Song-Yang Zhang,1 Ying Lv,1 Heng Zhang,2 Song Gao,3 Ting Wang,1 Juan Feng,1

Yuhui Wang,4 George Liu,4 Ming-Jiang Xu,1 Xian Wang,1 and Changtao Jiang1

Adrenomedullin 2 Improves EarlyObesity-Induced Adipose InsulinResistance by Inhibiting the Class IIMHC in AdipocytesDiabetes 2016;65:2342–2355 | DOI: 10.2337/db15-1626

MHC class II (MHCII) antigen presentation in adipocyteswas reported to trigger early adipose inflammation andinsulin resistance. However, the benefits of MHCII inhi-bition in adipocytes remain largely unknown. Here, weshowed that human plasma polypeptide adrenomedullin2 (ADM2) levels were negatively correlated with HOMA ofinsulin resistance in obese human. Adipose-specific hu-man ADM2 transgenic (aADM2-tg) mice were generated.The aADM2-tg mice displayed improvements in high-fatdiet–induced early adipose insulin resistance. This was as-sociated with increased insulin signaling and decreasedsystemic inflammation. ADM2 dose-dependently inhibitedCIITA-induced MHCII expression by increasing Blimp1 ex-pression in a CRLR/RAMP1-cAMP–dependent manner incultured adipocytes. Furthermore, ADM2 treatment re-stored the high-fat diet–induced early insulin resistancein adipose tissue, mainly via inhibition of adipocyte MHCIIantigen presentation and CD4+ T-cell activation. This studydemonstrates that ADM2 is a promising candidate for thetreatment of early obesity-induced insulin resistance.

Obesity, as a result of the expansion of adipose tissues,disturbs the insulin sensitivity of insulin target organs andinduces the onset of type 2 diabetes (1). There is a stronginteraction between the chronic innate immune responseand insulin resistance (2). Previous reports showed thatthe expression of the proinflammatory cytokines TNFa,mainly derived from macrophages, was upregulated in

obese mice and exerted an important role in adipose andhepatic insulin resistance (3–5). However, to date the roleof the adaptive immune response in adipose inflammationis largely unknown.

It has been reported that a short-term high-fat diet(HFD) elicited substantial numbers of CD4+ T cells inadipose tissues. Importantly, the increased percentagesof CD4+ T cells in adipose tissues occurred prior to mac-rophages, which suggests that the adaptive immune re-sponse may play an important role in early adiposeinflammation and insulin resistance (6). The resident na-ïve CD4+ T cells require antigens presented in an MHCclass II (MHCII)-dependent manner by antigen-presentingcells (APCs) as the first signaling. CD28 and CD40L on themembrane of T cells interact with the costimulatory mol-ecules CD80/86 and CD40 on APCs, forming the secondarysignaling. The naïve T cells secret IL2, which acts in anautocrine manner to activate T cells, resulting in T-cellactivation and proliferation (7). The activation of adiposeresident T cells triggers the infiltration of more T cells aswell as other immune cells, which initiate adipose inflam-mation (8).

Adrenomedullin 2 (ADM2)/intermedin is a widely ex-pressed bioactive peptide belonging to the calcitonin gene-related peptide (CGRP)/calcitonin family (9,10). Calcitoninreceptor–like receptor (CRLR) is an ADM2 receptor, andreceptor activity–modifying proteins (RAMPs) are thecoreceptors of CRLR (9). The interactions of CRLR with

1Department of Physiology and Pathophysiology, School of Basic Medical Sci-ences, Peking University, Key Laboratory of Molecular Cardiovascular Science,Ministry of Education and Beijing Key Laboratory of Cardiovascular ReceptorsResearch, Beijing, China2Department of Endocrinology, Beijing Chao-Yang Hospital, Capital Medical Uni-versity, Beijing, China3Department of General Surgery, Peking University First Hospital, Peking Univer-sity, Beijing, China4Institute of Cardiovascular Sciences, Peking University, Key Laboratory ofMolecular Cardiovascular Sciences, Ministry of Education, Beijing, China

Corresponding authors: Changtao Jiang, jiangchangtao@bjmu.edu.cn, andXian Wang, xwang@bjmu.edu.cn.

Received 3 December 2015 and accepted 26 April 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1626/-/DC1.

© 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.

2342 Diabetes Volume 65, August 2016

IMMUNOLOGY

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TRANSPLANTATIO

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different RAMPs (RAMP1, -2, and -3) form different re-ceptor complexes (11). ADM2 has been shown to exertprotective effects on cardiac ischemia/reperfusion injury,vascular calcification, and immunoglobulin A nephro-pathy by inhibiting inflammation, oxidative stress, andendoplasmic reticulum stress (12–14). We and othergroups reported that ADM2 treatment reduced athero-sclerosis by inhibiting foam cell formation and improvingdyslipidemia (15–17). Recently, our group discovered thatADM2 had a protective effect on HFD-induced establishedobesity by increasing thermogenesis in adipocytes (18).However, whether ADM2 has a protective effect on earlyinsulin resistance is still unknown. In this study, we dem-onstrate that adipose-specific overexpression of ADM2substantially improves early obesity-induced adipose in-flammation and insulin resistance in mice. Mechanisticstudies revealed that ADM2 inhibits adipose proinflammatoryT-cell activation mainly through decreasing MHCII-mediatedantigen presentation in adipocytes. Furthermore, ADM2treatment upregulated B lymphocyte–induced maturationprotein 1 (Blimp1) and inhibited the class II transactivator(CIITA)-MHCII axis in a CRLR/RAMP1-cAMP–dependentmanner.

