2342 Diabetes Volume 65, August 2016

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 Early Obesity-Induced Adipose Resistance by Inhibiting the Class II MHC in Adipocytes

Diabetes 2016;65:2342–2355 | DOI: 10.2337/db15-1626

MHC class II (MHCII) antigen presentation in adipocytes obese mice and exerted an important role in adipose and was reported to trigger early adipose inflammation and hepatic insulin resistance (3–5). However, to date the role insulin resistance. However, the benefits of MHCII inhi- of the adaptive immune response in adipose inflammation bition in adipocytes remain largely unknown. Here, we is largely unknown. showed that human plasma polypeptide It has been reported that a short-term high-fat diet 2 (ADM2) levels were negatively correlated with HOMA of (HFD) elicited substantial numbers of CD4+ T cells in fi insulin resistance in obese human. Adipose-speci c hu- adipose tissues. Importantly, the increased percentages man ADM2 transgenic (aADM2-tg) mice were generated. of CD4+ T cells in adipose tissues occurred prior to mac- The aADM2-tg mice displayed improvements in high-fat rophages, which suggests that the adaptive immune re- diet–induced early adipose insulin resistance. This was as- sponse may play an important role in early adipose sociated with increased insulin signaling and decreased inflammation and insulin resistance (6). The resident na- systemic inflammation. ADM2 dose-dependently inhibited ïve CD4+ T cells require antigens presented in an MHC CIITA-induced MHCII expression by increasing Blimp1 ex- pression in a CRLR/RAMP1-cAMP–dependent manner in class II (MHCII)-dependent manner by antigen-presenting fi cultured adipocytes. Furthermore, ADM2 treatment re- cells (APCs) as the rst signaling. CD28 and CD40L on the stored the high-fat diet–induced early insulin resistance membrane of T cells interact with the costimulatory mol- in adipose tissue, mainly via inhibition of adipocyte MHCII ecules CD80/86 and CD40 on APCs, forming the secondary + IMMUNOLOGY AND TRANSPLANTATION antigen presentation and CD4 T-cell activation. This study signaling. The naïve T cells secret IL2, which acts in an demonstrates that ADM2 is a promising candidate for the autocrine manner to activate T cells, resulting in T-cell treatment of early obesity-induced insulin resistance. activation and proliferation (7). The activation of adipose resident T cells triggers the infiltration of more T cells as well as other immune cells, which initiate adipose inflam- Obesity, as a result of the expansion of adipose tissues, mation (8). disturbs the insulin sensitivity of insulin target organs and Adrenomedullin 2 (ADM2)/intermedin is a widely ex- induces the onset of type 2 diabetes (1). There is a strong pressed bioactive peptide belonging to the - interaction between the chronic innate immune response related peptide (CGRP)/calcitonin family (9,10). Calcitonin and insulin resistance (2). Previous reports showed that receptor–like receptor (CRLR) is an ADM2 receptor, and the expression of the proinflammatory cytokines TNFa, receptor activity–modifying (RAMPs) are the mainly derived from macrophages, was upregulated in coreceptors of CRLR (9). The interactions of CRLR with

