Yang Sun,1 Yili Yang,2 Zhen Qin,1 Jinya Cai,3 Xiuming Guo,1 Yun Tang,3

Jingjing Wan,1 Ding-Feng Su,1 and Xia Liu1

The Acute-Phase Protein OrosomucoidRegulates Food Intake and EnergyHomeostasis via Leptin ReceptorSignaling PathwayDiabetes 2016;65:1630–1641 | DOI: 10.2337/db15-1193

The acute-phase protein orosomucoid (ORM) exhibits avariety of activities in vitro and in vivo, notably modulationof immunity and transportation of drugs. We found in thisstudy that mice lacking ORM1 displayed aberrant energyhomeostasis characterized by increased body weight andfat mass. Further investigation found that ORM, predom-inantly ORM1, is significantly elevated in sera, liver, andadipose tissues from the mice with high-fat diet (HFD)–induced obesity and db/db mice that develop obesityspontaneously due to mutation in the leptin receptor(LepR). Intravenous or intraperitoneal administration ofexogenous ORM decreased food intake in C57BL/6, HFD,and leptin-deficient ob/obmice, which was absent in db/dbmice and was significantly reduced in mice with arcu-ate nucleus (ARC) LepR knockdown, whereas enforcedexpression of ORM1 in ARC significantly decreased foodintake, body weight, and serum insulin level. Furthermore,we found that ORM is able to bind directly to LepR andactivate the receptor-mediated JAK2–STAT3 signaling inhypothalamus tissue and GT1-7 cells, which was derivedfrom hypothalamic tumor. These data indicated that ORMcould function through LepR to regulate food intake andenergy homeostasis in response to nutrition status. Mod-ulating the expression of ORM is a novel strategy for themanagement of obesity and related metabolic disorders.

Obesity is a condition marked by excess accumulation ofbody fat that results from an imbalance between calorie

intake and energy expenditure. Energy homeostasis in thebody is maintained by the integrated actions of multiplefactors (1,2), including adipose hormones (such as leptinand adiponectin), gastrointestinal hormones (such as in-sulin, ghrelin, and cholecystokinin), and nutrient-relatedsignals (such as free fatty acids). In addition to acting onperipheral tissues, these actions can also influence centralcircuits in the hypothalamus, brainstem, and limbic systemto modulate food intake and energy expenditure (1,3).Notably, the adipose tissue–produced leptin is a majorregulator of fat, and the level of leptin in circulation isproportional to body fat (4) and is a reflection of long-term nutrition status as well as acute energy balance.Furthermore, leptin deficiency or leptin receptor (LepR)mutation leads to hyperphagia, obesity, and insulin resis-tance (5), whereas administration of leptin causes weightloss and improved insulin resistance and hyperglycemia intype 2 diabetes mice (6,7). Patients with leptin deficiencyor LepR mutation also develop severe obesity (8,9). It isevident that hypothalamic LepR (10,11) is critical for leptin-mediated regulation of energy metabolism, as impairmentof LepR signaling in the hypothalamus selectively results inhyperphagia and adiposity (1,12,13).

Orosomucoid (ORM), also known as a1-acid glyco-protein (AGP), is one of the acute-phase proteins. Thereare two isoforms of ORM in human (ORM1 and ORM2),one isoform in rat (ORM), and three isoforms in mouse(ORM1, ORM2, and ORM3) (14). These genes have an

1Department of Pharmacology, School of Pharmacy, Second Military MedicalUniversity, Shanghai, China2Laboratory of Translational Medicine, Suzhou Institute of Systems Medicine,Center for Systems Medicine, Chinese Academy of Medical Sciences, Suzhou,China3Shanghai Key Laboratory of New Drug Design, School of Pharmacy, EastChina University of Science and Technology, Shanghai, China

Corresponding author: Xia Liu, lxflying@aliyun.com, or Ding-Feng Su, dfsu@smmu.edu.cn.

Received 26 August 2015 and accepted 11 March 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1193/-/DC1.

Y.S., Y.Y., and Z.Q. contributed equally to this study.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

1630 Diabetes Volume 65, June 2016

OBESITY

STUDIES

identical structure with six exons and five introns. Bothin humans and mice, constitutive level of ORM1 is muchhigher (fivefold) than ORM2, and only ORM1 can be in-duced by acute-phase stimuli (15). Although it is mainlysynthesized by the liver, many extrahepatic tissues, includ-ing adipocytes, heart, and brain, are capable of producingORM under myriad physiological and pathological condi-tions (16–19). A variety of activities have been attributedto ORM, which include modulating immunity, carryingdrugs, maintaining the capillary barrier, and mediatingsphingolipid metabolism (14,20–23). It has been reportedthat the effects of ORM on macrophages, neutrophils, andliver parenchymal cells are mediated by membrane receptorCCR5, Siglect-5, and hemoglobin b-chain, respectively(24–26). Interestingly, increase of serum ORM level hasbeen observed in obese humans, mice, and Ossabaw pigs(17,27–29). The increased level is correlated with BMI,body fat mass, serum leptin, and fasting plasma glucoselevel in human (27,30). In addition, adipose ORM level iscorrelated with adiponectin that regulates glucose leveland fatty acid breakdown and is regulated by insulin,high glucose, and free fatty acid in differentiated adipo-cytes (17,27). These results suggested that ORM mightparticipate in the regulation of energy balance.