RESEARCH DESIGN AND METHODS

ReagentsHuman ADM21–53, ADM217–47, CGRP8–37, ADM22–52, andthe anti-ADM2 antibody were purchased from Phoenix Phar-maceuticals (Belmont, CA). The anti-Blimp1 antibody waspurchased from Cell Signaling Technology (Boston, MA).The anti-MHCII (M5/114.15.2) and anti-MHCII (OX-6) an-tibodies were purchased from BD Biosciences (San Jose, CA).Human g-interferon (IFNg) was purchased from R&D Sys-tems (Minneapolis, MN). All other chemicals and drugs werepurchased from Sigma-Aldrich (St. Louis, MO).

Subject Sample CollectionThe study was approved by the ethics committee of BeijingChao-Yang Hospital and complied with the principlesoutlined in the Declaration of Helsinki. All subjects gavewritten informed consent prior to participation. The bloodsamples from all subjects were placed in tubes containingEDTA and aprotinin (500 kIU/mL) and centrifuged imme-diately. The plasma was stored at 280°C.

AnimalsThe wild-type (WT) mice, aP2-driven adipose-specifichuman ADM2 transgenic (aADM2-tg) mice (a mouseline constructed by the authors), systemic MHCII knock-out (MKO) mice (MARC, Nanjing, China), and OTII mice(a kind gift from Yu Zhang, Peking University) were on apure C57BL/6J background and housed as previously de-scribed (18). The animals were littermates, mixed housedin a cage, and fed a normal chow diet (NCD) (20% caloriesfrom fat) or HFD (60% calories from fat; Research Diets,New Brunswick, NJ) for 4 weeks. All the animal protocolswere approved by the Animal Care and Use Committee ofPeking University.

Cell CultureMature adipocytes and adipose precursor cells were isolatedas previously described (18).

Cytometric Bead Array and Flow CytometryThe inflammatory cytokine levels in plasma were inves-tigated using a cytometric bead array inflammation kit(BD Biosciences). For flow cytometry, the mature adipo-cytes or stromal vascular fractions (SVFs) were isolated asdescribed in cell culture. Cells were filtered and eliminatedred blood cells. Fixation and permeabilization were neededwhen intracellular protein was stained. Then cells werestained with different antibodies and analyzed by flowcytometer. When adipocytes were analyzed, the cellsshould be mixed constantly to avoid floating of adipocytes.The stained cells were analyzed using a FACSCaliber (BDBiosciences) and Beckman Gallios (Beckman Coulter, Brea,CA) with FlowJo software.

In Vitro Antigen Presentation AssayFor in vitro antigen presentation assay, the differentiated3T3-L1 adipocytes were seeded in 12-well plates andhandled as previously described (19).

Bone Marrow TransplantationThe murine total bone marrow hematopoietic progenitordonor cells were harvested and were transplanted via tailvein injection into the lethally irradiated WT and MKOmice. The transplanted mice were maintained for 6 weeksand treated with vehicle or ADM2 subcutaneously insaline through an Alzet Mini-osmotic Pump (DURECT,Cupertino, CA) at a rate of 300 ng/kg/h.

Quantitative PCR AnalysisThe extraction of total RNA, reverse transcription, and real-time PCR were undertaken as previously described (18).

Western Blot AnalysisThe Western blot analyses were undertaken as previouslydescribed (18).

Statistical AnalysisThe data were expressed as means 6 SEM and analyzedusing GraphPad Prism software as previously described(18). P , 0.05 was considered significant.

RESULTS

Adipose-Specific ADM2 Overexpression ImprovesEarly Obesity-Induced Inflammation and InsulinResistance in Adipose TissueFor investigation of the role of ADM2 during the path-ogenesis of insulin resistance, the plasma level of ADM2in human was firstly determined. The human plasmaADM2 level was negatively correlated with HOMA of insulinresistance (HOMA-IR) in human (Fig. 1A). Western blot andquantitative PCR (qPCR) analysis further showed thatADM2 was highly expressed in the epididymal white adiposetissues (eWATs) and was substantially downregulated in themice fed an HFD for 8 weeks, whereas the expression ofADM2 was much lower and remained unchanged in the

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Figure 1—Adipose-specific ADM2 overexpression improves early HFD-induced inflammation and insulin resistance in the adipose tissue.A: Correlation of the plasma ADM2 levels with the HOMA-IR in human (n = 41 total individuals). B: GTT (left panel) and the area under thecurve (AUC) (right panel). C: ITT (left panel) and the area under the curve (right panel). D: Fasting plasma glucose levels. E: Fasting plasmainsulin levels. F: HOMA-IR. G: Western blot analysis (top panel) and quantitation (bottom panel) of AKT phosphorylation (Tyr308) in theeWAT. The relative protein levels were normalized to those of the WT mice fed a 4w-HFD. The mice were fasted for 4 h and then treated ornot with insulin (2 IU/kg) for 5 min before sacrifice. H: Plasma levels of inflammatory cytokines. B–F and H: Seven-week-old WT andaADM2-tg mice were fed an NCD or a 4w-HFD. G: Seven-week-old WT and aADM2-tg mice were fed a 4w-HFD with or without insulintreatment. B–H: n = 5–7 mice per group. All data are presented as the means 6 SEM. B–F and H: One-way ANOVA with Tukey correc-tion, *P< 0.05, **P< 0.01, compared with the WT mice being fed an NCD; #P< 0.05, ##P < 0.01, compared with the WT mice being fed a4w-HFD. G: One-way ANOVA with Tukey correction, *P < 0.05 compared with the WT mice being fed a 4w-HFD without insulin treatment;#P < 0.05 compared with the WT mice being fed a 4w-HFD with insulin treatment.