1Department of Physiology and Pathophysiology, School of Basic Medical Sci- Corresponding authors: Changtao Jiang, [email protected], and ences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Xian Wang, [email protected]. Ministry of Education and Beijing Key Laboratory of Cardiovascular Receptors Received 3 December 2015 and accepted 26 April 2016. Research, Beijing, China This article contains Supplementary Data online at http://diabetes 2Department of Endocrinology, Beijing Chao-Yang Hospital, Capital Medical Uni- .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1626/-/DC1. versity, Beijing, China 3Department of General Surgery, Peking University First Hospital, Peking Univer- © 2016 by the American Diabetes Association. Readers may use this article as sity, Beijing, China long as the work is properly cited, the use is educational and not for profit, and 4Institute of Cardiovascular Sciences, Peking University, Key Laboratory of the work is not altered. Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China diabetes.diabetesjournals.org Zhang and Associates 2343 different RAMPs (RAMP1, -2, and -3) form different re- Cell Culture ceptor complexes (11). ADM2 has been shown to exert Mature adipocytes and adipose precursor cells were isolated protective effects on cardiac ischemia/reperfusion injury, as previously described (18). vascular calcification, and immunoglobulin A nephro- Cytometric Bead Array and Flow Cytometry pathy by inhibiting inflammation, oxidative stress, and The inflammatory cytokine levels in plasma were inves- endoplasmic reticulum stress (12–14). We and other tigated using a cytometric bead array inflammation groups reported that ADM2 treatment reduced athero- (BD Biosciences). For flow cytometry, the mature adipo- sclerosis by inhibiting foam cell formation and improving cytes or stromal vascular fractions (SVFs) were isolated as dyslipidemia (15–17). Recently, our group discovered that described in cell culture. Cells were filtered and eliminated ADM2 had a protective effect on HFD-induced established red blood cells. Fixation and permeabilization were needed obesity by increasing thermogenesis in adipocytes (18). when intracellular was stained. Then cells were However, whether ADM2 has a protective effect on early stained with different antibodies and analyzed by flow insulin resistance is still unknown. In this study, we dem- cytometer. When adipocytes were analyzed, the cells onstrate that adipose-specific overexpression of ADM2 should be mixed constantly to avoid floating of adipocytes. substantially improves early obesity-induced adipose in- The stained cells were analyzed using a FACSCaliber (BD flammation and insulin resistance in mice. Mechanistic Biosciences) and Beckman Gallios (Beckman Coulter, Brea, studies revealed that ADM2 inhibits adipose proinflammatory CA) with FlowJo software. T-cell activation mainly through decreasing MHCII-mediated antigen presentation in adipocytes. Furthermore, ADM2 In Vitro Antigen Presentation Assay treatment upregulated B lymphocyte–induced maturation For in vitro antigen presentation assay, the differentiated protein 1 (Blimp1) and inhibited the class II transactivator 3T3-L1 adipocytes were seeded in 12-well plates and (CIITA)-MHCII axis in a CRLR/RAMP1-cAMP–dependent handled as previously described (19). manner. Bone Marrow Transplantation RESEARCH DESIGN AND METHODS The murine total bone marrow hematopoietic progenitor donor cells were harvested and were transplanted via tail Reagents vein injection into the lethally irradiated WT and MKO Human ADM2 – ,ADM2 – ,CGRP– ,ADM – ,and 1 53 17 47 8 37 22 52 mice. The transplanted mice were maintained for 6 weeks the anti-ADM2 antibody were purchased from Phoenix Phar- and treated with vehicle or ADM2 subcutaneously in maceuticals (Belmont, CA). The anti-Blimp1 antibody was saline through an Alzet Mini-osmotic Pump (DURECT, purchased from Cell Signaling Technology (Boston, MA). Cupertino, CA) at a rate of 300 ng/kg/h. The anti-MHCII (M5/114.15.2) and anti-MHCII (OX-6) an- tibodies were purchased from BD Biosciences (San Jose, CA). Quantitative PCR Analysis Human g-interferon (IFNg) was purchased from R&D Sys- The extraction of total RNA, reverse transcription, and real- tems(Minneapolis,MN).Allother chemicals and drugs were time PCR were undertaken as previously described (18). purchased from Sigma-Aldrich (St. Louis, MO). Western Blot Analysis Subject Sample Collection The Western blot analyses were undertaken as previously The study was approved by the ethics committee of Beijing described (18). Chao-Yang Hospital and complied with the principles Statistical Analysis outlined in the Declaration of Helsinki. All subjects gave The data were expressed as means 6 SEM and analyzed written informed consent prior to participation. The blood using GraphPad Prism software as previously described samples from all subjects were placed in tubes containing (18). P , 0.05 was considered significant. EDTA and aprotinin (500 kIU/mL) and centrifuged imme- 2 diately. The plasma was stored at 80°C. RESULTS Animals Adipose-Specific ADM2 Overexpression Improves The wild-type (WT) mice, aP2-driven adipose-specific Early Obesity-Induced Inflammation and Insulin human ADM2 transgenic (aADM2-tg) mice (a mouse Resistance in Adipose Tissue line constructed by the authors), systemic MHCII knock- For investigation of the role of ADM2 during the path- out (MKO) mice (MARC, Nanjing, China), and OTII mice ogenesis of insulin resistance, the plasma level of ADM2 (a kind gift from Yu Zhang, Peking University) were on a in human was firstly determined. The human plasma pure C57BL/6J background and housed as previously de- ADM2 level was negatively correlated with HOMA of insulin scribed (18). The animals were littermates, mixed housed resistance (HOMA-IR) in human (Fig. 1A). Western blot and in a cage, and fed a normal chow diet (NCD) (20% calories quantitative PCR (qPCR) analysis further showed that from fat) or HFD (60% calories from fat; Research Diets, ADM2 was highly expressed in the epididymal white adipose New Brunswick, NJ) for 4 weeks. All the animal protocols tissues (eWATs) and was substantially downregulated in the were approved by the Animal Care and Use Committee of mice fed an HFD for 8 weeks, whereas the expression of Peking University. ADM2 was much lower and remained unchanged in the 2344 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