In this study, we found alterations of energy homeo-stasis in mice deficient of ORM1, which accounts for themajority of serum ORM as well as most of the changesinduced by acute-phase stimuli (15,31). The aberrant en-ergy homeostasis is characterized by significant elevationin body weight and fat mass, increased serum total cho-lesterol (TC), fatty liver, and insulin and leptin resistance.We also found that ORM derived from adipose and livertissues is regulated by short- or long-term nutrition sig-nals and administration of ORM affects feeding behavior.Furthermore, we demonstrate that ORM binds to LepRand activates the Janus kinase (JAK)2–signal transducerand activator of transcription (STAT)3 pathway in hypo-thalamus. Thus, ORM could function as an agonist forLepR and is an important regulator in food intake andenergy homeostasis.

RESEARCH DESIGN AND METHODS

ReagentsORM was purchased from Sigma-Aldrich (St. Louis, MO).BSA was obtained from Boguang Biological Technology(Shanghai, China). IgG was from Beyotime Institute of Bio-technology (Shanghai, China). Fluorescein isothiocyanate(FITC)-labeled ORM and BSA were made by Youke BiologicalTechnology (Shanghai, China). Antibodies against ORM (rat)and LepR were purchased from Abcam (Cambridge, U.K.).Antibodies against JAK, phosphorylated (p)-JAK, STAT3,and p-STAT3 were from Cell Signaling Technology (Dan-vers, MA). Antibody against ORM (mouse) was obtainedfrom Genway (San Diego, CA). Antibodies against GAPDHand tubulin were from Beyotime Institute of Biotechnology.Secondary antibodies conjugated with IRDye 800CW werefrom Rockland Immunochemicals, Inc. (Limerick, PA). The

LepR small interfering RNA and its control small interferingRNA were from Santa Cruz Biotechnology (Dallas, TX).Lentivirus carrying full-length ORM1 or LepR short hairpin(sh)RNA was constructed by Shanghai GenePharma Co.,Ltd. (Shanghai, China). The sequence used for LepR shRNAis 59-GCTGAAATTGTCTCAGCTACA-39. The 60% high-fatdiet (HFD) and standard chow were purchased from ShanghaiSLAC Laboratory Animal Co., Ltd. (Shanghai, China).

Cell Culture and TransfectionMouse hypothalamic GT1-7 cells were generously pro-vided by Professor Xiao-Ying Li from the Shanghai ClinicalCenter for Endocrine and Metabolic Diseases at ShanghaiJiaotong University School of Medicine (Shanghai, China).C2C12 cells (mouse muscle myoblasts) were obtained fromShanghai Institutes for Biological Sciences, Chinese Acad-emy of Sciences. These cells were cultured in DMEM (Gibco)supplemented with 10% FBS (Gibco). All cells were in-cubated at 37°C in a 5% CO2 incubator. For knockdownstudies, these cells were transfected with Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufac-turer’s instructions.

AnimalsEight-week-old male db/db and ob/ob mice were purchasedfrom Shanghai SLAC Laboratory Animal Co., Ltd. MaleC57BL/6 mice (18–22 g) and Sprague-Dawley rats (180–200 g) were purchased from Sino-British SIPPR/BK Lab-oratory Animals (Shanghai, China). ORM1 knockout micewere generated as previously described (32) and werebackcrossed 10 times with C57BL/6 mice before theywere characterized. All animal experiments were under-taken in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals and withthe approval of the Scientific Investigation Board of theSecond Military Medical University.

RNA QuantificationTotal RNA was extracted with TRIzol reagent (Invitrogen)following manufacturer’s instructions. Real-time quanti-tative RT-PCR analysis was performed using the SYBR RT-PCR kits (Takara, Otsu, Japan). Primer sequences wereshown in Supplementary Table 1.

Blood ParametersSerum levels of ORM, leptin, and insulin were detected byELISA kit according to the manufacturer’s instruction. Arat ORM ELISA kit was obtained from Abcam. A leptinELISA kit was bought from R&D Systems (Minneapolis,MN). An insulin ELISA kit was bought from Millipore(Billerica, MA). Total plasma cholesterol and triglyceride(TG) were measured by the Clinical Biochemical Labora-tory in Changhai Hospital (Shanghai, China).

Food IntakeThe effect of ORM on eating behavior was evaluated bythe amount of food intake. For fasting-induced foodintake, mice were starved overnight (no drinking limited),and the weight of consumed food at 2 h, 8 h, and 24 h wasrecorded 30 min after tail vein injection of vehicle or

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ORM (100 mg/kg). For spontaneous food intake, micewere intraperitoneally injected with vehicle or ORM (50mg/kg/day) for 4 or 7 consecutive days as indicated, andthe weight of daily consumed food was recorded.

Oil Red O StainingFrozen 20-mm-thick mouse liver sections were fixed in 4%paraformaldehyde. After removal of formalin and washingagain with PBS, slides were incubated with Oil Red Oworking solution (Sigma-Aldrich) at room temperaturefor 10 min. Then, slides were differentiated in 60% eth-anol solution and rinsed three times with distilled water,stained in hematoxylin for 30 s, and washed thoroughly.Photos were taken under the optical microscope (Olym-pus, Tokyo, Japan).