2344 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

liver and skeletal muscle (Supplementary Fig. 1A and B).The plasma level of ADM2 was also decreased after anHFD (Supplementary Fig. 1C).

For determination of whether ADM2 exerts a role ineWAT during insulin resistance, aADM2-tg mice weredeveloped (18). Western blot and qPCR analysis showedthat the expression of ADM2 was robustly induced in theeWAT of the aADM2-tg mice, while there were no differ-ences in the macrophages, liver, and skeletal muscle (Sup-plementary Fig. 1D and E). Then, the eWATs from the WTand aADM2-tg mice were separated into mature adipo-cytes and SVFs, and the expression of ADM2 was exam-ined. The ADM2 level was enhanced in the matureadipocytes of aADM2-tg mice but not in the SVFs (Sup-plementary Fig. 1F).

For further validation of the role of adipose ADM2 ininsulin resistance, WT and aADM2-tg mice were fed anNCD or an HFD for 4 weeks (4w-HFD). The aADM2-tgmice fed an NCD or a 4w-HFD displayed a body weightgain similar to that of the WT mice fed an NCD or a4w-HFD (Supplementary Figs. 1G and 2A). However, theglucose tolerance test (GTT) and insulin tolerance test (ITT)revealed that adipose-specific ADM2 overexpression no-ticeably improved the HFD-induced systemic insulin re-sistance (Fig. 1B and C) but not in the aADM2-tg mice fedan NCD (Supplementary Fig. 2B and C). Although thefasting plasma level of glucose was not changed in theWT or aADM2-tg mice fed an NCD or a 4w-HFD (Fig.1D and Supplementary Fig. 2D), the 4w-HFD–inducedincreased fasting plasma insulin level and HOMA-IRwere significantly reversed in the aADM2-tg mice fed a4w-HFD (Fig. 1E and F) but not in the aADM2-tg mice fedan NCD (Supplementary Fig. 2E and F). Insulin signalingwas further investigated in the eWAT. The 4w-HFD–induced insulin signaling impairment in the eWAT of theWT mice was substantially improved, as revealed by anincreased level of AKT phosphorylation (Fig. 1G), whichwas not observed in the aADM2-tg mice fed an NCD (Sup-plementary Fig. 2G). The plasma levels of inflammatorycytokines were also measured to determine the extent ofthe systemic inflammation. The 4w-HFD–induced eleva-tion of plasma proinflammatory cytokines levels, includingthose of IL2, IL17a, IL6, IL12p70, TNFa, and MCP1, in theWT mice was markedly restored in the aADM2-tg mice(Fig. 1H). These results suggest that adipose ADM2 pro-tects against early obesity-induced insulin resistance andinflammation in eWAT of mice.

aADM2-tg Mice Display Lower Susceptibilityto 4w-HFD–Induced Adipose T-Cell Inflammationand Adipocyte MHCII ExpressionThe increased percentage of immune cells in white adiposetissue (WAT) is one of the important causes of adiposeinflammation (20). Thus, the percentages of immune cells ineWAT were determined by flow cytometry. The increasedproportion of CD45+CD3+ T cells in the eWAT of the WTmice induced by a 4w-HFD was substantially reversed in the

aADM2-tg mice (Fig. 2A). However, the proportions ofother immune cells, such as CD45+F4/80+ macrophages,CD45+CD19+ B cells, and CD45+CD11c+ dendritic cells,were not reduced (Fig. 2A). Furthermore, the proportionof CD3+CD4+ T cells was increased by a 4w-HFD treatmentand was noticeably blunted in the aADM2-tg mice comparedwith that of the WT mice (Fig. 2B), whereas there was nosignificant alteration in the proportion of CD3+CD8+ T cells(Fig. 2B). The percentages of different subsets of CD4+ T cellsin eWAT were further assessed. The 4w-HFD–induced in-creased percentages of proinflammatory CD4+IFNg+ T helper(Th)1 and CD4+IL17a+ Th17 cells, as well as the decreased per-centages of anti-inflammatory CD4+IL4+ Th2 and CD4+Foxp3+

regulatory T cells in the eWAT, were dramatically reversedin the aADM2-tg mice (Fig. 2C).

The activation and proliferation of CD4+ Th cells de-pend on the MHCII-mediated antigen presentation func-tion in APCs (21). Thus, the expression of MHCII ineWAT was determined. The mRNA levels of the relatedMHCII family genes H2-Eb1 and Ciita as well as levels ofthe MHCII (H2-A/E) protein were substantially induced inthe eWAT of the WT mice treated with a 4w-HFD (Fig. 2Dand E) but were downregulated in the eWAT of theaADM2-tg mice fed an NCD or a 4w-HFD (Fig. 2F andG and Supplementary Fig. 3A and B). Flow cytometryanalysis of SVFs and adipocytes in eWAT was performed tofurther investigate the type of cells responsible for the de-creased MHCII expression in aADM2-tg mice. The 4w-HFD–induced increased proportion of MHCII+ adipocytes wassubstantially blunted in the aADM2-tg mice but not theMHCII+ SVFs (Fig. 2H). Immunofluorescence staining ofeWAT further confirmed that the MHCII protein level wasdownregulated in the aADM2-tg mice compared withthose of the WT mice on a 4w-HFD (Fig. 2I). Taken to-gether, these results suggest that adipose-specific ADM2overexpression noticeably reduces adipocyte-derived MHCIIexpression and T-cell activation in eWAT.

