicle control-

Fig. 1. M257 and Y258 residues are required for activation of Olfr43 by (−)-carvone. (A) Olfr43 was expressed in mouse livers confirmed by RT-PCR (219 bp; n=3).M, mock (a RT-PCR reaction with RNA samples without reverse transcription reaction); L32, a reference gene ribosomal protein L32. (B) Model of the structure ofOlfr43:(−)-carvone. The homology model of Olfr43 is shown in wheat color and has seven transmembrane (TM) helices. The binding pocket comprises the hy-drophobic residues surrounding the (−)-carvone ligand. Y258 in TM helix VI forms a hydrogen bond with (−)-carvone. (C) A hydrophobicity simulation showed that(−)-carvone was found in the hydrophobic ligand binding pocket of Olfr43. (D), (E) CRE-luciferase activity in Hana3A cells transfected with Olfr43 wild-type (WT)or mutations or mock vector (lacking Olfr43 coding sequence) (n=3–4). Data are the mean ± SEM. Different letters indicate a significant difference at P < 0.05 byone-way ANOVA followed by Tukey's HSD test.

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stimulated cells, possibly due to disruption of the hydrogen bond be-tween Y258 and (−)-carvone but with an intact hydrophobic pocket.To determine whether the hydrophobic pocket is crucial for (−)- car-vone binding to Olfr43, M257 was mutated to alanine (A) to generateM257A Olfr43, and a M257A/Y258F double-mutant Olfr43 was alsoproduced. The M257A change resulted in loss of a hydrophobic sidechain, thereby greatly reducing the size of the hydrophobic ligandbinding pocket. Although the CREB activity of the M257A mutant wasslightly higher than that of the control, the M257A/Y258F doublemutant lacked CREB activity (Fig. 1E). The EC50 value of (−)-carvonefor Olfr43 mutants were also examined in Hana3A cell. The EC50 valuesof (−)-carvone for Y258F and M257A mutants were much higher thanthat of (−)-carvone for Olfr43 WT, and the induction of (−)-carvoneon CREB activity were abolished in M257A/Y258F double mutant.Therefore, Olfr43 activation by (−)-carvone requires M257 and Y258to activate the cAMP-PKA-CREB pathway.

3.3. (−)-Carvone administration reduces hepatic steatosis and adiposity inHFD-fed mice

As previously noted, (−)-carvone is an agonist of Olfr43 [21,29,30].To assess the metabolic function of (−)-carvone–activated Olfr43 invivo, HFD-fed C57BL/6 J WT mice were orally administered (−)-car-vone for 5 weeks. (−)-Carvone significantly reduced the body weights

of the mice but did not affect their food intake (Fig. 2A and Table S2).Liver and adipose tissues of mice in the (−)-carvone group showed anormal morphology; however, liver and adipose tissue weights weresignificantly reduced compared to those of the controls (Fig. 2B and C).Hematoxylin and eosin staining of liver sections showed a substantialreduction in hepatic lipid accumulation in mice in the (−)-carvonegroup (Fig. 2D). Hepatic and plasma triglyceride contents were alsosignificantly reduced (Fig. 2E). In contrast, the plasma cholesterol levelwas marginally affected (P=0.65), and HDL-to-LDL-cholesterol ratiowas slightly increased by (−)-carvone administration (Fig. 2F). Plasmaaspartate aminotransferase and alanine aminotransferase levels werenot different between the two groups, which suggests that (−)-carvoneadministration may not have liver toxicity (Fig. 2G). Collectively, theseresults demonstrate that (−)-carvone administration enhances hepaticlipid metabolism in HFD-fed mice.

3.4. (−)-Carvone administration improves glucose and insulin tolerance inHFD-fed mice

We next evaluated the in vivo effects of (−)-carvone on glucose andinsulin tolerance in HFD-fed mice. (−)-Carvone administration sig-nificantly improved glucose and insulin tolerance (Fig. 3A and B).Fasting plasma glucose and insulin levels were significantly decreased,and the insulin resistance index was increased, in

Fig. 2. (−)-Carvone administration reduces hepatic steatosis in HFD-fed mice. (A) Body, (B) liver tissue, and (C) adipose tissue weights in HFD-fed mice administered(−)-carvone for 5 weeks. (D) Histological analysis of the liver tissue by hematoxylin and eosin staining (×200 magnification). (E) Hepatic triglyceride and plasmatriglyceride in HFD-fed mice administered (−)-carvone for 5 weeks. (F) Plasma total cholesterol levels and HDL-to-LDL-cholesterol ratio. (G) Plasma aspartatetransaminase (AST) and alanine transaminase (ALT) levels. HFD, high-fat diet; CV20, (−)-carvone 20mg/kg body weight. Data are the mean ± SEM (n=6–7;*P < 0.05; **P < 0.01 vs. vehicle controls by Student's t-test).