Proximity Ligation AssayGT1-7 cells or C2C12 cells were treated with ORM(10 mg/mL) or vehicle for 3 h and fixed with 4% paraformal-dehyde. Cells were incubated with primary antibodiesagainst ORM (1:300; rabbit, Genway) and LepR (1:50;goat, Santa Cruz Biotechnology, Santa Cruz, CA) overnight.Proximity ligation assay (PLA) was performed using theDuolink in situ PLA kit (Olink Bioscience, Uppsala, Sweden)with PLA PLUS or MINUS probes for rabbit or goatantiserum. The nuclei of cells were stained using DAPI(Olink Bioscience).

Immunoblotting and ImmunoprecipitationCells and tissues were lysed with Cell & Tissue ProteinExtraction Reagent (Kangchen, Shanghai, China) supple-mented with a protease inhibitor mixture (Kangchen). Forimmunoblotting, cell and tissue lysates (30–50 mg pro-tein) in the supernatant were separated by SDS-PAGEand transferred to nitrocellulose membranes. For immu-noprecipitation, cell or tissue lysates (100–200 mg) wereincubated with 2 mg either polyclonal antibody againstORM (Abcam) or polyclonal LepR antibody (Abcam) cou-pled to protein G–sepharose (Invitrogen) overnight at 4°C.Immunoprecipitates were washed and separated by SDS-PAGE and transferred to nitrocellulose membranes. Themembranes were then probed for specific proteins.

ImmunohistochemistryImmunohistochemistry was carried out according to thestandard protocol using the slides from brain tissues. Afterovernight incubation at 4°C with the anti-ORM antibody(1:50; rat, MyBioSource) or anti-LepR antibody (1:50; goat,Santa Cruz Biotechnology), the slides were washed and in-cubated with Cy3-conjugated anti-rat IgG or Alexa Fluor488–conjugated anti-goat IgG (Jackson ImmunoResearch)for 2 h at room temperature. After extensive washing, thenuclei were stained with DAPI. Negative controls were runconcurrently, except that the antibody dilution buffer wasused to substitute the primary antibody.

Flow CytometryThe interaction of ORM with the surface of GT1-7 cellswas analyzed using flow cytometry. Briefly, cells wereblocked with 5% BSA and incubated with FITC-conjugated

BSA, FITC-conjugated ORM (10 mg/mL), FITC-conjugatedORM in combination with LepR antibody (mouse-blockingpeptide, 10 mg/mL) (Alpha Diagnostic International, SanAntonio, TX), or FITC-conjugated ORM in combinationwith leptin (10 mg/mL; Abcam) for 1 h and washed withPBS three times and then analyzed by flow cytometry.

Stereotaxic Lentivirus InjectionMale C57BL/6 mice (25–30 g) were fixed in an ALC-Hmotorized digital stereotaxic instrument (Shanghai AlcottBiotech Co., Ltd., Shanghai, China). Lentiviruses express-ing ORM1, shRNA silencing LepR, or their correspondingcontrols were injected into arcuate nucleus (ARC) (bilater-ally, 0.5 mL/side; 109 transduction units/mL) with microlitersyringes (Hamilton Company, Reno, NV). The coordinatesfor lateral ventricular injection were as follows: anterior-posterior (from bregma), –2 mm; dorsal-ventral (from skullsurface), –6 mm; and medial-lateral, 0.24 mm. All the micewith ARC injection were examined via immunofluorescencemicroscopy to verify the correct placement after experi-ments were completed. Virus in ARC occasionally spreadover other nucleus (usually ventromedial nucleus) in thehypothalamus. Data from mice with proven injection inARC were collected.

Intraperitoneal Glucose Tolerance TestingIntraperitoneal glucose tolerance testing was performedaccording to the protocol recommended by the AnimalModels of Diabetic Complications Consortium. Mice werefasted for 6 h by removal to a clean cage without food atthe end of their dark (feeding) cycle. After 6 h of fasting,the basal blood glucose derived from tail venous blood wasdetected. Then 2 mg/g body wt i.p. glucose was injected.Blood samples were collected at 15, 30, 60, and 120 minpostinjection to measure blood glucose levels using anOneTouch Ultra glucometer (LifeScan, Milpitas, CA).

Molecular ModelingThe model of the leptin-binding domain of mouse LepR(LepR-LBD) was built by the homology modeling protocolin Discovery Studio, version 3.5 (Accelrys, San Diego, CA).The X-ray crystal structure of human LepR-LBD (33) wasobtained from the Protein Data Bank (34) (PDB) (PDBentry code 3V6O) and used as the template for modelbuilding. They share ;75.7% of sequence identity, andgood superimposition was obtained between the crystalstructure and the constructed model (Supplementary Fig.1A and B). In total, 20 models were generated. The bestmodel verified by MODELER and Profiles-3D (Supplemen-tary Fig. 1C) was chosen as the mouse LepR-LBD struc-ture. The X-ray crystal structure of human ORM (i.e.,AGP) was taken from PDB (PDB entry code 3KQ0) (35).Both structures were then prepared with the ProteinPreparation Wizard workflow in Maestro, version 9.3(Schrödinger, L.L.C., New York, NY). Docking of LepR-LBD with ORM was performed using ZDOCK, version2.3 (36). Two thousand outputs of the ORM poses weregenerated for each docking run. All poses were clustered

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based on an all-against-all root mean square deviationmatrix.

Statistical AnalysisData are presented as means 6 SEM. Statistical analyseswere performed using GraphPad Prism. Two-tailed Stu-dent t tests were used to compare two distinct groups.One-way ANOVA followed by Bonferroni test was used tocompare more than two groups. Interactions were ana-lyzed using two-way ANOVA followed by Bonferroni test.P , 0.05 was considered statistically significant.

