Fen Zhuge,1 Yinhua Ni,1 Mayumi Nagashimada,1 Naoto Nagata,1 Liang Xu,1

Naofumi Mukaida,2 Shuichi Kaneko,3 and Tsuguhito Ota1,3

DPP-4 Inhibition by LinagliptinAttenuates Obesity-RelatedInflammation and Insulin Resistanceby Regulating M1/M2 MacrophagePolarizationDiabetes 2016;65:2966–2979 | DOI: 10.2337/db16-0317

Dipeptidyl peptidase 4 (DPP-4) cleaves a large numberof chemokine and peptide hormones involved in theregulation of the immune system. Additionally, DPP-4may also be involved in macrophage-mediated in-flammation and insulin resistance. Thus, the currentstudy investigated the effect of linagliptin, an inhibitorof DPP-4, on macrophage migration and polarizationin white adipose tissue (WAT) and liver of high-fatdiet–induced obese (DIO) mice. DPP-4+ macrophages inlean and obese mice were quantified by fluorescence-activated cell sorting (FACS) analysis. DPP-4 waspredominantly expressed in F4/80+ macrophages incrown-like structures compared with adipocytes inWAT of DIO mice. FACS analysis also revealed that,compared with chow-fed mice, DIO mice exhibited asignificant increase in DPP-4+ expression in cells withinadipose tissue macrophages (ATMs), particularly M1 ATMs.Linagliptin showed a greater DPP-4 inhibition andantioxidative capacity than sitagliptin and reducedM1-polarized macrophage migration while inducingan M2-dominant shift of macrophages within WATand liver, thereby attenuating obesity-induced inflam-mation and insulin resistance. Loss of macrophageinflammatory protein-1a, a chemokine and DPP-4substrate, in DIO mice abrogated M2 macrophage-polarizing and insulin-sensitizing effects of linagliptin.Therefore, the inhibition of DPP-4 by linagliptin reducedobesity-related insulin resistance and inflammation byregulating M1/M2 macrophage status.

Obesity activates the innate immune system with sub-sequent recruitment of immune cells such as macrophagesand T cells into metabolic tissues, leading to the devel-opment of insulin resistance (1). Macrophage recruitmentand polarization are pivotal in obesity-induced inflamma-tion and insulin resistance (2–5). However, treatment op-tions that target immune cells with the aim of halting thedevelopment of insulin resistance and type 2 diabetes re-main limited.

Dipeptidyl peptidase 4 (DPP-4) inhibitors are ef-fective in the treatment of type 2 diabetes, as theymaintain blood glucose levels through degradation ofincretin peptides, glucagon-like peptide 1 (GLP-1) andglucose-dependent insulinotropic polypeptide (6). Nume-rous investigations have detailed the effects of DPP-4inhibitors on insulin and/or glucagon secretion, butlittle evidence indicates that DPP-4 inhibitors directlyimprove chronic inflammation. Although several animalstudies suggest that DPP-4 inhibition may attenuateobesity-associated inflammation and cardiovascular dis-ease (7,8), mechanisms describing these actions have yetto be elicited.

DPP-4, originally known as T-cell surface marker CD26,is widely expressed in many cells, including immune cells(9). DPP-4 cleaves a large number of chemokine and pep-tide hormones involved in the regulation of the immunesystem, inferring a role for DPP-4 in the pathogenesis of

1Department of Cell Metabolism and Nutrition, Brain/Liver Interface MedicineResearch Center, Kanazawa University, Kanazawa, Ishikawa, Japan2Division of Molecular Bioregulation, Cancer Research Institute, KanazawaUniversity, Kanazawa, Ishikawa, Japan3Department of Disease Control and Homeostasis, Kanazawa University GraduateSchool of Medical Science, Kanazawa, Ishikawa, Japan

Corresponding author: Tsuguhito Ota, tota@staff.kanazawa-u.ac.jp.