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(−)-carvone–administered mice compared to the controls (Fig. 3C).These results demonstrate that (−)-carvone administration sig-nificantly improves glucose and insulin tolerance in HFD-fed mice.Taken together with the reduction in body weight and hepatic lipidaccumulation, these results suggest that (−)-carvone improves meta-bolic syndrome in HFD-fed obese mice.

3.5. Olfr43 activation by (−)-carvone attenuates hepatic lipidaccumulation via the CREB-hairy and enhancer of split 1 (HES1)-PPARγsignaling pathway and the induction of FAO

To investigate the mechanism of the reduction in hepatic lipid levelsby (−)-carvone-activated Olfr43, we investigated hepatic lipid meta-bolism signaling pathways. The administration of (−)-carvone sig-nificantly increased the phosphorylation level of CREB on Ser133 inliver tissues compared to the controls (Fig. 4A). This may have been dueto an increase in the intracellular cAMP level and subsequent activationof PKA, which is a canonical signaling pathway of ORs. CREB activatesHES1, which suppresses the expression of PPARγ in hepatocytes [34].Hes1 gene expression was significantly increased in the livers of miceadministered (−)-carvone, while Pparg gene expression was decreased(Fig. 4B). PPARγ regulates the expression of the genes encoding two keyenzymes in triglyceride synthesis: mitochondrial glycerol-3-phosphateacyltransferase (GPAM) and diacylglycerol acyltransferase (DGAT)[35,36]. In liver tissue of mice administered (−)-carvone, the mRNAlevel of Dgat2 was significantly reduced, but the level of Gpam wasunchanged, compared to the controls (Fig. 4C). Immunoblot analysisconsistently showed a significant induction in the HES1 protein level,while a significant reduction in the PPARγ and DGAT2 protein levels inthe (−)-carvone group compared to the control group (Fig. 4D).Moreover, we analyzed FAO in the HFD-fed mouse livers. The resultsrevealed that (−)-carvone administration significantly increased he-patic FAO in HFD-fed mouse livers (Fig. 4E). The level of hepatic β-hydroxybutyrate was significantly increased in the HFD-fed mouse li-vers with (−)-carvone administration (Fig. 4E), indicating stimulationof ketogenesis, potentially due to increased FAO by (−)-carvone ad-ministration. We therefore conclude that (−)-carvone administration

results in attenuation of hepatic triglyceride metabolism by modulatingthe activity of the CREB-HES1-PPARγ signaling pathway to reduce TGbiosynthesis, and inducing of hepatic FAO.

3.6. (−)-Carvone attenuates hepatic lipid accumulation by activatingOlfr43

We then investigated the effects of (−)-carvone on culturedHepa1c1c-7 cells. Olfr43 was expressed in cultured mouse hepatocytes(Fig. 5A). Consistent with the results in the livers of HFD-fed mice,Olfr43 activation by (−)-carvone activated the CREB-HES1-PPARγsignaling pathway in cultured hepatocytes (Fig. 5B and C) but had noeffect on intracellular calcium concentrations (Fig. 5D). To assess thespecific effects of (−)-carvone on hepatic lipid metabolism, we per-formed Olfr43 small interfering RNA (siRNA) experiments in culturedmouse hepatocytes. Olfr43 expression was suppressed in cultured he-patocytes transfected with Olfr43 siRNA (Fig. 5E). In Olfr43 knockdownhepatocytes, the CREB phosphorylation level, Hes1, Pparg, and Dgat2expression, the intracellular triglyceride levels and hepatic FAO wereunaffected by (−)-carvone (Fig. 5F–H). Collectively, these results de-monstrate that Olfr43 activation by (−)-carvone stimulates the CREB-HES1-PPARγ signaling pathway, which reduces triglyceride accumula-tion in the livers of HFD-fed mice.