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 the curve (AUC) (right panel). C: ITT (left panel) and the area under the curve (right panel). D: Fasting plasma glucose levels. E: Fasting plasma insulin levels. F: HOMA-IR. G: Western blot analysis (top panel) and quantitation (bottom panel) of AKT phosphorylation (Tyr308) in the eWAT. 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 or not with insulin (2 IU/kg) for 5 min before sacrifice. H: Plasma levels of inflammatory cytokines. B–F and H: Seven-week-old WT and aADM2-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 insulin treatment. 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 a 4w-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. diabetes.diabetesjournals.org Zhang and Associates 2345 liver and skeletal muscle (Supplementary Fig. 1A and B). aADM2-tg mice (Fig. 2A). However, the proportions of The plasma level of ADM2 was also decreased after an other immune cells, such as CD45+F4/80+ macrophages, HFD (Supplementary Fig. 1C). CD45+CD19+ Bcells,andCD45+CD11c+ dendritic cells, For determination of whether ADM2 exerts a role in were not reduced (Fig. 2A). Furthermore, the proportion eWAT during insulin resistance, aADM2-tg mice were of CD3+CD4+ T cells was increased by a 4w-HFD treatment developed (18). Western blot and qPCR analysis showed and was noticeably blunted in the aADM2-tg mice compared that the expression of ADM2 was robustly induced in the with that of the WT mice (Fig. 2B), whereas there was no eWAT of the aADM2-tg mice, while there were no differ- significant alteration in the proportion of CD3+CD8+ Tcells ences in the macrophages, liver, and skeletal muscle (Sup- (Fig. 2B). The percentages of different subsets of CD4+ Tcells plementary Fig. 1D and E). Then, the eWATs from the WT in eWAT were further assessed. The 4w-HFD–induced in- and aADM2-tg mice were separated into mature adipo- creased percentages of proinflammatory CD4+IFNg+ Thelper cytes and SVFs, and the expression of ADM2 was exam- (Th)1 and CD4+IL17a+ Th17 cells, as well as the decreased per- ined. The ADM2 level was enhanced in the mature centages of anti-inflammatory CD4+IL4+ Th2 and CD4+Foxp3+ adipocytes of aADM2-tg mice but not in the SVFs (Sup- regulatory T cells in the eWAT, were dramatically reversed plementary Fig. 1F). in the aADM2-tg mice (Fig. 2C). For further validation of the role of adipose ADM2 in The activation and proliferation of CD4+ Th cells de- insulin resistance, WT and aADM2-tg mice were fed an pend on the MHCII-mediated antigen presentation func- NCD or an HFD for 4 weeks (4w-HFD). The aADM2-tg tion in APCs (21). Thus, the expression of MHCII in mice fed an NCD or a 4w-HFD displayed a body weight eWAT was determined. The mRNA levels of the related gain similar to that of the WT mice fed an NCD or a MHCII family H2-Eb1 and Ciita as well as levels of 4w-HFD (Supplementary Figs. 1G and 2A). However, the the MHCII (H2-A/E) protein were substantially induced in glucose tolerance test (GTT) and insulin tolerance test (ITT) the eWAT of the WT mice treated with a 4w-HFD (Fig. 2D revealed that adipose-specific ADM2 overexpression no- and E) but were downregulated in the eWAT of the ticeably improved the HFD-induced systemic insulin re- aADM2-tg mice fed an NCD or a 4w-HFD (Fig. 2F and sistance (Fig. 1B and C) but not in the aADM2-tg mice fed G and Supplementary Fig. 3A and B). Flow cytometry an NCD (Supplementary Fig. 2B and C). Although the analysis of SVFs and adipocytes in eWAT was performed to fasting plasma level of glucose was not changed in the further investigate the type of cells responsible for the de- WT or aADM2-tg mice fed an NCD or a 4w-HFD (Fig. creased MHCII expression in aADM2-tg mice. The 4w-HFD– 1D and Supplementary Fig. 2D), the 4w-HFD–induced induced increased proportion of MHCII+ adipocytes was increased fasting plasma insulin level and HOMA-IR substantially blunted in the aADM2-tg mice but not the were significantly reversed in the aADM2-tg mice fed a MHCII+ SVFs (Fig. 2H). Immunofluorescence staining of 4w-HFD (Fig. 1E and F) but not in the aADM2-tg mice fed eWAT further confirmed that the MHCII protein level was an NCD (Supplementary Fig. 2E and F). Insulin signaling downregulated in the aADM2-tg mice compared with was further investigated in the eWAT. The 4w-HFD– those of the WT mice on a 4w-HFD (Fig. 2I). Taken to- induced insulin signaling impairment in the eWAT of the gether, these results suggest that adipose-specificADM2 WT mice was substantially improved, as revealed by an overexpression noticeably reduces adipocyte-derived MHCII increased level of AKT phosphorylation (Fig. 1G), which expression and T-cell activation in eWAT. was not observed in the aADM2-tg mice fed an NCD (Sup- plementary Fig. 2G). The plasma levels of inflammatory ADM2 Inhibits Adipocyte MHCII Expression, cytokines were also measured to determine the extent of Adipocyte-Mediated MHCII Antigen Presentation, the systemic inflammation. The 4w-HFD–induced eleva- and T-Cell Activation In Vitro g tion of plasma proinflammatory cytokines levels, including It was reported that IFN induced MHCII expression in g those of IL2, IL17a, IL6, IL12p70, TNFa,andMCP1,inthe adipocytes (19,22). Similarly, IFN treatment dramati- WT mice was markedly restored in the aADM2-tg mice cally increased the mRNA and protein level of MHCII in (Fig. 1H). These results suggest that adipose ADM2 pro- cultured adipocytes in a dose-dependent manner (Supple- mentary Fig. 4A–C). Flow cytometry analysis confirmed that tects against early obesity-induced insulin resistance and + inflammation in eWAT of mice. the proportion of membrane MHCII adipocytes was sub- stantially expanded after IFNg stimulation (Supplementary aADM2-tg Mice Display Lower Susceptibility Fig. 4D). Immunofluorescence staining also demonstrated to 4w-HFD–Induced Adipose T-Cell Inflammation an increase of MHCII expression in the adipocytes treated and Adipocyte MHCII Expression with IFNg (Supplementary Fig. 4E). The increased percentage of immune cells in white adipose Primary adipocytes treated with ADM2 were investigated tissue (WAT) is one of the important causes of adipose by RNA-Seq to explore the mechanism by which adipose- inflammation (20). Thus, the percentages of immune cells in specific ADM2 overexpression markedly downregulated eWAT were determined by flow cytometry. The increased MHCII expression. The RNA-Seq analysis showed that proportion of CD45+CD3+ TcellsintheeWAToftheWT ADM2 stimulation reduced the mRNA levels of related mice induced by a 4w-HFD was substantially reversed in the MHCII genes, Rt1-B, Rt1-D, Rt1-DM, Rt1-DO, Cd74, and 2346 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