Received 12 March 2016 and accepted 6 July 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0317/-/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, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

2966 Diabetes Volume 65, October 2016

OBESITY

STUDIES

inflammation (6,10). Earlier work has focused on the func-tion of DPP-4 as a surface protease involved in T cell activa-tion (11). Recent studies show that DPP-4 release correlateswith adipocyte size or visceral adiposity, and thus, DPP-4 hascome to be considered as an adipokine with potential toimpair insulin sensitivity (12,13). In addition, DPP-4 on den-dritic cell/macrophages contributes to potentiating inflam-mation of adipose tissue in obesity (14). However, the roleof DPP-4 in macrophage-mediated inflammation and insulinresistance remains largely unknown.

Linagliptin is a DPP-4 inhibitor with a high affinity forDPP-4 in various tissues (15–17). In rodents, linagliptinmore effectively reduces DPP-4 activity in tissue and atten-uates inflammatory properties under conditions of vasculardysfunction than does sitagliptin (18,19). Therefore, wehypothesized that DPP-4 plays a role in regulating macro-phage activation in response to obesity and that DPP-4inhibition may attenuate obesity-induced inflammation.In the current study, we demonstrated that DPP-4 is pre-dominantly expressed in M1-polarized macrophages inwhite adipose tissue (WAT) of high-fat (HF) diet–inducedobese (DIO) mice. Furthermore, we present evidence sug-gesting that DPP-4 inhibition attenuates obesity-relatedinsulin resistance and inflammation by regulating bothmacrophage recruitment and M1/M2 status in DIO mice.

RESEARCH DESIGN AND METHODS

Mice and DietsEight-week-old male C57BL/6J mice (Charles River Lab-oratories, Yokohama, Japan) were divided into fourgroups and fed for 8 weeks as follows: 1) normal chow(NC) with 10% of calories from fat (CRF-1) (Charles RiverLaboratories); 2) NC containing 0.003% linagliptin (NC+Lina)(Boehringer Ingelheim Pharma GmbH & Co. KG, Biberachan der Riss, Germany); 3) HF diet, consisting of 60% fat(Research Diets, New Brunswick, NJ); and 4) HF dietwith 0.003% linagliptin (HF+Lina). To compare the ef-fects of linagliptin and sitagliptin on DIO mice, groups of8-week-old C57BL/6J mice were fed with NC, HF, HF+Lina,or an HF diet with 0.01% sitagliptin (HF+Sita) (Sequoia,Oxford, U.K.) for 14 weeks. MIP-1a2/2 mice were providedby N. Mukaida (Kanazawa University, Kanazawa, Japan)(20). Eight-week-old male MIP-1a2/2 mice were fed withthe HF or HF+Lina for 8 weeks. All animal procedures wereperformed in accordance with the standards set forth inthe Guidelines for the Care and Use of Laboratory Animalsat Kanazawa University.

DPP-4 Activity and GLP-1 DeterminationsC57BL/6J mice were dosed with vehicle, linagliptin (3 mg/kg),or sitagliptin (10 mg/kg) by gavage, once daily for 5 days.Blood was collected before or 2 h and 24 h after drugadministration. Tissue and plasma DPP-4 activities weredetected with DPP-4 assay kit (BioVision, Milpitas, CA).Plasma active GLP-1 concentration was determined usingenzyme-linked immunosorbent assay ELISA (Linco Research,St. Charles, MO).

Histological and Immunofluorescence StainingParaffin wax–embedded epididymal WAT (eWAT) and liversections were stained with hematoxylin and eosin (H&E) andimmunohistochemically stained for F4/80, as described pre-viously (5,21). For immunofluorescence staining, epididymalfat pads were stained with perilipin (Sigma-Aldrich, Tokyo,Japan), F4/80 (Abcam, Cambridge, U.K.), CD11c (AbD Sero-tec, Hercules, CA), CD206 (AbD Serotec), and DPP-4 (R&DSystems, Minneapolis, MN), followed by secondary antibodies(Alexa Fluor 488, Alexa Fluor 594, Cy3, and CF 594; JacksonImmunoResearch Laboratories, West Grove, PA) using stan-dard techniques.