4. Discussion

ORs are primarily expressed in the olfactory epithelium; however,many reports during the last decade have indicated that ORs are alsopresent in most mammalian tissues, and the biological functions of ORsin non-nasal tissues are now beginning to be understood. For instance,ectopic ORs mitigate airway obstruction and mediate oxygen home-ostasis [8,37]; regulate glucose homeostasis and renal blood pressure[7,38]; regulate cellular energy metabolism and obesity [9]; andmodulate cell migration, proliferation and apoptosis [39], gut motilityand secretion, and angiogenesis [40,41]. We previously reported thatOR1A1 activation by (−)-carvone reduces hepatic lipid accumulationin cultured human hepatocytes [10]. We have demonstrated that

Fig. 3. (−)-Carvone administration improves glucose and insulin tolerance in HFD-fed mice. Plasma glucose levels determined by (A) oral glucose tolerance test, and(B) intraperitoneal insulin sensitivity test in HFD-fed mice. (C) Plasma glucose, insulin levels, and insulin sensitivity indices in HFD-fed mice after oral administrationof (−)-carvone for 5 weeks. HFD, high-fat diet; CV20, (−)-carvone 20mg/kg body weight; AUC, area under the curve; HOMA-IR, homeostatic model assessment-insulin resistance. Data are the mean ± SEM (n=6–7; *P < 0.05; **P < 0.01 vs. vehicle controls by Student's t-test).

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Olfr43, a mouse homolog of OR1A1, plays a key role in the regulationof hepatic steatosis in HFD-fed mice. This result suggests that a mousehomolog of ectopic OR plays the same metabolic role in the liver asectopic ORs in humans. Activation of OR1A1 by geraniol stimulatesglucagon-like peptide-1 secretion in cultured human enteroendocrinecells [42]. Oral administration of geraniol also improves glucosehomeostasis in type 2 diabetic mice [42], which may be carried out atleast in part by Olfr43.

We investigated the functionality of ectopic ORs in hepatic lipid

metabolism based on our hypothesis that dietary aroma compoundsmay function as ligands for ectopic ORs since all odorants can bind toand activate OR signaling pathways. This hypothesis was proposedbased on the findings from natural aroma substances, such as EOs. EOsare a mixture of aroma compounds synthesized as secondary metabo-lites in plants, and these substances display diverse biological functionssuch as anti-inflammatory, antioxidant and antimicrobial abilities[43,44]. More importantly, several studies have suggested that the oralintake of EOs could trigger physiological activities such as

Fig. 4. Olfr43 activation attenuates hepatic lipid accumulation via the CREB-HES1-PPARγ signaling pathway. (A) Total CREB, phospho-CREB levels, and the p-CREB/CREB ratio, in the livers of HFD-fed mice after oral administration of (−)-carvone for 5 weeks (n= 3). (B) Hes1 and Pparg and (C) Dgat2 and Gpam expressions in thelivers of HFD-fed mice (n= 3). (D) HES1, PPARγ and DGAT2 levels in the livers of HFD-fed mice after oral administration of (−)-carvone for 5 weeks (n= 3). The α-tubulin levels in (A) and (D) were from a single control experiment. (E) Liver fatty acid oxidation and β-hydroxybutyrate (ketone body) levels in HFD-fed mice(n=6–7). HFD, high-fat diet; CV20, (−)-carvone 20mg/kg body weight. Data are the mean ± SEM (*P < 0.05; **P < 0.01 vs. vehicle controls by Student's t-test).

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hypolipidemic and anti-diabetic effects [45–47]. Thus, we hypothesizedthat dietary aroma compounds, such as (−)-carvone in EOs, mayfunction as a ligand for certain ectopic ORs in non-nasal tissue in mice.