ADM2 Inhibits Adipocyte MHCII Expression,Adipocyte-Mediated MHCII Antigen Presentation,and T-Cell Activation In VitroIt was reported that IFNg induced MHCII expression inadipocytes (19,22). Similarly, IFNg treatment dramati-cally increased the mRNA and protein level of MHCII incultured adipocytes in a dose-dependent manner (Supple-mentary Fig. 4A–C). Flow cytometry analysis confirmed thatthe proportion of membrane MHCII+ adipocytes was sub-stantially expanded after IFNg stimulation (SupplementaryFig. 4D). Immunofluorescence staining also demonstratedan increase of MHCII expression in the adipocytes treatedwith IFNg (Supplementary Fig. 4E).

Primary adipocytes treated with ADM2 were investigatedby RNA-Seq to explore the mechanism by which adipose-specific ADM2 overexpression markedly downregulatedMHCII expression. The RNA-Seq analysis showed thatADM2 stimulation reduced the mRNA levels of relatedMHCII genes, Rt1-B, Rt1-D, Rt1-DM, Rt1-DO, Cd74, and

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Figure 2—aADM2-tg mice display lower susceptibility to 4w-HFD–induced adipose T-cell inflammation and adipocyte MHCII expres-sion. A: The proportions of CD45+F4/80+ macrophages, CD45+CD19+ B cells, CD45+CD11c+ dendritic cells, and CD45+CD3+ T cells inthe SVFs of the eWAT were analyzed by flow cytometry. B: The proportions of CD3+CD4+ and CD3+CD8+ T cells in the SVFs of theeWAT were analyzed by flow cytometry. C: The proportions of CD4+IFNg+, CD4+IL4+, CD4+IL17a+, and CD4+Foxp3+ T cells in the SVFsof the eWAT were analyzed by flow cytometry. D: qPCR analysis of the mRNA levels of H2-Eb1 and Ciita in the eWAT. E: Western blotanalysis (top panel) and quantitation (bottom panel) of the MHCII protein in the eWAT. The relative protein levels were normalized tothose of the WT mice being fed an NCD. F: qPCR analysis of the mRNA levels of H2-Eb1, Cd74, and Ciita in the eWAT. G: Western blotanalysis (top panel) and quantitation (bottom panel) of the MHCII protein in the eWAT. The relative protein levels were normalized tothose of the WT mice fed a 4w-HFD. H: The proportions of MHCII+ cells in the SVFs and adipocytes of the eWAT were analyzed by flowcytometry. I: Representative immunofluorescence staining of the MHCII and perilipin-1 protein in the eWAT. MHCII was stained in red,perilipin-1 was stained in green, and the merge signal was stained in yellow. A–C and H: Seven-week-old WT and aADM2-tg mice werefed an NCD or 4w-HFD. F, G, and I: Seven-week-old WT and aADM2-tg mice were fed a 4w-HFD. D and E: Seven-week-old WT micewere fed an NCD or a 4w-HFD. A–I: n = 5–8 mice per group. For qPCR analysis, the expression was normalized to b-actin. All the dataare presented as means6 SEM. A–C and H: One-way ANOVA with Tukey correction, *P < 0.05, **P < 0.01, compared with the WT micebeing fed an NCD; #P < 0.05, ##P < 0.01, compared with the WT mice being fed a 4w-HFD. D–G: Two-tailed Student t test, *P < 0.05,**P < 0.01, compared with the WT mice being fed a 4w-HFD.

2346 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

Ciita, as well as the costimulatory molecules, Cd80 andIcam1 (Fig. 3A). qPCR analysis confirmed that ADM2 sub-stantially reversed the IFNg-induced expression of theMHCII family genes, including H2-Aa, H2-Ab1, H2-Eb1,H2-Eb2, H2-Ma, H2-Mb, H2-Oa, Cd74, and Ciita, but notthe costimulatory factors and adherent molecule, includ-ing Cd40, Cd80, Cd86, and Icam1, in 3T3-L1 adipocytes(Fig. 3B). Furthermore, the ADM2 treatment inhibitedthe expression of MHCII mRNA and protein levels inthe primary adipocytes in a dose-dependent manner un-der both basal and IFNg-induced conditions (Fig. 3C–F).Immunofluorescence staining further validated that theIFNg-induced upregulation of MHCII in adipocytes wasabolished after ADM2 treatment (Fig. 3G). The image-captureflow cytometry analysis also showed that the ADM2 treat-ment markedly reversed the IFNg-induced upregulation ofMHCII protein level at the adipocyte membrane (Fig. 3H).

For further identification of the role of ADM2 inadipocyte-mediated antigen presentation function, an anti-gen presentation assay was performed in vitro. 3T3-L1adipocytes were pretreated with IFNg and ADM2 and weresubsequently cocultured with naïve T cells isolated fromOTII mice with ovalbumin (OVA). The ability of adipocytesto activate naïve T cells was assessed by the levels of IL2 andIFNg secreted into the culture media by the T cells. TheIFNg-induced secretion of IL2 and IFNg was partially re-versed by ADM2 treatment (Fig. 3I and J). Overall, theseresults indicate that ADM2 prevents the activation of T cellsin WAT primarily by downregulating MHCII expression inadipocytes.