Fluorescence-Activated Cell Sorting AnalysisStromal vascular fraction (SVF) and nonparenchymal cellswere isolated as described previously (5,21,22) and incu-bated with Fc-Block (BD Bioscience), followed by incubationwith fluorochrome-conjugated antibodies (SupplementaryTable 1). Cells were analyzed using a FACSAria II (BD Bio-sciences, San Jose, CA). Data analysis and compensationwere performed using FlowJo (Tree Star, Ashland, OR).

Lipid, Glucose, and Insulin DeterminationsPlasma triglycerides (TG), total cholesterol (TC), nones-terified fatty acids (NEFA), aspartate aminotransferase(AST), alanine aminotransferase (ALT), glucose, insulinlevels and hepatic TG, thiobarbituric acid reactive sub-strates (TBARS) concentrations, and urine 8-hydroxy-29-deoxyguanosine (8-OHdG) content were measured asdescribed previously (21,22). Glucose tolerance test (GTT)was conducted after an overnight fast, and then micewere injected with 2 g/kg i.p. glucose. Insulin tolerancetest (ITT) was performed after a 4-h fast, and mice wereinjected with 0.5 units/kg i.p. human insulin.

Quantitative Real-Time PCRQuantitative real-time PCR was performed as describedpreviously (21,22). Primers used for real-time PCR areshown in Supplementary Table 2.

ImmunoblotsImmunoblots were conducted as described previously(5,21). Antibodies are shown in Supplementary Table 3.

Culture of Peritoneal MacrophagesPeritoneal macrophages were isolated and cultured as de-scribed previously (5,21). After starving for 6 h, the cellswere coincubated with 100 ng/mL lipopolysaccharide (LPS)(Sigma-Aldrich) or 10 ng/mL interleukin (IL)-4 (Sigma-Aldrich)and DPP-4 (100, 200, and 500 ng/mL) or linagliptin (50, 100,or 200 nmol/L) for 16 h and then harvested. Intracellularreactive oxygen species (ROS) formation was determinedby 5-(and-6)-chloromethyl-29,79-dichlorodihydrofluoresceindiacetateacetylester fluorescent probe as described previously(23).

Statistical AnalysisAll data are presented as means 6 SEM. Differences be-tween the mean values of two groups were assessed usinga two-tailed Student t test. Differences in mean values

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between more than two groups were determined usingANOVA. A P value ,0.05 was considered significant.

RESULTS

Accumulation of DPP-4+ Macrophages Increasesin Fat of DIO miceDPP-4 activity in both the plasma and eWAT signifi-cantly increased in HF-fed mice compared with NC-fedmice (Fig. 1A). Expression of mRNA for Dpp-4 was higher inthe SVF than adipocyte fraction from both NC and HF-fedmice (Fig. 1B). Moreover, Dpp-4 expression in the SVF in

HF-fed mice was markedly higher than that in NC-fedmice.

Immunofluorescence analysis of eWAT in HF-fed micerevealed that DPP-4 was expressed by F4/80+ macrophagesin crown-like structures, but it was poorly expressed byperilipin+ adipocytes (Fig. 1C). Interestingly, most ofCD11c+ M1 macrophages expressed DPP-4, whereas fewerCD206+ M2 macrophages expressed DPP-4 (Fig. 1C).Fluorescence-activated cell sorting (FACS) analysis revealedthat the total number of adipose tissue macrophages(ATMs) was 3.3-fold higher in HF-fed mice than the

Figure 1—DPP-4+ ATMs increase in HF diet–induced obesity. C57BL/6J mice fed with NC or HF diet for 8 weeks. A: DPP-4 activity inplasma and eWAT. B: DPP-4 mRNA expression in adipocyte fraction and SVF. C: Immunofluorescence staining for F4/80, CD11c, CD206,perilipin (green), and DPP-4 (red) in eWAT from DIO mice. Scale bars, 50 mm. D: Quantification of DPP-4+ ATMs and DPP-4+ M1 or M2ATMs in eWAT from NC diet– or HF diet–fed mice by FACS analysis. Gating strategies to determine ATMs are depicted in SupplementaryFig. 1, and fluorescence-minus-one (FMO) controls were used for gating highly pure populations of DPP-4+ macrophages. Black arrowsindicate DPP-4+ macrophages with high fluorescent intensity. E: Relative abundance of DPP-42 M1 or DPP-42 M2 and DPP-4+ M1 or DPP-4+ M2 ATMs in eWAT. n = 6–8. *P < 0.05, **P < 0.01 vs. mice fed the NC diet.