Figure 2—aADM2-tg mice display lower susceptibility to 4w-HFD–induced adipose T-cell inflammation and adipocyte MHCII expres- sion. A:TheproportionsofCD45+F4/80+ macrophages, CD45+CD19+ B cells, CD45+CD11c+ dendritic cells, and CD45+CD3+ T cells in the SVFs of the eWAT were analyzed by flow cytometry. B:TheproportionsofCD3+CD4+ and CD3+CD8+ T cells in the SVFs of the eWAT were analyzed by flow cytometry. C: The proportions of CD4+IFNg+,CD4+IL4+,CD4+IL17a+,andCD4+Foxp3+ T cells in the SVFs of the eWAT were analyzed by flow cytometry. D: qPCR analysis of the mRNA levels of H2-Eb1 and Ciita in the eWAT. E:Westernblot analysis (top panel) and quantitation (bottom panel) of the MHCII protein in the eWAT. The relative protein levels were normalized to those of the WT mice being fed an NCD. F: qPCR analysis of the mRNA levels of H2-Eb1, Cd74,andCiita in the eWAT. G:Westernblot analysis (top panel) and quantitation (bottom panel) of the MHCII protein in the eWAT. The relative protein levels were normalized to those 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 flow cytometry. 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 were fed an NCD or 4w-HFD. F, G,andI: Seven-week-old WT and aADM2-tg mice were fed a 4w-HFD. D and E: Seven-week-old WT mice were 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 data are presented as means 6 SEM. A–C and H: One-way ANOVA with Tukey correction, *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 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. diabetes.diabetesjournals.org Zhang and Associates 2347