Inhibition of MHCII Expression in Adipocytesby ADM2 Results From the Downregulation of CiitaTranscription via an Increase in Blimp1 ExpressionIn atypical APCs, IFNg is the only well-characterized in-ducer of MHCII expression (23). For exploration of themechanism by which ADM2 reduced the expression ofMHCII in adipocytes, whether the ADM2-inhibited expres-sion of MHCII depended on IFNg pretreatment was exam-ined. ADM2 was observed to inhibit the basal expression ofMHCII in primary adipocytes without IFNg treatment(Fig. 3C–E). Moreover, the effect of ADM2 on the IFNgsignaling pathway was also detected. The IFNg-inducedphosphorylation of signal transducer and activator oftranscription 1 (STAT1) remained similar after ADM2treatment (Supplementary Fig. 5A), suggesting that theADM2-mediated inhibition of MHCII was indepen-dent of the IFNg signaling pathway. CYT387, an IFNgreceptor adaptor protein Jak1/2 inhibitor, was also usedto block the IFNg signal pathway (Supplementary Fig.5B). CYT387 blocked both the basal and IFNg-inducedMHCII expression in adipocytes (Supplementary Fig.5C–E). However, ADM2 had a further inhibitory effecton MHCII expression after pretreatment of CYT387 (Sup-plementary Fig. 5C–E), which suggesting that the inhibi-tion of MHCII expression by ADM2 is independent ofIFNg.

CIITA was shown to be the core transcription factor ofMHCII with three types of transcripts, Ciita-PI, Ciita-PIII,and Ciita-PIV, expressing in different types of cells (23).Therefore, the expression of different Ciita transcriptswas examined. The expression levels of Ciita-PI and Ciita-PIV in adipocytes were restrained by ADM2 under thebasal and IFNg-treated conditions (Fig. 4A). These resultsindicate that ADM2 inhibits MHCII expression via directdownregulation of Ciita-PI and Ciita-PIV transcription.

The precise molecular mechanism by which ADM2downregulated CIITA expression in adipocytes was thendetermined. It is reported that Blimp1 is a powerfultranscription factor that negatively regulates MHCIIexpression and downregulates all three Ciita transcripts(24–26). Our results showed that ADM2 markedly in-creased the expression of Blimp1 mRNA and protein inadipocytes in a time-dependent manner (Fig. 4B and C).Consistently, the aADM2-tg mice displayed a striking in-crease of Blimp1 mRNA and protein levels in the eWATcompared with those of the WT mice on an NCD or a4w-HFD (Fig. 4D and E and Supplementary Fig. 6A and B).For determination of whether Blimp1 is involved inADM2 inhibition of CIITA expression, Blimp1 expres-sion was knocked down in adipocytes with a specificsmall interfering (si)RNA. The knockdown efficiency ofBLIMP1 expression in adipocytes was .50% at the pro-tein level (Fig. 4F). The ADM2-mediated inhibition ofthe IFNg-induced MHCII expression was totally abol-ished after the Blimp1 siRNA treatment (Fig. 4G). Col-lectively, these results suggest that the downregulationof the CIITA transcription via the increase of Blimp1expression mediates the ADM2-mediated inhibition ofMHCII expression in adipocytes.

ADM2 Restrained the Blimp1-CIITA-MHCII AxisThrough the CRLR/RAMP1-cAMP PathwayFor validation that the CRLR/RAMPs that are responsiblefor the ADM2-mediated inhibition of MHCII expression,three different peptide antagonists—ADM217–47 for theinhibition of CRLR/RAMP1, -2, and 3; CGRP8–37 for theinhibition of CRLR/RAMP1; and ADM22–52 for the inhi-bition of CRLR/RAMP2 and -3—were used. Pretreatmentwith ADM217–47 and CGRP8–37 eliminated the ADM2-mediated inhibition of the IFNg-induced MHCII expres-sion as well as increased Blimp1 expression in adipocytesbut not ADM22–52 (Fig. 5A–D). These results imply thatCRLR/RAMP1 might mainly mediate the effects of ADM2on the Blimp1-CIITA-MHCII axis. It is reported that theactivation of CRLR/RAMP1 triggers various signal trans-duction pathways (27). In the current study, two signalingpathways were firstly determined with the following in-hibitors: LY294002 to inhibit Akt and compound C toinhibit AMPK. The ADM2-mediated downregulation ofMHCII and upregulation of Blimp1 were not inhibitedby LY294002 or compound C (Fig. 5F–I). CRLR/RAMP1,as a G-protein–coupled receptor complex, increased thecellular cAMP level (Fig. 5E). Blocking the cAMP pathway

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Figure 3—ADM2 inhibits adipocyte MHCII expression, adipocyte-mediated MHCII antigen presentation, and T-cell activation in vitro.A: RNA-Seq analysis of the MHCII family genes and costimulatory molecules. The differentiated adipocytes were treated with ADM2 (20 nmol/L)for 8 h. B: qPCR analysis of the mRNA levels of the MHCII family genes and costimulatory molecules. The differentiated 3T3-L1adipocytes were treated with control (Con), IFNg (5 ng/mL), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) for 24 h (n = 6). C–E: qPCR analysisof the mRNA levels of Rt1-Db (C ), Cd74 (D), and Ciita (E ). The primary adipocytes were treated with control or indicated doses of ADM2,IFNg (5 ng/mL), or IFNg (5 ng/mL) + indicated doses of ADM2 for 16 h (n = 10). F: Western blot analysis (top panel) and quantitation (bottompanel) of the MHCII protein. The differentiated adipocytes were treated with control, IFNg (5 ng/mL), or IFNg (5 ng/mL) + indicated doses of

2348 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

by Rp-cAMPS eliminated the ADM2-mediated inhibition ofthe MHCII expression and Blimp1 upregulation (Fig. 5F–I).These results reveal that ADM2 inhibits the Blimp1-CIITA-MHCII axis in a CRLR/RAMP1-cAMP–dependent manner.