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NC-fed mice (Supplementary Fig. 1). When gated for DPP-4/CD26, ATMs exhibited a higher level of DPP-4 expres-sion in response to HF diet (Fig. 1D). Furthermore, HF-fedmice exhibited a significant increase in the percentage ofDPP-4+ cells within CD11c+CD2062 (M1) ATMs comparedwith NC-fed mice. However, the percentage of DPP-4+

cells within CD11c2CD206+ (M2) ATMs was unaffectedby HF feeding (Fig. 1D). Among ATMs, both DPP-42 M1and DPP4+ M1 ATMs increased with HF feeding, andyet DPP-42 M2 ATMs decreased, with the expression ofDPP4+ M2 ATMs remaining unchanged (Fig. 1E). Collec-tively, these results support the proposition that DPP-4+

macrophages infiltrate the WAT of obese mice and thatDPP-4+ M1 ATMs accumulate in response to obesity.

DPP-4 Regulates M1/M2 Polarization in MacrophagesGiven the association between ATM accumulation andDPP-4 activation in obese mice, we next determined whetherDPP-4 directly regulates macrophage activation and/orpolarization. Exposure of cultured peritoneal macrophages toDPP-4 increased mRNA expression for M1 markers (TNFa,MCP-1, and RANTES) and intracellular ROS production uponstimulating with LPS in a dose-dependent fashion (Fig. 2A).The M1 polarization and increased ROS generation in mac-rophages were diminished in the presence of linagliptin (Fig.2B). In contrast, exposure of macrophages to DPP-4 reducedM2 markers of mRNA expression (Arg1, Chi3l3, and Mgl2)and increased intracellular ROS production in a dose-dependent manner when incubated with IL-4 (Fig. 2C).Exposure to linagliptin increased expression of M2 mac-rophage markers and decreased ROS generation (Fig. 2D).DPP-4 alone had no effect on mRNA expression of macro-phage markers or ROS production (Supplementary Fig. 2Aand B). Additionally, linagliptin (50–200 nmol/L) decreasedthe expression of LPS-induced M1 markers mRNA expressionand ROS generation. In contrast, linagliptin augmented IL-4–induced M2 marker expression and decreased ROS produc-tion in a dose-dependent manner (Supplementary Fig. 2C andD). Taken together, these results suggest that DPP-4 directlyregulates M1/M2 macrophage polarization following LPS orIL-4 stimulation, and linagliptin can suppress M1-polarizedactivation and induce M2-polarized activation, at least inpart, by regulating intracellular ROS generation.

Linagliptin Ameliorates HF Diet–Induced InsulinResistance in a Dose-Dependent MannerTo determine the effective doses of linagliptin, C57BL/6Jmice were fed the NC, HF, or HF diets containing 0.003%or 0.01% linagliptin for 8 weeks. Administrating linagliptindid not affect weight or adiposity in HF-fed mice (Supple-mentary Table 4 and Supplementary Fig. 3A). Treatmentwith linagliptin decreased plasma TG, TC, AST, and ALTlevels in a dose-dependent fashion and tended to decreaseplasma NEFA levels in HF mice (Supplementary Table 4).Increased plasma DPP-4 activity in HF-fed mice wasinhibited by 89.8% and 98.4% in the two doses used,respectively (Supplementary Fig. 3B). Evidence of HFdiet–induced glucose intolerance, insulin resistance, and

hyperinsulinemia appeared to follow a dose-dependentpattern of inhibition by linagliptin (Supplementary Fig.3C–E). Greater than 80% inhibition of DPP-4 is effective,as the DPP-4 inhibitor class (24) and insulin-sensitizingeffect were observed in the 0.003% linagliptin-treatedgroup; therefore, subsequent experiments were performedusing that dose (named HF+Lina).