Ciita, as well as the costimulatory molecules, Cd80 and CIITA was shown to be the core transcription factor of Icam1 (Fig. 3A). qPCR analysis confirmed that ADM2 sub- MHCII with three types of transcripts, Ciita-PI, Ciita-PIII, stantially reversed the IFNg-induced expression of the and Ciita-PIV, expressing in different types of cells (23). MHCII family genes, including H2-Aa, H2-Ab1, H2-Eb1, Therefore, the expression of different Ciita transcripts H2-Eb2, H2-Ma, H2-Mb, H2-Oa, Cd74,andCiita, but not was examined. The expression levels of Ciita-PI and Ciita- the costimulatory factors and adherent molecule, includ- PIV in adipocytes were restrained by ADM2 under the ing Cd40, Cd80, Cd86, and Icam1, in 3T3-L1 adipocytes basal and IFNg-treated conditions (Fig. 4A). These results (Fig. 3B). Furthermore, the ADM2 treatment inhibited indicate that ADM2 inhibits MHCII expression via direct the expression of MHCII mRNA and protein levels in downregulation of Ciita-PI and Ciita-PIV transcription. the primary adipocytes in a dose-dependent manner un- The precise molecular mechanism by which ADM2 der both basal and IFNg-induced conditions (Fig. 3C–F). downregulated CIITA expression in adipocytes was then Immunofluorescence staining further validated that the determined. It is reported that Blimp1 is a powerful IFNg-induced upregulation of MHCII in adipocytes was transcription factor that negatively regulates MHCII abolished after ADM2 treatment (Fig. 3G). The image-capture expression and downregulates all three Ciita transcripts flow cytometry analysis also showed that the ADM2 treat- (24–26). Our results showed that ADM2 markedly in- ment markedly reversed the IFNg-induced upregulation of creased the expression of Blimp1 mRNA and protein in MHCII protein level at the adipocyte membrane (Fig. 3H). adipocytes in a time-dependent manner (Fig. 4B and C). For further identification of the role of ADM2 in Consistently, the aADM2-tg mice displayed a striking in- adipocyte-mediated antigen presentation function, an anti- crease of Blimp1 mRNA and protein levels in the eWAT genpresentationassaywasperformed in vitro. 3T3-L1 compared with those of the WT mice on an NCD or a adipocytes were pretreated with IFNg and ADM2 and were 4w-HFD (Fig. 4D and E and Supplementary Fig. 6A and B). subsequently cocultured with naïve T cells isolated from For determination of whether Blimp1 is involved in OTII mice with ovalbumin (OVA). The ability of adipocytes ADM2 inhibition of CIITA expression, Blimp1 expres- to activate naïve T cells was assessed by the levels of IL2 and sion was knocked down in adipocytes with a specific IFNg secreted into the culture media by the T cells. The small interfering (si)RNA. The knockdown efficiency of IFNg-induced secretion of IL2 and IFNg was partially re- BLIMP1 expression in adipocytes was .50% at the pro- versed by ADM2 treatment (Fig. 3I and J). Overall, these tein level (Fig. 4F). The ADM2-mediated inhibition of results indicate that ADM2 prevents the activation of T cells the IFNg-induced MHCII expression was totally abol- in WAT primarily by downregulating MHCII expression in ished after the Blimp1 siRNA treatment (Fig. 4G). Col- adipocytes. lectively, these results suggest that the downregulation of the CIITA transcription viatheincreaseofBlimp1 Inhibition of MHCII Expression in Adipocytes expression mediates the ADM2-mediated inhibition of by ADM2 Results From the Downregulation of Ciita MHCIIexpressioninadipocytes. Transcription via an Increase in Blimp1 Expression In atypical APCs, IFNg is the only well-characterized in- ADM2 Restrained the Blimp1-CIITA-MHCII Axis ducer of MHCII expression (23). For exploration of the Through the CRLR/RAMP1-cAMP Pathway mechanism by which ADM2 reduced the expression of For validation that the CRLR/RAMPs that are responsible MHCII in adipocytes, whether the ADM2-inhibited expres- for the ADM2-mediated inhibition of MHCII expression, sion of MHCII depended on IFNg pretreatment was exam- three different peptide antagonists—ADM217–47 for the ined. ADM2 was observed to inhibit the basal expression of inhibition of CRLR/RAMP1, -2, and 3; CGRP8–37 for the MHCII in primary adipocytes without IFNg treatment inhibition of CRLR/RAMP1; and ADM22–52 for the inhi- (Fig. 3C–E). Moreover, the effect of ADM2 on the IFNg bition of CRLR/RAMP2 and -3—were used. Pretreatment signaling pathway was also detected. The IFNg-induced with ADM217–47 and CGRP8–37 eliminated the ADM2- phosphorylation of signal transducer and activator of mediated inhibition of the IFNg-induced MHCII expres- transcription 1 (STAT1) remained similar after ADM2 sion as well as increased Blimp1 expression in adipocytes treatment (Supplementary Fig. 5A), suggesting that the but not ADM22–52 (Fig. 5A–D). These results imply that ADM2-mediated inhibition of MHCII was indepen- CRLR/RAMP1 might mainly mediate the effects of ADM2 dent of the IFNg signaling pathway. CYT387, an IFNg on the Blimp1-CIITA-MHCII axis. It is reported that the receptor adaptor protein Jak1/2 inhibitor, was also used activation of CRLR/RAMP1 triggers various signal trans- to block the IFNg signal pathway (Supplementary Fig. duction pathways (27). In the current study, two signaling 5B). CYT387 blocked both the basal and IFNg-induced pathways were firstly determined with the following in- MHCII expression in adipocytes (Supplementary Fig. hibitors: LY294002 to inhibit Akt and compound C to 5C–E). However, ADM2 had a further inhibitory effect inhibit AMPK. The ADM2-mediated downregulation of on MHCII expression after pretreatment of CYT387 (Sup- MHCII and upregulation of Blimp1 were not inhibited plementary Fig. 5C–E), which suggesting that the inhibi- by LY294002 or compound C (Fig. 5F–I). CRLR/RAMP1, tion of MHCII expression by ADM2 is independent of as a G-protein–coupled receptor complex, increased the IFNg. cellular cAMP level (Fig. 5E). Blocking the cAMP pathway 2348 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