Inhibition of Adipocyte MHCII Expression MainlyMediates the Improvements of Early Obesity-InducedAdipose Insulin Resistance by ADM2For further identification of the role of adipocyte MHCII inthe ADM2-induced early adipose insulin resistance improve-ments, MKO mice were used. The levels of the MHCIImRNA and protein were almost undetectable in the macro-phages and eWAT of the MKO mice (Supplementary Fig. 7Aand B). However, adipose macrophages, B cells, and den-dritic cells as APCs derived from bone marrow infiltrateinto adipose tissue under physiological and pathological con-ditions (28–30). The antigen-presenting function of thesecells in the eWAT should be considered. Therefore, the he-matopoietic cells from the WT mice were transplanted intothe WT and MKO mice to generate the chimeric WT andMKO mice (WT-WT and WT-MKO) (Supplementary Fig.7C). PCR analysis confirmed the highly chimeric tissue inWT-MKO mice (Supplementary Fig. 7D).

The WT-WT and WT-MKO mice were treated with vehicleor ADM2 subcutaneously via mini-pumps and concurrentlyfed a 4w-HFD. The ADM2 treatment substantially increasedthe plasma ADM2 level in the WT-WT and WT-MKO mice atthe end of the fourth week (Supplementary Fig. 7E). And theADM2 treatment had no effect on the body weight gain ofthe WT-WT and WT-MKO mice fed a 4w-HFD (Supplemen-tary Fig. 7F). However, ADM2 noticeably ameliorated insulinresistance in the WT-WT mice but not in the WT-MKO mice(Fig. 6A and B). No significant difference in the fastingplasma glucose levels was observed in the WT-WT andWT-MKO mice after the ADM2 treatment (Fig. 6C). Thefasting plasma insulin level and HOMA-IR were substantiallyreduced in the ADM2-treated WT-WT mice comparatively(Fig. 6D and E). However, the WT-MKO mice were relativelyunresponsive to the metabolic benefits of the ADM2 treat-ment (Fig. 6D and E). ADM2 treatment markedly increasedthe basal level of AKT phosphorylation in the WT-WT mice.Although the AKT phosphorylation level was increased inWT-MKO mice compared with WT-WT mice, ADM2 treat-ment cannot further increase the phosphorylation level of

AKT (S.Y.-Z., Y.L., H.Z., C.J., X.W., unpublished data). Takentogether, these results indicate that the protective role ofADM2 in 4w-HFD–induced insulin resistance in eWAT de-pends primarily on the inhibition of MHCII in adipocytes.

DISCUSSION

In the current study, we found that ADM2 exerts metabolicbenefits on early obesity-induced adipose inflammationand insulin resistance by inhibiting MHCII expression inadipocytes (Fig. 7). During the pathogenesis of obesity,adipose CD4+ proinflammatory Th1 cells were activatedand secreted large amounts of IFNg (6,19,31). IFNg stim-ulation substantially enhanced the expression of MHCII inadipocytes. Subsequently, adipocyte MHCII-mediated anti-gen presentation activated more proinflammatory CD4+ Thcells. ADM2 was observed to upregulate Blimp1 expres-sion by activating the CRLR/RAMP1-cAMP pathway. TheBlimp1-CIITA-MHCII axis mediated ADM2-induced im-provement in early obesity-induced adipose inflammationand insulin resistance. These findings demonstrate thatADM2 is a potential drug candidate for early obesity-induced inflammation and insulin resistance.

In obese mice, the adipose T cells exhibited antigen-specific expansion, and the Th1 proportion was strikinglyincreased (32). The MHCII, CD40L, and CD80/86 sys-temic knockout mice displayed a lower susceptibility toHFD-induced adipose inflammation, suggesting that APC-induced CD4+ T-cell activation is required for adipose in-flammation (19,33,34). However, the exact type of APCthat presents the antigen to the adipose T cells in earlyobesity remains largely unknown. Previous results showthat adipocytes have antigen presentation function andmight be the APCs in WAT (19,22). Consistently, we alsofound the expression of MHCII in adipocytes.

Preadipocytes share a lot of features with fibroblasts andmacrophages (35,36). Both macrophages and fibroblasts havebeen shown to express MHCII after IFNg treatment (23).Therefore, it is difficult to exclude whether the expression ofMHCII induced by IFNg in differentiated adipocytes is derivedfrom undifferentiated preadipocytes. Here we isolated primaryadipocytes from eWAT of rat and treated with IFNg. IFNgmarkedly upregulated MHCII expression. MHCII expressionwas induced in the eWAT of mice fed a 4w-HFD. However,

ADM2 for 16 h (n = 6). The relative protein levels were normalized to that of the control. G: Representative immunofluorescence staining ofMHCII proteins. MHCII was stained in green, lipid was stained in red, and nucleus was stained in blue. The differentiated adipocytes weretreated with control, IFNg (5 ng/mL), ADM2 (20 nmol/L), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) for 24 h (n = 5). H: Representative im-munofluorescence staining (top panel) and quantitation (bottom panel) of the MHCII+ adipocytes were analyzed by image-capture flowcytometry. The primary adipocytes were treated with control, ADM2 (20 nmol/L), IFNg (5 ng/mL), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) for16 h (n = 7). MFI, mean fluorescence intensity. I and J: Levels of IL-2 (I) and IFNg (J) in the supernatants from the antigen presentation assay.The differentiated 3T3-L1 adipocytes were treated with control, ADM2 (20 nmol/L), IFNg (5 ng/mL), or ADM2 (20 nmol/L) + IFNg (5 ng/mL)for 24 h, washed and changed to fresh media, and then cocultured with OTII T cells treated or not with 500 mg/mL OVA for another 24 h(n = 6). For qPCR analysis, the expression was normalized to b-actin. B–J: All the data are presented as means 6 SEM. B–H: One-wayANOVA with Tukey correction, *P< 0.05, **P< 0.01, compared with control; #P< 0.05, ##P< 0.01, compared with IFNg. I and J: One-wayANOVA with Tukey correction, *P< 0.05, **P< 0.01, compared with control; ##P< 0.01, compared with OVA; and §P< 0.05, compared withOVA + IFNg.