The identification of a protein structure is critical to understandingthe characteristics of the protein and its interaction with other smallmolecules such as ligands. Although the crystal structure of GPCRs hasbeen characterized, none of the OR protein structures has yet beendemonstrated. To understand how a ligand binds to Olfr43, we per-formed in silico modeling, ligand docking, combined with site-directedmutagenesis and cell-based functional experiments. Correction of thesequence alignment between the target protein and template structuresis a crucial step in homology modeling. OR has an approximately 25%sequence identity with class A GPCR proteins; thus, homologous modelsof OR based on class A GPCRs have limited structure quality. The use of

multiple templates combining the truncation of the first flexible 20 N-terminal amino acids can reduce this ambiguity. For our research, wemodeled the structure of Olfr43 based on sequence alignments withthree active GPCR conformations using Modeller 11.1 [23] to create aprobability density function of the location of each atom in the proteinstructure. The Olfr43 (−)-carvone complex structure consisted of ahydrophobic pocket formed by portions of transmembrane domains III,V, and VI, and EL2. Eight amino acids within the presumptive bindingpocket were involved in receptor activation; overlap 50% with thosefrom Geithe et al. [30] and 75% from Man et al. [48] (Fig. 1C and D).The observation that odorant binding is dominated by hydrophobiccontacts is consistent with other ORs such as hOR1G1 [49] and mOR-EG [50]. Our model was functionally characterized and subsequentlyvalidated by point mutagenesis. The results newly showed that

Fig. 5. (−)-Carvone attenuates hepatic lipid accumulation by activating Olfr43. (A) Olfr43 was expressed in Hepa1c1c-7 cells (n= 3). M, mock (a RT-PCR reactionwith RNA samples without reverse transcription reaction); L32, a reference gene ribosomal protein L32. (B) Total CREB, phospho-CREB levels, and the p-CREB/CREBratio in Hepa1c1c-7 cells treated with (−)-carvone (n= 3). (C) Hes1, Pparg, and Dgat2 expression in Hepa1c1c-7 cells (n= 4). (D) Calcium assay in Hepa1c1c-7 cells(n=3). (E) Olfr43 expression in Hepa1c1c-7 cells transfected with Olfr43 siRNA by RT-PCR (n= 3). (F) Total CREB and phospho-CREB levels, the p-CREB/CREBratio, and (G) Hes1, Pparg, and Dgat2 expression following transfection of Olfr43 siRNA (n= 3). (H) Intracellular triglyceride levels and hepatic fatty acid oxidationfollowing transfection of Olfr43 siRNA (n=4). C, vehicle control; CV, 100 μM (−)-carvone; A-23187, 10 μM (a calcium-ionophore, positive control); Scr siRNA,scrambled siRNA; LL, lipid loading. Data are the mean ± SEM. Student's t-test or one-way ANOVA followed by Tukey's HSD test were used for comparisons of two ormultiple groups, respectively. Different letters or * indicate a significant difference at P < 0.05.

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activation of Olfr43 by (−)-carvone required the M257 and Y258 re-sidues, two amino acids in transmembrane helix domain VI. This out-come suggests that a change in the microenvironment of transmem-brane domain VI causes major disruption of the ligand binding pocket,which can negate the ligand-mediated signaling pathway. Geithe et al.also recently confirmed that (−)-carvone is a ligand for Olfr43, al-though the affinity is lower for Olfr43 than for human OR1A1 [30],which is consistent with our study.

The Olfr43 gene was highly expressed in several non-olfactory tis-sues including the liver (Fig. S1). (−)-Carvone activated CREB activityin Olfr43-overexpressed Hana3A cells, mouse livers, and cultured he-patocytes. In animal experiment, long-term oral administration of(−)-carvone reduced the body weight, and the liver and adipose tissueweights of HFD-fed C57BL/6 J mice, suggesting that (−)-carvone exertsa beneficial effect on obesity and hepatic steatosis. Importantly, trans-fection with siRNA against Olfr43 in cultured hepatocytes abrogatedthe hepatic triglyceride-lowering effect of (−)-carvone. These resultsdemonstrate that activation of Olfr43 reduces hepatic lipid accumula-tion.

Olfr43 activation stimulated by (−)-carvone induced CREB activitybut not intracellular calcium concentration, suggesting that Olfr43 mayselectively increase cAMP level and PKA activity. CREB plays criticalroles in various biological processes, including cell proliferation andhepatic lipid and glucose homeostasis [51,52]. In lipid metabolism,CREB regulates the expression of HES1 [34], which is a basic helix-loop-helix transcriptional repressor [53,54] that suppresses the ex-pression of downstream targets, such as PPARγ [34]. PPARγ is a nuclearreceptor that plays a critical role as a lipogenic transcription factoractivating transcription of key genes in glucose and lipid metabolism[55]. Treatment with thiazolidinediones, which are ligands for PPARγ,stimulates hepatic steatosis in cell culture and rodent models [56,57].And these effects were negated in liver-specific PPARγ knockout mice[58,59]. In this study, oral administration of (−)-carvone induced theexpression of Hes1, a repressor of PPARγ, leading to a reduction inPPARγ expression in the livers. This effect is of particular interest be-cause PPARγ regulates hepatic lipid metabolism by stimulating lipiduptake and lipogenesis [60].