Linagliptin Protects Mice From HF Diet–InducedImpaired Glucose Homeostasis and Hepatic SteatosisNC and NC+Lina mice had similar body, liver, and eWATweights as did the HF and HF+Lina mice (Fig. 3A). How-ever, the histological analysis revealed severe lipid accu-mulation, which was decreased markedly by linagliptin, inthe livers of mice fed the HF diet (Fig. 3B). Linagliptinconsistently reduced liver TG in the HF-fed mice (Fig. 3C).These findings were associated with the suppression oflipogenic genes expression (SREBP-1c, FAS, and SCD1)and upregulation of mitochondrial fatty acid b-oxidationgenes (PPARa, Cpt1a, and LCAD) (Fig. 3D).

GTT indicated that linagliptin had no effect on glucosetolerance in NC-fed mice. However, HF diet–induced glu-cose intolerance was suppressed significantly by linagliptin(Fig. 3E). ITT also showed that HF+Lina mice had increasedinsulin sensitivity when compared with HF mice (Fig. 3F).Linagliptin also suppressed hyperinsulinemia in both fast-ing and fed states (Fig. 3G) as well as enhancing insulinsignaling in the eWAT and liver of HF-fed mice (Fig. 3H).Thus, linagliptin protected mice against diet-induced he-patic steatosis, insulin resistance, and glucose intolerance.

Linagliptin Attenuates Inflammation in Fat and Liverof DIO MiceWe next investigated the effect of linagliptin on adiposetissue inflammation. Infiltration of macrophages into hy-pertrophied adipose tissue and crown-like structures thatwere induced by HF diet decreased markedly in HF+Linamice according to immunostaining and mRNA expression ofF4/80 (Fig. 4A and B). Inflammatory cytokines derived fromM1 macrophages, including TNFa, IL-6, and IL-1b, weredecreased in eWAT of HF+Lina mice compared with HFmice (Fig. 4B). In addition, the aberrant expression of adi-pokines such as adiponectin and leptin were improved bylinagliptin (Fig. 4B). These findings were associated withattenuated nuclear factor-kB (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK), Jun NH2-terminal kinase(JNK), and extracellular signal–related kinase (ERK) phos-phorylation in eWAT of DIO mice (Fig. 4C and Supplemen-tary Fig. 4A). Linagliptin also markedly decreased thenumber of F4/80+ cells (Fig. 4D and E) and reduced geneexpression of proinflammatory cytokines and inflammatorysignaling in liver (Fig. 4E and F and Supplementary Fig. 4B).

Effects of Linagliptin and Sitagliptin on DPP-4 Activityand Oxidative StressStudies have shown that linagliptin at doses of 3–5 mg/kgand sitagliptin at doses of 10–50 mg/kg produce equivalentreductions in plasma glucose, with linagliptin eliciting agreater effect on vascular dysfunction in rodents (18,19).

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Figure 2—DPP-4 enhances M1 polarization and inhibits M2 polarization in macrophages in an ROS-dependent manner. A: DPP-4upregulates LPS-induced M1 marker mRNA expression and increases intracellular ROS production in peritoneal macrophages. B: Lina-gliptin (Lina) suppresses the increase of DPP-4–induced (500 ng/mL) M1 marker mRNA expression and ROS production by LPS stimu-lation. C: DPP-4 inhibits IL-4–induced M2 marker mRNA expression and increases ROS in M2-polarized macrophages. D: Linagliptinrestores the decrease of DPP-4–induced (500 ng/mL) M2 marker mRNA expression and decreases ROS production in the presence of IL-4.Peritoneal macrophages were isolated and coincubated with LPS (100 ng/mL) or IL-4 (10 ng/mL) and DPP-4 (100–500 ng/mL) or linagliptin(200 nmol/L) for 16 h. n = 6. *P < 0.05, **P < 0.01 vs. control incubations; †P < 0.05, ††P < 0.01 vs. LPS- or IL-4–stimulated incubations;‡P < 0.05, ‡‡P < 0.01 vs. LPS/IL-4 and DPP-4 and/or linagliptin-stimulated incubations.