RESULTS

ORM1-Deficient Mice Display Alteration of EnergyHomeostasisIn previous study, we generated a strain of ORM1-deficientmice to explore the biological function of ORM1 (Supple-mentary Fig. 2A–D). Interestingly, they had a significantlyincreased serum insulin level at the age of 4 weeks com-pared with wild-type littermates, although these mice hadsimilar serum leptin, TC, TG, body weight, fat mass, andglucose tolerance (Supplementary Fig. 2E–K). When ex-amined at the age of 24 weeks, ORM1-deficient miceshowed markedly elevated serum levels of insulin andleptin (Fig. 1A and B), indicatives of insulin and leptinresistance. TC was also increased in these mice, whereas TGlevels were not significantly different between wild-type

and ORM1-deficient mice (Fig. 1C and D). Of note, com-pared with wild-type mice, ORM1-deficient mice had asignificant increase in fat mass (1.32 6 0.20 g vs. 2.27 60.27 g, P , 0.05) and a mild increase in body weight(31.3 6 0.75 g vs. 34.6 6 1.05 g, P , 0.05) (Fig. 1E andF and Supplementary Fig. 2L and M). While there wereno increases in the weights of heart, kidney, pancreas,and spleen (Supplementary Fig. 2L), the weight of liverwas markedly raised, accompanied by increased adiposedeposition (Fig. 1G). In addition, ORM1-deficient miceshowed impaired glucose tolerance (Fig. 1H). Therefore,deficiency of ORM1 in mice impaired energy homeostasis,which prompted us to further investigate the mechanismsand potential physiological and pathological significanceof the ORM1 action.

ORM Expression Is Elevated in the Obese MiceTo further clarify the relationship between ORM andobesity, we examined ORM expression in mice with HFD-induced obesity and in LepR-defected db/db obese mice.As shown in Fig. 2, these mice had significantly elevatedserum levels of leptin and insulin (Fig. 2A–D) as well asincreased ORM levels in serum, liver, and subcutaneousadipose tissues (Fig. 2E–J). It is worth noting that theanti-ORM antibody we used for immunoblotting cannotdistinguish the three murine ORM isoforms (ORM1 andORM2 were single polypeptide chains of 207 amino acids

Figure 1—ORM1-deficient mice display the disturbance of energy homeostasis. A–E: Serum level of insulin (A), leptin (B), TC (C), TG(D), and mass of adipose tissues (E ) in 24-week-old male ORM1+/+ (n = 5) or ORM12/2 mice (n = 11). Mass of adipose tissues was the sumof inguinal fat, perirenal fat, mesenteric fat, and epididymal fat. F: Body weight in 24-week-old male ORM1+/+ (n = 13) or ORM12/2 (n = 20)mice. G and H: Representative hematoxylin-eosin (HE) and Oil Red O staining of liver (G) and blood glucose level after intraperitonealglucose tolerance testing (H) in 24-week-old male ORM1+/+ (n = 5) or ORM12/2 (n = 11) mice. Bar = 100 mmol/L. Data are presented asmeans 6 SEM. *P < 0.05, **P < 0.01 vs. ORM1+/+ mice by Student t test.

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each, and ORM3 contains 206 amino acids). Real-time PCRwas then used to determine the level of each isoform. Asshown in Fig. 2K and L, ORM1 is the most abundant

isoform that is likely responsible for the majority of ORMincrease in the liver and subcutaneous adipose tissues(Fig. 2M–P). Interestingly, it was shown that HFD feeding

Figure 2—ORM expression is elevated in obese mice. A and B: Serum level of leptin (A) and insulin (B) in C57BL/6 mice fed with chow dietor 60% HFD for 7 weeks (n = 10 per group). C and D: Serum level of leptin (C) and insulin (D) in C57BL/6 or db/db mice (n = 8 per group).E–G: Representative of Western blot of ORM in serum (E), liver (F ), and subcutaneous adipose tissue (SAT) (G) derived from control or HFDmice treated as described for A and B (n = 10 per group). H–J: Representative of Western blot of ORM in serum (H), liver (I), and SAT(J) derived from C57BL/6 or db/db mice (n = 8 per group). K and L: Quantitative PCR detection of relative ORM1, ORM2, or ORM3 mRNAsin liver (K) or subcutaneous adipose tissue (L) derived from C57BL/6 mice (n = 6). The mRNA level of ORM1 was calculated as 100%.M andN: Quantitative PCR detection of relative ORM1, ORM2, or ORM3 mRNAs in liver (M) or SAT (N ) derived from control or HFD mice (n = 6 pergroup). The mRNA level of ORM isoform in control mice was calculated as 100%. O and P: Quantitative PCR detection of relative ORM1,ORM2, or ORM3 mRNAs in liver (O) or SAT (P) derived from C57BL/6 or db/db mice (n = 6 per group). The mRNA level of ORM isoform inC57BL/6 mice was calculated as 100%. For E and H, the Ponceau S–stained blot was used as the loading control. Data are presented asmeans 6 SEM. For A–D and M–P, *P < 0.05, **P < 0.01 by Student t test. For K and L, *P < 0.05, **P < 0.01 by one-way ANOVA withBonferroni test. Con, control; n.s., not significant.