PPARγ regulates the expression of mitochondrial GPAM and DGAT,which are associated with the outer mitochondrial membrane and theendoplasmic reticulum, respectively [35,36]. Mitochondrial GPAM,mainly expressed in hepatocytes, catalyzes the first committed step intriglyceride biosynthesis for the formation of lysophosphatidic acidfrom glycerol-3-phosphate [61]. DGAT catalyzes the transfer of an acylgroup from acyl-CoA to diacylglycerol, which is the last and committedstep in triglyceride biosynthesis [62,63]. DGAT has two forms: DGAT1and DGAT2; the latter reportedly plays a crucial role in triglyceridemetabolism in DGAT2 knockout mice [64]. In this study, (−)-carvoneadministration significantly reduced Dgat2 expression but did not affectthat of Gpam, leading to reduced hepatic lipid accumulation.

The Olfr43-dependent induction of the CREB-HES1-PPARγ signalingaxis by (−)-carvone was confirmed in Olfr43 knockdown hepatocytes.The increase in phospho-CREB and Hes1 levels and the decrease inPparg and Dgat2 expression levels were ameliorated in Olfr43 knock-down hepatocytes. These results demonstrate that (−)-carvone medi-ated activation of Olfr43 activated the CREB-HES1-PPARγ signalingpathway, which modulated hepatic lipid metabolism.

Insulin and glucose tolerance in HFD-fed mice improved after oraladministration of (−)-carvone for 5 weeks, which was confirmed by theimproved insulin sensitivity index. (−)-Carvone administration mayinduce glucagon-like peptide-1 secretion through Olfr43 to regulateglucose homeostasis, similar to OR1A1 [42], which would be an in-teresting topic for future study. The reduction in adiposity by(−)-carvone administration may also improve the insulin sensitivity ofHFD-fed mice.

The dosage of (−)-carvone (20mg/kg body weight) in mouse ex-periments was calculated using the highest daily exposure level of

(−)-carvone of 1.87mg/kg bw/day [22] and the formula for dosetranslation from human to animal study as previously reported [65]. Inhumans, (−)-carvone was reported to metabolized into several oxi-dized compounds including dihydrocarvonid acid, carvonic acid, anduroterpenolone [66], however, somewhat different metabolites weredetected in rats [67]. No data are available in mice.

There are limitations in the study. We were unable to measure theconcentrations of (−)-carvone in blood or in tissues in the presentstudy, however, it was reported that high levels of (−)-carvone wasdetected in human liver microsome [67], which suggest that (−)-car-vone in liver tissue may be sufficiently high to activate Olfr43. Phar-macokinetic quantification of (−)-carvone in blood and different tis-sues, and identification of all metabolites in vivo will be topics forfuture works. In addition, we cannot rule out the possibility that some(−)-carvone metabolites might also activate Olfr43 and this should beinvestigated in the future as well.

In conclusion, our results demonstrate that Olfr43 is ectopicallyexpressed in the cultured hepatocytes and liver of mice, where itfunctions as a non-redundant receptor regulating the CREB-HES1-PPARγ signaling axis and modulating hepatic lipid metabolism via itsresponse to the food odorant (−)-carvone. Olfr43 therefore is a po-tential target for the treatment of disorders of lipid metabolism, such ashypertriglyceridemia.

Author contributions

C.W., T.T.T., and S.J.L. designed the research; C.W., T.T.T., andY.J.K. performed the research and analyzed the data; and C.W., T.T.T.,and S.J.L. wrote the manuscript.

Conflict of interest

The authors have no conflicts of interest to declare.

Transparency document

The Transparency document associated with this article can befound, in online version.

Acknowledgments

This work was supported by a National Research Foundation ofKorea (NRF) grant (NRF-2016R1A2A2A05005483), the Basic ResearchLab Program through the NRF funded by the MSIT (NRF-2018R1A4A1022589) and a grant from Cooperative Research Program,Rural Development Administration, Republic of Korea (No.PJ011253042018).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbalip.2019.01.004.

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