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Figure 3—Linagliptin alleviates diet-induced insulin resistance and hepatic steatosis. A: Weight gain and tissue weights of C57BL/6J micefed an NC or HF diet without or with 0.003% linagliptin (Lina) from age 8 to 16 weeks. B: H&E-stained liver sections from mice. Scale bars,100 mm. C: Liver TG content. D: mRNA expression of lipogenic regulator genes, fatty acid synthesis genes, and b-oxidation genes in theliver. E: GTT in mice fed NC or NC+Lina diet (top, n = 5) and HF or HF+Lina diet (bottom, n = 8) at 16 weeks of age. F: ITT in mice fed the NCor NC+Lina (top, n = 5) and HF or HF+Lina (bottom, n = 8) diet. G: Plasma insulin levels. H: Immunoblots of phosphorylated Tyr1146 insulinreceptor b subunit (p-IRb), IRb, phosphorylated Ser473 Akt (p-Akt), and Akt in the eWAT and liver of mice. The levels of p-IRb and p-Aktwere normalized to those of IRb and Akt, respectively. n = 5–8. *P < 0.05, **P < 0.01 vs. NC group; †P < 0.05, ††P < 0.01 vs. HF group.

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Therefore, the effects of linagliptin and sitagliptin were in-vestigated in mice. Liver and eWAT weights were unaffectedby linagliptin or sitagliptin in HF-fed mice (Table 1 and Fig.5A). Plasma lipid, AST, and ALT were decreased with lina-gliptin and similarly with sitagliptin (Table 1). Linagliptinproduced a marked reduction in hepatic steatosis and TGaccumulation in DIO mice, whereas there were only slightreductions following sitagliptin treatment (Fig. 5B). TheGTT and ITT data showed that linagliptin and sitagliptinlowered blood glucose by similar amounts (Fig. 5C and D).Hyperinsulinemia and homeostasis model assessment of

insulin resistance were also suppressed considerably byboth agents (Fig. 5E). Plasma active GLP-1 levels were in-creased by both linagliptin and sitagliptin treatment (Fig.5E).

Although DPP-4 activities in plasma and eWAT weredecreased by 94% and 92%, respectively, following linaglip-tin treatment and by 13% and 69%, respectively, followingsitagliptin treatment (Fig. 5F), hepatic DPP-4 activity wasinhibited only by linagliptin. To better understand thedifferences between linagliptin and sitagliptin on DPP-4inhibition, we conducted a short-term study (Fig. 5G).

Figure 4—Linagliptin (Lina) attenuates adipose tissue and liver inflammation in DIO mice. A: Macrophage infiltration in the eWAT of DIOmice, assessed by F4/80 immunostaining. Scale bars, 100 mm. B: mRNA expression of F4/80 and inflammatory cytokines and adipokinesin eWAT. C: Immunoblots of phosphorylated p38 MAPK (p-p38 MAPK), phosphorylated NF-kB p65 (p–NF-kB p65), phosphorylated JNK(p-JNK), phosphorylated ERK (p-ERK), and their total proteins in eWAT of mice. D: F4/80 immunostaining in the liver of mice. Scale bars,100 mm. E: mRNA expression of F4/80 and inflammatory cytokines in the liver. F: Immunoblot of p-p38 MAPK, p–NF-kB p65, p-JNK,p-ERK, and their total proteins in the liver of mice. n = 5–8. *P < 0.05, **P < 0.01 vs. NC group; †P < 0.05, ††P < 0.01 vs. HF group.

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After 5 days’ administration of each DPP-4 inhibitor,plasma DPP-4 activity was almost completely eliminated2 h after the last treatment with linagliptin and decreasedby 68% by sitagliptin. Furthermore, significant reduction ofplasma DPP-4 activity was observed only in the linagliptingroup 24 h after the last treatment. Similarly, linagliptinshowed significant reduction of DPP-4 activities in botheWAT and liver (Fig. 5G).