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caused significant increases in both ORM1 and -2 mRNAlevels in fat tissue without significant changes in hepaticORM1 and -2 mRNA levels (17). The differences in HFDfeeding period (7 days in the report vs. 7 weeks in ourstudy) and fat tissue source (epididymal fat tissue in thereport vs. subcutaneous fat tissue in our study) might beresponsible for the inconsistency. Furthermore, we foundthat ORM was widely expressed in the brain includinghypothalamic area as detected by an antibody that recog-nizes all forms of ORM (Supplementary Fig. 3A). Isoform-specific quantitative PCR revealed that ORM2 is the majorisoform (Supplementary Fig. 3B) in hypothalamus, and theexpression of ORMs in the hypothalamus tissue did notchange in db/db mice or in response to HFD (Supplemen-tary Fig. 3C and D).

ORM Responds to Acute Nutritional StatusShort-term acute nutritional changes (24-h fasting and2-h refeeding) are known to affect serum leptin andinsulin levels independent of adiposity (37–39), whichillustrates how the body maintains energy homeostasis.We used this model to explore the potential role of ORMin the process. Similar to leptin and insulin, serum ORMlevel significantly decreased in fasted animals, and itincreased markedly after refeeding in both mice and rats(Fig. 3A–C and Supplementary Fig. 4). The level of ORMwas further assessed in tissues. As shown in Fig. 3D, whileORM expression in liver showed a pattern of changesimilar to that of serum, the amount of ORM in sub-cutaneous adipose tissue and hypothalamus tissue (Fig.3E and Supplementary Fig. 3E) did not have such a pat-tern. These data support the notion that liver is respon-sible for producing ORM under an acute nutritionalalteration condition.

ORM Decreases Food Intake Dependent onHypothalamic LepRThe close association of ORM and energy homeostasisprompted us to ask whether ORM is involved the regula-tion of feeding behavior. We examined the effect of purifiedORM on food intake in C57BL/6 mice. Tail vein injection ofORM significantly decreased fasting-induced food intakeat 2 h, 8 h, and 24 h (Fig. 4A). Intraperitoneal injection ofORM for consecutive 4 days also markedly reduced spon-taneous daily food consumption (Fig. 4B). Interestingly,these effects were still found in HFD-induced obese miceand ob/ob obese mice that are insulin and/or leptin re-sistant (Fig. 4C–F), whereas they were not present inLepR-defected db/db mice (Fig. 4G and H), indicatingthe involvement of LepR in the process. The ARC of thehypothalamus plays a pivotal role in the integration ofsignals regulating appetite. The ARC lies in close proxim-ity to the median eminence that lacks a complete bloodbrain barrier (40) and thus is uniquely placed to respondto circulating hormonal signals. It has been shown thatthe ARC is essential for the regulation of energy balancethrough LepR (1,41). We therefore asked whether block-ade of LepR signaling in hypothalamic ARC affects theaction of ORM on energy homeostasis. Lentivirus encodingshRNA targeting LepR (shLepR) or control was injectedinto hypothalamic ARC stereotaxically (SupplementaryFig. 5A–C). Seven days later, as expected, LepR knock-down in ARC resulted in a significant increase in fasting-induced food intake and spontaneous daily food intake(Fig. 4J and K). While ORM administration still reducedfasting-induced food intake significantly at indicatedtimes in mice injected with control lentivirus, its effectwas significantly attenuated in mice treated with shLepR(Fig. 4I). ORM-induced reduction in spontaneous daily

Figure 3—ORM responds to acute nutritional status in mice. A and B: Serum leptin (A) and insulin (B) level in C57BL/6 mice after fasting(starving) for 24 h and refeeding for 2 h (n = 6 per group). C–E: Representative of Western blot of ORM in serum (C), liver (D), andsubcutaneous adipose tissue (SAT) (E ) in C57BL/6 mice treated as described in A and B (n = 6 per group). The Ponceau S–stained blotwas used as the loading control for serum samples. Data are presented as means 6 SEM. *P < 0.05 by one-way ANOVA with Bonferronitest. con, control.

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food intake and serum insulin level was also reversedwith LepR knockdown in ARC (Fig. 4J and K). Addition-ally, injection of lentiviral vector LV-ORM1 into ARC

to overexpress ORM1 (Supplementary Fig. 5D and E)resulted in a significant decrease in food intake com-pared with control mice (Fig. 4L), and this effect lasted

Figure 4—ORM decreases food intake dependent on hypothalamic LepR. A, C, E, and G: Fasting-induced food intake in C57BL/6 (A) (n = 10per group), HFD (C) (n = 12–13 per group), ob/ob (E) (n = 10 per group), or db/db (G) (n = 7 per group) mice after tail vein injection with vehicle(control) or ORM (100 mg/kg). B, D, F, and H: Spontaneous food intake in C57BL/6 (B) (n = 10 per group), HFD (D) (n = 12–13 per group), ob/ob(F) (n = 8 per group), or db/db (H) (n = 5–7 per group) mice treated with intraperitoneal injection of vehicle or ORM (50 mg/kg/day) for4 consecutive days. I–K: ARC was injected with lentivirus carrying shLepR or matched control (sh-control) in C57BL/6 mice. One week later,100 mg/kg/day ORM was tail vein injected and fasting-induced food intake was evaluated (I) (n = 10 per group) or 50 mg/kg/day i.p. ORMwas injected for 7 consecutive days and spontaneous food intake in the last 4 days was evaluated (J) (n = 8–10 per group) and correspondingserum insulin level was detected after the experiment was over (K) (n = 8–10 per group). L–N: ARC was injected with LV-ORM1 or matchedcontrol (LV-GFP) in C57BL/6 mice (n = 12 per group). Weekly food intake (L), body weight (M), and serum insulin level (N) were then observed.Data are presented as means 6 SEM. For A–H and L–N, *P < 0.05, **P < 0.01 by Student t test. For I–K, two-way ANOVA with Bonferronitest was adopted: *P < 0.05, **P < 0.01; sh-control+ORM vs. sh-control, #P < 0.05, ##P < 0.01; shLepR+ORM vs. shLepR, $P < 0.05,$$P< 0.01. Interaction for I: 8 h (F = 134.1, P< 0.01), 24 h (F = 12.59, P< 0.01); interaction for J: 1 day (F = 4.72, P< 0.05), 2 day (F = 10.79,P < 0.01), 3 day (F = 4.79, P < 0.05), 4 day (F = 5.78, P < 0.05); interaction for K: F = 9.74, P < 0.01. n.s., not significant.