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-L1 adipocytes 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 analysis of 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 (bottom panel) of the MHCII protein. The differentiated adipocytes were treated with control, IFNg (5 ng/mL), or IFNg (5 ng/mL) + indicated doses of diabetes.diabetesjournals.org Zhang and Associates 2349 by Rp-cAMPS eliminated the ADM2-mediated inhibition of AKT (S.Y.-Z., Y.L., H.Z., C.J., X.W., unpublished data). Taken the MHCII expression and Blimp1 upregulation (Fig. 5F–I). together, these results indicate that the protective role of These results reveal that ADM2 inhibits the Blimp1-CIITA- ADM2 in 4w-HFD–induced insulin resistance in eWAT de- MHCII axis in a CRLR/RAMP1-cAMP–dependent manner. pends primarily on the inhibition of MHCII in adipocytes.

Inhibition of Adipocyte MHCII Expression Mainly Mediates the Improvements of Early Obesity-Induced DISCUSSION Adipose Insulin Resistance by ADM2 In the current study, we found that ADM2 exerts metabolic For further identification of the role of adipocyte MHCII in benefits on early obesity-induced adipose inflammation the ADM2-induced early adipose insulin resistance improve- and insulin resistance by inhibiting MHCII expression in ments, MKO mice were used. The levels of the MHCII adipocytes (Fig. 7). During the pathogenesis of obesity, mRNA and protein were almost undetectable in the macro- adipose CD4+ proinflammatory Th1 cells were activated phages and eWAT of the MKO mice (Supplementary Fig. 7A and secreted large amounts of IFNg (6,19,31). IFNg stim- and B). However, adipose macrophages, B cells, and den- ulation substantially enhanced the expression of MHCII in dritic cells as APCs derived from bone marrow infiltrate adipocytes. Subsequently, adipocyte MHCII-mediated anti- into adipose tissue under physiological and pathological con- gen presentation activated more proinflammatory CD4+ Th ditions (28–30). The antigen-presenting function of these cells. ADM2 was observed to upregulate Blimp1 expres- cells in the eWAT should be considered. Therefore, the he- sion by activating the CRLR/RAMP1-cAMP pathway. The matopoietic cells from the WT mice were transplanted into Blimp1-CIITA-MHCII axis mediated ADM2-induced im- the WT and MKO mice to generate the chimeric WT and provement in early obesity-induced adipose inflammation MKO mice (WT-WT and WT-MKO) (Supplementary Fig. and insulin resistance. These findings demonstrate that 7C). PCR analysis confirmed the highly chimeric tissue in ADM2 is a potential drug candidate for early obesity- WT-MKO mice (Supplementary Fig. 7D). induced inflammation and insulin resistance. The WT-WT and WT-MKO mice were treated with vehicle In obese mice, the adipose T cells exhibited antigen- or ADM2 subcutaneously via mini-pumps and concurrently specific expansion, and the Th1 proportion was strikingly fed a 4w-HFD. The ADM2 treatment substantially increased increased (32). The MHCII, CD40L, and CD80/86 sys- the plasma ADM2 level in the WT-WT and WT-MKO mice at temic knockout mice displayed a lower susceptibility to the end of the fourth week (Supplementary Fig. 7E). And the HFD-induced adipose inflammation, suggesting that APC- ADM2 treatment had no effect on the body weight gain of induced CD4+ T-cell activation is required for adipose in- the WT-WT and WT-MKO mice fed a 4w-HFD (Supplemen- flammation (19,33,34). However, the exact type of APC tary Fig. 7F). However, ADM2 noticeably ameliorated insulin that presents the antigen to the adipose T cells in early resistance in the WT-WT mice but not in the WT-MKO mice obesity remains largely unknown. Previous results show (Fig. 6A and B). No significant difference in the fasting that adipocytes have antigen presentation function and plasma glucose levels was observed in the WT-WT and might be the APCs in WAT (19,22). Consistently, we also WT-MKO mice after the ADM2 treatment (Fig. 6C). The found the expression of MHCII in adipocytes. fasting plasma insulin level and HOMA-IR were substantially Preadipocytes share a lot of features with fibroblasts and reduced in the ADM2-treated WT-WT mice comparatively macrophages (35,36). Both macrophages and fibroblasts have (Fig. 6D and E).However,theWT-MKOmicewererelatively been shown to express MHCII after IFNg treatment (23). unresponsive to the metabolic benefits of the ADM2 treat- Therefore, it is difficult to exclude whether the expression of ment (Fig. 6D and E). ADM2 treatment markedly increased MHCII induced by IFNg in differentiated adipocytes is derived the basal level of AKT phosphorylation in the WT-WT mice. from undifferentiated preadipocytes. Here we isolated primary Although the AKT phosphorylation level was increased in adipocytes from eWAT of rat and treated with IFNg.IFNg WT-MKO mice compared with WT-WT mice, ADM2 treat- markedly upregulated MHCII expression. MHCII expression ment cannot further increase the phosphorylation level of was 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 of MHCII proteins. MHCII was stained in green, lipid was stained in red, and nucleus was stained in blue. The differentiated adipocytes were treated 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 flow cytometry. The primary adipocytes were treated with control, ADM2 (20 nmol/L), IFNg (5 ng/mL), or IFNg (5 ng/mL) + ADM2 (20 nmol/L) for 16 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-way ANOVA 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-way ANOVA with Tukey correction, *P < 0.05, **P < 0.01, compared with control; ##P < 0.01, compared with OVA; and §P < 0.05, compared with OVA + IFNg. 2350 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