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Figure 4—The inhibition of MHCII expression in adipocytes by ADM2 results from the downregulation of Ciita transcription via an increasein Blimp1 expression. A: qPCR analysis of the mRNA levels of different transcripts of Ciita. The differentiated 3T3-L1 adipocytes weretreated with control (Con), ADM2 (20 nmol/L), IFNg (5 ng/mL), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) for 24 h (n = 4). B: qPCR analysis of themRNA levels of Blimp1. The differentiated 3T3-L1 adipocytes were treated with ADM2 (20 nmol/L) for indicated hours (n = 6). C: Westernblot analysis (top panel) and quantitation (bottom panel) of the BLIMP1 protein. The differentiated 3T3-L1 adipocytes were treated withADM2 (20 nmol/L) for indicated hours (n = 5). The relative protein levels were normalized to that of the control. D: qPCR analysis of themRNA levels of Blimp1 in the eWAT. E: Western blot analysis (top panel) and quantitation (bottom panel) of the BLIMP1 protein in theeWAT. The relative protein levels were normalized to that of the WT mice fed a 4w-HFD. F: Western blot analysis (top panel) andquantitation (bottom panel) of the BLIMP1 protein. The differentiated adipocytes were transfected with scramble siRNA (si-Scramble) orBlimp1 siRNA (si-Blimp1) for 36 h (n = 4). The relative protein levels were normalized to that of the scramble siRNA. G: qPCR analysis of themRNA levels of Rt1-Db, Cd74, and Ciita. The differentiated adipocytes were transfected with scramble siRNA or Blimp1 siRNA for 24 h andwere treated with control, IFNg (5 ng/mL), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) for another 24 h (n = 6). D and E: Seven-week-old WT andaADM2-tg mice were fed a 4w-HFD. n = 5–7 mice per group. For qPCR analysis, the expression was normalized to b-actin. All the data arepresented as means 6 SEM. A and G: One-way ANOVA with Tukey correction, **P < 0.01 compared with control; #P < 0.05, ##P < 0.01,compared with the IFNg. B and C: One-way ANOVA with Tukey correction, **P < 0.01 compared with the control. D and E: Two-tailedStudent t test, *P < 0.05, **P < 0.01, compared with the WT mice being fed a 4w-HFD. F: Two-tailed Student t test, *P < 0.05 comparedwith the scramble siRNA.

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Figure 5—ADM2 restrained the Blimp1-CIITA-MHCII axis through the CRLR/RAMP1-cAMP pathway. A–C: qPCR analysis of the mRNAlevels of Rt1-Db (A), Cd74 (B), and Ciita (C ). The primary adipocytes were treated with control, IFNg (5 ng/mL), IFNg (5 ng/mL) + ADM2(20 nmol/L), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) + indicated CRLR/RAMPs antagonists (200 nmol/L) for 24 h (n = 6). D: qPCR analysis ofthe mRNA levels of Blimp1. The primary adipocytes were treated with control, ADM2 (20 nmol/L), or ADM2 (20 nmol/L) + indicated CRLR/RAMP antagonists (200 nmol/L) for 24 h (n = 6). E: Levels of intracellular cAMP. The differentiated adipocytes were treated with control orADM2 (20 nmol/L) for 5 min (n = 6). F–H: qPCR analysis of the mRNA levels of Rt1-Db (F ), Cd74 (G), and Ciita (H). The primary adipocyteswere treated with control, IFNg (5 ng/mL), IFNg (5 ng/mL) + ADM2 (20 nmol/L), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) + indicated inhibitors

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other groups did not observe this phenotype in the mice fedan NCD (28,37). This might result from the fact that thesemice were not fed with an HFD and the expression of MHCIIwas too low to be observed. In addition, WAT is not uniform

and not all adipocytes express MHCII. The immunofluores-cence staining might miss the MHCII expression in WAT.

Although adipocytes might be the APC-presentingantigen in WAT, the exact antigen presented by adipocytes

(10 mmol/L Rp-cAMPS, 10 mmol/L LY294002, and 2 mg/mL compound C) for 24 h (n = 6). I: qPCR analysis of the mRNA levels of Blimp1.The primary adipocytes were treated with control, ADM2 (20 nmol/L), or ADM2 (20 nmol/L) + indicated inhibitors (10 mmol/L Rp-cAMPS,10 mmol/L LY294002, and 2 mg/mL compound C) for 24 h (n = 6). For qPCR analysis, the expression was normalized to b-actin. All the dataare presented as means 6 SEM. A–C and F–H: One-way ANOVA with Tukey correction, *P < 0.05, **P < 0.01 compared with the control;#P < 0.05, ##P < 0.01 compared with IFNg; §P < 0.05, §§P < 0.01 compared with IFNg + ADM2. D and I: One-way ANOVA with Tukeycorrection, **P < 0.01 compared with the control; #P < 0.05, ##P < 0.01 compared with ADM2. E: Two-tailed Student t test, *P < 0.05compared with the control.