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up to 6 weeks. Meanwhile, body weight of these mice andtheir serum insulin level began to decrease 2 weeks afterLV-ORM1 injection (Fig. 4M and N). These data indicatedthat ORM acts on hypothalamus to regulate food intake ina LepR-dependent manner. Despite its effect in reducingfood intake, 4 days of ORM treatment did not affect bodyweight, serum insulin, TC, or TG levels in HDF or ob/obmice (Supplementary Fig. 6). Since these two obese animalmodels have insulin and/or leptin resistance, it is likelythat longer time of ORM administration is needed to fur-ther significantly change energy homeostasis.

ORM Specifically Binds to the LepR in HypothalamusWe then examined whether there exists a direct interactionbetween ORM and LepR. From rat hypothalamus tissue, anORM/LepR complex could be coimmunoprecipitated, espe-cially after ORM tail vein injection (Fig. 5A). Use was alsomade of PLAs, which showed that the ORM and LepRinteract on membrane of GT1-7 cells (Fig. 5B). Further-more, flow cytometry analysis found that FITC-labeledORM bound to GT1-7 cell membrane and the binding wascompletely inhibited by a LepR-blocking peptide (Fig. 5C).Taken together, these data demonstrated that ORM in-teracts directly with LepR. Intriguingly, this binding wasnot competitively blocked by leptin (Fig. 5D). Structuralstudies have indicated that leptin binds to LepR-LBD,likely in the middle of the LBD’s outer side (33). Basedon molecular docking, we predicted that the binding site

of ORM is in the middle of the LBD’s inner side (Fig. 5E),which may explain the lack of competition between ORMand leptin. Four clusters were further identified as the mostpossible binding poses out of a total 2,000 output from thedocking (Supplementary Table 2). Among them, clusters 1and 7 provided us with the detailed interactions betweenORM and LepR-LBD (Supplementary Fig. 7A and B). Theywill be used as basic models for further mutational analysis.

In mice and humans, only the long isoform of LepR(LepR isoform b) has an elongated intracellular domaincoupled to downstream signaling cascades (42). AlthoughLepR isoform b is primarily expressed in hypothalamic regionsand ARC (11), it can also be detected in a broad range of othercell types in line with the pleiotropic effects of leptin in pe-ripheral tissues (43). We further observed the interaction ofORM and LepR in rat muscle and mouse myoblast C2C12 cellsand achieved similar results (Supplementary Fig. 7C and D).

ORM Acts as an Agonist for LepR to Activate ItsDownstream JAK2-STAT3 Pathway in HypothalamusIt has been shown that binding of leptin to LepR activatesthe associated JAK2 tyrosine kinase that phosphorylatesLepR on tyrosine residues (44,45), which in turn leads tothe recruitment and phosphorylation of STAT3. Disrup-tion of the STAT3-binding site on LepR resulted in hyper-phagia and obesity (46). Therefore, activation of STAT3 isa critical event for LepR-mediated signaling and is a com-monly used marker. As shown in Fig. 6, ORM treatment

Figure 5—ORM specifically binds to the LepR in hypothalamus. A: Representative coimmunoprecipitation assay of the interaction of ORMand LepR in the hypothalamic lysates of rats treated with control or tail vein injection of 70 mg/kg ORM for 30 min (n = 3). IB, immuno-blotting; IP, immunoprecipitation. B: Representative immunofluorescence images of mouse hypothalamic cell line GT1-7 treated withvehicle or 10 mg/mL ORM for 3 h (n = 3). Each red spot represents an ORM-LepR interaction. Nuclei were stained with DAPI. Bar = 20mmol/L. C: Representative histogram of hypothalamic GT1-7 cells incubated with FITC-labeled BSA or FITC-labeled ORM (10 mg/mL) in theabsence or presence of IgG or anti-LepR antibody (10 mg/mL) (n = 3). D: Representative histogram of hypothalamic GT1-7 cells incubatedwith FITC-labeled BSA or FITC-labeled ORM (10 mg/mL) in the absence or presence of leptin (10 mg/mL) (n = 3). E: Top poses in largeclusters of ORM within LepR-LBD. Red balls indicate the highest value poses of ORM.