Figure 4—The inhibition of MHCII expression in adipocytes by ADM2 results from the downregulation of Ciita transcription via an increase in Blimp1 expression. A: qPCR analysis of the mRNA levels of different transcripts of Ciita. The differentiated 3T3-L1 adipocytes were treated 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 the mRNA levels of Blimp1. The differentiated 3T3-L1 adipocytes were treated with ADM2 (20 nmol/L) for indicated hours (n = 6). C: Western blot analysis (top panel) and quantitation (bottom panel) of the BLIMP1 protein. The differentiated 3T3-L1 adipocytes were treated with ADM2 (20 nmol/L) for indicated hours (n = 5). The relative protein levels were normalized to that of the control. D: qPCR analysis of the mRNA levels of Blimp1 in the eWAT. E: Western blot analysis (top panel) and quantitation (bottom panel) of the BLIMP1 protein in the eWAT. The relative protein levels were normalized to that of the WT mice fed a 4w-HFD. F: Western blot analysis (top panel) and quantitation (bottom panel) of the BLIMP1 protein. The differentiated adipocytes were transfected with scramble siRNA (si-Scramble) or Blimp1 siRNA (si-Blimp1) for 36 h (n = 4). The relative protein levels were normalized to that of the scramble siRNA. G: qPCR analysis of the mRNA levels of Rt1-Db, Cd74, and Ciita. The differentiated adipocytes were transfected with scramble siRNA or Blimp1 siRNA for 24 h and were 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 and aADM2-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 are presented 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-tailed Student 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 compared with the scramble siRNA. diabetes.diabetesjournals.org Zhang and Associates 2351

Figure 5—ADM2 restrained the Blimp1-CIITA-MHCII axis through the CRLR/RAMP1-cAMP pathway. A–C: qPCR analysis of the mRNA levels 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 of the 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 or ADM2 (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 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 inhibitors 2352 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

Figure 6—The inhibition of adipocyte MHCII expression mainly mediates the improvements of early obesity-induced adipose insulin resistance 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 mice transplanted 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 6 SEM. Two-tailed Student t test, **P < 0.01, compared with the vehicle-treated mice. other groups did not observe this phenotype in the mice fed and not all adipocytes express MHCII. The immunofluores- an NCD (28,37). This might result from the fact that these cence staining might miss the MHCII expression in WAT. mice were not fed with an HFD and the expression of MHCII Although adipocytes might be the APC-presenting was too low to be observed. In addition, WAT is not uniform antigen 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 data are 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 Tukey correction, **P < 0.01 compared with the control; #P < 0.05, ##P < 0.01 compared with ADM2. E: Two-tailed Student t test, *P < 0.05 compared with the control. diabetes.diabetesjournals.org Zhang and Associates 2353