Figure 6—The inhibition of adipocyte MHCII expression mainly mediates the improvements of early obesity-induced adipose insulinresistance by ADM2. A: GTT of the WT-WT mice (left panel) and the WT-MKO mice (middle panel) and the area under the curve (AUC)(right panel). B: ITT of the WT-WT mice (left panel) and the WT-MKO mice (middle panel) and the area under the curve (right panel). C:Fasting plasma glucose levels. D: Fasting plasma insulin levels. E: HOMA-IR. A–E: The vehicle- and ADM2-treated WT and MKO micetransplanted with WT mouse bone marrow were fed a 4w-HFD and subsequently treated with vehicle or ADM2 via subcutaneous mini-pumps. A–E: n = 5–8 mice per group. For qPCR analysis, the expression was normalized to b-actin. All the data are presented as means 6SEM. Two-tailed Student t test, **P < 0.01, compared with the vehicle-treated mice.

2352 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

during obesity is still unknown. MHCII is believed topresent exogenous antigens, but nearly 20–30% of anti-gens presented by APCs in a MHCII-dependent mannerare self-antigens (38). Previous results showed that inatherosclerosis heat shock protein 60, oxidized LDL andb2-glycoprotein I act as self-antigens (39–41). During theonset of adipose expansion in obesity, oxidative stressand endoplasmic reticulum stress induce the generationof lipid-binding protein, oxidative modified protein, andmisfolded protein, which might be the self-antigen toMHCII.

The inhibition of adipocyte MHCII antigen presen-tation led to the ADM2-mediated improvements in 4w-HFD–induced early adipose inflammation, as revealedby the attenuated benefits of ADM2 in the WT-MKOmice. The WT-MKO mice also displayed an improve-ment of insulin resistance compared with that in theWT-WT mice, which is similar to that of the WT-WTmice treated with ADM2 and cannot be further im-proved by ADM2. This observation is consistent withprevious results and suggests that the protective effectof ADM2 is at least partially through the inhibition ofMHCII expression in adipocytes (19). However, otherevidence revealed that adipose macrophages also pre-sent antigen and play a key role in the late stage ofobesity-induced adipose T-cell activation and insulin re-sistance (28,42). It should be noted that the role ofmacrophages in adipose antigen presentation cannotbe excluded in obesity-induced adipose inflammation

and insulin resistance. Therefore, the role of MHCII indifferent cells and under different conditions needs tobe investigated further.

ADM2 is highly expressed in WAT (43). Our unpub-lished data showed that ADM2 was expressed in adipo-cytes. In addition, ADM2 is expressed in vascular tissue(44), which might be another source of ADM2 in WAT.ADM2 shares receptor complexes with CGRP and ADM.Here, ADM2 was demonstrated to inhibit MHCII expres-sion in adipocytes primarily through CRLR/RAMP1, whichis the classical pharmacological CGRP1 receptor (45). Inagreement with our present findings, some reports haveshown that CGRP reduced MHCII expression in dendriticcells and hair follicle dermal papilla (46,47), although theexact mechanism remains unclear.

The related MHCII family genes are tightly regulatedprimarily at the transcription levels by CIITA. Three CIITAtranscripts were expressed in different cell types. In additionto the constitutive Ciita-PI and Ciita-PIII expression by pro-fessional APCs, IFNg was shown to increase Ciita-PIV ex-pression (48). Ciita-PI and Ciita-PIV were expressed inadipocytes, suggesting that adipocytes may have some fea-tures of professional APCs. Previous studies have shownthat IL4, IL10, and TGF-b inhibit MHCII expression, whilethe exact molecular mechanism has not been described(49,50). In this study, ADM2 downregulated MHCII expres-sion as a result of increased Blimp1 expression through theCRLR/RAMP1-cAMPpathway, which provided the exactmole-cular mechanism of MHCII downregulation.

Figure 7—Model of the inhibitory effects of ADM2 on HFD-induced adipocyte MHCII antigen presentation function, adipose inflammation,and insulin resistance. The activated adipose proinflammatory T cell secretes IFNg in response to HFD (1), induces MHCII expression (2),activates more naïve T cells (3), and aggravates HFD-induced adipose inflammation (4). 5 and 6: ADM2 significantly increases Blimp1expression in a CRLR/RAMP1-cAMP–dependent manner. 7: The upregulated Blimp1 inhibits MHCII expression and improves earlyobesity-induced adipose inflammation and insulin resistance.

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Funding. This work was supported by the National Natural Science Founda-tion of the P.R. of China (91439206 and 31230035 to X.W. and 81470554 toC.J.), the National Basic Research Program (973 Program) of the P. R. of China(2012CB518002 to M.-J.X.), the 111 Project of the Chinese Ministry of Edu-cation (B07001), and the Center for Molecular and Translational Medicine(BMU20140475 to G.L.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. S.-Y.Z., Y.L., and T.W. designed and performedthe experiments and analyzed data. H.Z. and S.G. contributed to the humansample collection and analysis. J.F. designed the immunological experiments.Y.W. and G.L. constructed the aADM2-tg mouse line. M.-J.X., X.W., and C.J.designed and supervised the research. S.-Y.Z., X.W., and C.J. wrote the manu-script. All authors approved the final manuscript. C.J. is the guarantor of this workand, as such, had full access to all the data in the study and takes responsibility forthe integrity of the data and the accuracy of the data analysis.

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