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Figure 6—ORM acts as an agonist for LepR to activate its downstream JAK2-STAT3 pathway in hypothalamus. A–C: RepresentativeWestern blot of p-STAT3/STAT3 and p-JAK2/JAK2 in the hypothalamus of C57BL/6, HFD, ob/ob or db/db mice treated as described forFig. 4B, D, F, and H (n = 6 per group). D: Representative Western blot of p-STAT3/STAT3 and p-JAK2/JAK2 in the hypothalamus of micetreated as described for Fig. 4J (n = 6 per group). E–G: Representative Western blot of p-STAT3/STAT3 and p-JAK2/JAK2 in mouse GT1-7hypothalamic cell line treated with the indicated doses of ORM for 3 h (E) (n = 3), 10 mg/mL ORM for the time indicated (F) (n = 3), or 10 mg/mLORM or vehicle for 3 h in the presence or absence of LepR small interfering RNA (siRNA) (G) (n = 3). Western blots are representative of threeindependent experiments. Quantification data were shown as a ratio of phosphorylated protein to total protein, and the ratio of vehicletreatment in each group was regarded as 100%. Data are presented as means6 SEM. For A and B, *P < 0.05, **P< 0.01 by Student t test.

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induced a significant increase in the phosphorylation ofJAK2 and STAT3 in the hypothalamus of C57BL/6, HFD,and ob/ob mice (Fig. 6A and B), which was largely atten-uated in the db/db mice (Fig. 6C) and in the mice withARC LepR knockdown (Fig. 6D). In vitro, ORM treatmentinduced dose- and time-dependent JAK2/STAT3 phos-phorylation in GT1-7 cells (Fig. 6E and F), and the effectswere inhibited by LepR knockdown (Fig. 6G and Supple-mentary Fig. 8A). Similarly, the dose- and time-dependentincrease of JAK2/STAT3 phosphorylation induced byORM in myoblast C2C12 cells was largely blocked whenLepR was knocked down (Supplementary Fig. 8B–E).

DISCUSSION

A variety of biological activities have been attributed toproteins of the ORM family. It has been shown that cellmembrane proteins CCR5, Siglect-5, and HBB can bindwith ORM and mediate its action in vitro (24–26). However,the signal transduction pathways for these interactionsand their biological significance in vivo have not beenfurther explored. In the current study, we showedthat ORM interacts with LepR specifically both in centraland peripheral tissues and activates its downstreamJAK-STAT3 signal pathway. We also found that ORM1-deficiency resulted in changes of energy homeostasis,characterized by adiposity, hyperleptinemia, hyperin-sulinemia, hypercholesterolemia, and impaired glucosetolerance, whereas administration of exogenous ORM, bothcentrally and peripherally, reduced food intake and bodyweight and improved insulin resistance in a LepR-dependentmanner. These data indicated that ORM could act as anagonist for LepR. Similar to leptin, ORM activates LepRto regulate food intake and energy homeostasis. Inter-estingly, the data also raised the question of why ORMbinds to multiple cell membrane receptors. As suggestedby the finding that the interaction with CCR5 was par-tially due to its glycosylation (24), it is likely that theheavy glycosylation of ORM might also contribute to itsbinding with LepR and other receptors.

The “negative feedback” model of energy homeostasishas been well established. Circulating signals inform thebrain of changes in body fat mass. In response to thisinput, the brain mounts adaptive adjustments of energybalance to stabilize fat storage. Leptin and insulin are twowell-known negative feedback signals that circulate at lev-els proportional to body fat content and can act on thebrain to promote weight loss (1). Our data showed thatORM is regulated by nutritional conditions and activatesthe LepR pathway in the hypothalamus to affect foodintake, indicating that it could be another negativefeedback signal and could play an important role in energy

homeostasis (Fig. 7). Because of the existence of hypotha-lamic ORM, the question of whether central ORM isalso important in the regulation of metabolism wasraised. We found that ORM2, not ORM1, is the majorisoform of ORM in hypothalamus and that there was noresponse of central ORM to short- or long-term nutri-tional signals, indicating that it may not be responsiblefor ORM-induced change of energy homeostasis underthese conditions.

In the present study, we found that in addition to thefat tissue, liver is also the main organ sensing long-termnutritional state, such as HFD and obese, to produceORM. ORM is a dual signal both from adipose tissue andfrom gastrointestinal tract. Interestingly, it is the liver,not the fat tissue, that sensed a short-term nutritionalsignal, with ORM decreased after the fasting and in-creased after refeeding. The ORM derived from gastro-intestinal tract is more important for the regulationof food intake in response to acute nutritional signals.This may be related to the specific regulation of liver inprotein production. The liver can respond to various

For E and F, *P < 0.05, **P < 0.01 vs. control by one-way ANOVA with Bonferroni test. For C, D, and G, *P < 0.05, **P < 0.01 by two-wayANOVA with Bonferroni test. Interaction for C: p-STAT3/STAT, F = 443.63, ##P < 0.01, and p-JAK2/JAK2, F = 443.1, ##P < 0.01; in-teraction for D: p-STAT3/STAT3, F = 287.1, ##P < 0.01, and p-JAK2/JAK2, F = 41.48, ##P < 0.01; interaction for G: p-STAT3/STAT3,F = 106.7, ##P < 0.01, and p-JAK2/JAK2, F = 84.32, ##P < 0.01. n.s., not significant.

Figure 7—Proposed working model for ORM in anorexigenicsignaling in the hypothalamus. Long- and short-term nutritionalstate (such as obese, HFD, and nutritional changes) could affectORM expression, mainly ORM1, in metabolic organs such as liverand fat as well as circulation level. Circulating ORM acting as anendogenous agonist for LepR could negatively regulate food in-take and energy balance via a LepR/JAK2/STAT3 pathway in thehypothalamus.