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 Blimp1 expression in a CRLR/RAMP1-cAMP–dependent manner. 7: The upregulated Blimp1 inhibits MHCII expression and improves early obesity-induced adipose inflammation and insulin resistance.

during obesity is still unknown. MHCII is believed to and insulin resistance. Therefore, the role of MHCII in present exogenous antigens, but nearly 20–30% of anti- different cells and under different conditions needs to gens presented by APCs in a MHCII-dependent manner be investigated further. are self-antigens (38). Previous results showed that in ADM2 is highly expressed in WAT (43). Our unpub- atherosclerosis heat shock protein 60, oxidized LDL and lished data showed that ADM2 was expressed in adipo- b2-glycoprotein I act as self-antigens (39–41). During the cytes. In addition, ADM2 is expressed in vascular tissue onset of adipose expansion in obesity, oxidative stress (44), which might be another source of ADM2 in WAT. and endoplasmic reticulum stress induce the generation ADM2 shares receptor complexes with CGRP and ADM. of lipid-binding protein, oxidative modified protein, and Here, ADM2 was demonstrated to inhibit MHCII expres- misfolded protein, which might be the self-antigen to sion in adipocytes primarily through CRLR/RAMP1, which MHCII. is the classical pharmacological CGRP1 receptor (45). In The inhibition of adipocyte MHCII antigen presen- agreement with our present findings, some reports have tation led to the ADM2-mediated improvements in 4w- shown that CGRP reduced MHCII expression in dendritic HFD–induced early adipose inflammation, as revealed cells and hair follicle dermal papilla (46,47), although the by the attenuated benefits of ADM2 in the WT-MKO exact mechanism remains unclear. mice. The WT-MKO mice also displayed an improve- The related MHCII family genes are tightly regulated ment of insulin resistance compared with that in the primarily at the transcription levels by CIITA. Three CIITA WT-WT mice, which is similar to that of the WT-WT transcripts were expressed in different cell types. In addition mice treated with ADM2 and cannot be further im- to the constitutive Ciita-PI and Ciita-PIII expression by pro- proved by ADM2. This observation is consistent with fessional APCs, IFNg was shown to increase Ciita-PIV ex- previous results and suggests that the protective effect pression (48). Ciita-PI and Ciita-PIV were expressed in of ADM2 is at least partially through the inhibition of adipocytes, suggesting that adipocytes may have some fea- MHCII expression in adipocytes (19). However, other tures of professional APCs. Previous studies have shown evidence revealed that adipose macrophages also pre- that IL4, IL10, and TGF-b inhibit MHCII expression, while sent antigen and play a key role in the late stage of the exact molecular mechanism has not been described obesity-induced adipose T-cell activation and insulin re- (49,50). In this study, ADM2 downregulated MHCII expres- sistance (28,42). It should be noted that the role of sion as a result of increased Blimp1 expression through the macrophagesinadiposeantigenpresentationcannot CRLR/RAMP1-cAMP pathway, which provided the exact mole- be excluded in obesity-induced adipose inflammation cular mechanism of MHCII downregulation. 2354 Adrenomedullin 2 Inhibits MHCII in Adipocytes Diabetes Volume 65, August 2016

15. Dai XY, Cai Y, Mao DD, et al. Increased stability of phosphatase and tensin Funding. This work was supported by the National Natural Science Founda- homolog by intermedin leading to scavenger receptor A inhibition of macro- tion of the P.R. of China (91439206 and 31230035 to X.W. and 81470554 to phages reduces atherosclerosis in apolipoprotein E-deficient mice. J Mol Cell C.J.), the National Basic Research Program (973 Program) of the P. R. of China Cardiol 2012;53:509–520 (2012CB518002 to M.-J.X.), the 111 Project of the Chinese Ministry of Edu- 16. Dai XY, Cai Y, Sun W, et al. Intermedin inhibits macrophage foam-cell for- cation (B07001), and the Center for Molecular and Translational Medicine mation via tristetraprolin-mediated decay of CD36 mRNA. Cardiovasc Res 2014; (BMU20140475 to G.L.). 101:297–305 Duality of Interest. No potential conflicts of interest relevant to this article 17. Zhang X, Gu L, Chen X, et al. 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