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stresses and induce the quick production of acute-phaseproteins, whereas ORM is one of the acute-phase proteinsmainly produced by the liver (47). In addition, liver he-patic nuclear bile acid receptor farnesoid X receptor (FXR)may be involved in the quick regulation of ORM in re-sponse to an acute nutritional signal. FXR regulates bileacid homeostasis, while bile acid secretion is affected byimmediate food intake (48). ORM is reported to be adirect target gene of hepatic FXR, and hepatic FXR de-letion in mice affects the expression of ORM (48). Fast-ing and refeeding may promptly affect bile acid secretionand therefore decrease or increase ORM expression inliver via FXR. Interestingly, vertical sleeve gastrectomy(VSG) is at present the most effective therapy for thetreatment of obesity, while the mechanism remains largelyunclear. Recently, bile acids and FXR signaling have beenreported to be important molecular targets for the effectsof VSG. In the absence of FXR, the ability of VSG to reducebody weight and improve glucose tolerance is substantiallyreduced (49). Whether ORM, the downstream target of FXR,is involved in the role of VSG is worthy of being explored.

It should be noted that although ORM-induced phos-phorylation of STAT3 was largely impaired when LepRdeficient or knockdown, there still exists the activationof the STAT3 pathway, suggesting the involvement ofother unknown receptor(s). Evidences showed that ORMmight bind to the macrophages via CCR5 (24), which isalso expressed in the hypothalamus and skeletal muscle.As a member of the cytokine receptors, CCR5 activationalso results in STAT3 phosphorylation in T cells (50). Inaddition, CCR5 deficiency suppressed lung tumor develop-ment through the inhibition of nuclear factor-kB/STAT3pathways (51). Therefore, the remaining phosphoryla-tion of STAT3 triggered by ORM might be the resultof CCR5 signaling. Interestingly, while we found that theeffect of ORM on decreased serum insulin levels (mostlikely the result of reducing food intake and weight loss)is LepR dependent, Lee et al. (17) reported that ORMimproved glucose tolerance (oral glucose tolerance test)and increased the sensitivity to insulin in decreasing bloodglucose (insulin tolerance test) in db/db mice. These resultsindicate that ORM affects insulin action at least at twolevels, controlling food intake and body weight with sub-sequent improvement of insulin resistance via the centralLepR pathway and increasing peripheral insulin sensitivityin a LepR-independent pathway. Additional receptors suchas CCR5 may also be involved in this process, since it wasreported that CCR5 activation enhanced glucose uptake inactivated T cells, and our previous studies found that ORMincreased muscle glycogen content via the CCR5 pathway(32,52). One of our ongoing efforts is analyzing the ORM1and LepR interaction through site-directed mutagenesis.

ORM also showed a variety of functions in peripheraltissues. It has been reported that ORM1 increases glucoseuptake activity in 3T3-L1 adipocytes and relieves hyper-glycemia-induced insulin resistance as well as tumor necrosisfactor-a–mediated lipolysis in adipocytes (17). Accordingly,

ORM improved glucose and insulin tolerance in obeseand diabetic db/db mice and could protect adipose tissuefrom excessive inflammation and subsequent metabolicdysfunction (17). We further found that ORM also inter-acted with LepR in peripheral skeletal muscle and acti-vated the JAK2-STAT3 signaling. Skeletal muscle, liver, andadipose tissue are the tissues with great metabolic activityand may constitute important targets for ORM in periph-eral tissue and be involved in the regulation of insulinsensitivity as well as glucose and lipid metabolism. Thefunction of ORM in peripheral tissues needs further inves-tigation. Additionally, it has been shown that the level ofORM2 is increased under certain stress conditions (19).Therefore, the function of central ORM and other isoformsof ORM, which are not found to be affected by nutritionalsignals, remains to be further investigated.

In summary, we found that the expression of ORM,one of the acute-phase proteins, is regulated by long- orshort-term nutritional signals and that ORM affectsfood intake and energy homeostasis through activatingthe LepR pathway in the hypothalamus. Thus, it is anegative feedback molecule in energy homeostasis and anovel target for the management of obesity and relatedmetabolic disorders.

Acknowledgments. The authors thank Professor Xiao-Ying Li of ShanghaiClinical Center for Endocrine and Metabolic Diseases, Shanghai Jiaotong UniversitySchool of Medicine (Shanghai, China) for the hypothalamic GT1-7 cells.Funding. This study was supported by grants from the National NaturalScience Foundation of China (81230083 to D.-F.S. and 81273606 and 81473259to X.L.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Y.S. wrote the manuscript. Y.Y., D.-F.S., and X.L.revised the manuscript. Y.S., Y.Y., D.-F.S., and X.L. designed the study. D.-F.S.and X.L. supervised the experiments. Y.S., Z.Q., and X.G. carried out most of thestudies. J.C. and Y.T. carried out molecular modelling and docking analysis. X.G.carried out lentivirus injection experiments. Y.S. and X.G. bred and maintainedORM1 knockout mice. J.W. helped with some of the experiments. All authorsanalyzed and interpreted experimental data. X.L. is the guarantor of this workand, as such, had full access to all the data in the study and takes responsibilityfor the integrity of the data and the accuracy of the data analysis.

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