In parallel to the activity of DPP-4, 8-OHdG, a marker ofoxidized DNA damage, was significantly increased in theurine of HF-fed mice, but linagliptin and sitagliptin reducedthese levels by 49% and 34%, respectively (Fig. 5H). Inaddition, increased lipid peroxidation, assessed by TBARSin eWAT and liver, was suppressed by linagliptin but un-affected by sitagliptin (Fig. 5H). These findings occurred inassociation with increased mRNA expression of mitochon-drial fatty acid b-oxidation genes (PPARa, Cpt1a, LCAD,and Acox1) and decreased mRNA expression of NADPHoxidase subunits (gp91phox, p22phox, p67phox, and p47phox)in eWAT and liver of DIO mice (Supplementary Fig. 5).

DPP-4 Inhibition Causes Reciprocal Decrease in M1Macrophages and Increase in M2 Macrophages in Fatand Liver of MiceThe effect of DPP-4 inhibition on macrophage polarizationin vivo was examined. The increased total numbers ofATMs associated with HF feeding were not significantly af-fected by either linagliptin or sitagliptin treatment (Fig. 6Aand B). HF-fed mice showed a significantly higher M1 andlower M2 expression within ATMs. Linagliptin administra-tion resulted in a 28% decrease in M1 ATMs and a33% increase in M2 ATMs compared with HF feeding,which resulted in macrophage polarization toward ananti-inflammatory phenotype. In contrast, sitagliptin hadlittle effect on ATM phenotype (Fig. 6A and B).

The total number of liver macrophages (LMs) alsoincreased in mice fed the HF diet compared with NC-fedmice (Fig. 6C and D). In addition to reduced total LMcontent, both linagliptin- and sitagliptin-treated mice hadfewer M1 LMs and more M2 LMs than HF-fed mice, whichresulted in macrophage polarization toward an anti-

inflammatory phenotype in the liver (Fig. 6C and D).The total numbers of CD3+, CD4+, and CD8+ T cells ineWAT and liver increased with HF feeding, and this effectwas significantly decreased by linagliptin, whereas it wasslightly decreased by sitagliptin (Supplementary Fig. 6). Incontrast, there was a significant increase in the Ly6Chi

monocyte population in the blood of HF-fed mice relativethat of NC-fed mice (Supplementary Fig. 7A), indicatingthat there was an enhanced recruitment of inflammatorymonocytes into the eWAT and liver following HF feeding.However, a predominance of the Ly6C2 over the Ly6Chi

monocyte population was not observed in either theperipheral blood or the bone marrow of linagliptin- orsitagliptin-treated mice (Supplementary Fig. 7).

Loss of Protective Effects of DPP-4 Inhibitor onObesity-Related Inflammation and Insulin Resistancein Chemokine-Deficient MiceMuch like incretin peptides, chemokines also act as DPP-4substrates (10). Among the chemokines, macrophage in-flammatory protein (MIP)-1a becomes the most effi-cient monocyte chemoattractant after cleavage by DPP-4(25,26), which indicates that DPP-4 may regulate inflam-mation by increasing the activity of MIP-1a. In the currentstudy, HF diet–induced glucose intolerance, insulin resis-tance, and hyperinsulinemia were significantly improved inMIP-1a2/2 mice compared with wild-type mice (Supple-mentary Fig. 8A–C). Moreover, macrophage accumulationin the eWAT and liver were decreased by an MIP-1a de-ficiency with polarization toward an anti-inflammatoryphenotype (Supplementary Fig. 8D and E). To further in-vestigate whether the linagliptin-mediated amelioration ofmacrophage polarization and insulin resistance dependedon MIP-1a, MIP-1a2/2 mice were fed an HF diet eitherwith or without linagliptin for 8 weeks. Linagliptin didnot affect body weight (Fig. 7A) but significantly reducedDPP-4 activities in the plasma, eWAT, and liver by 92%,84%, and 78%, respectively (Fig. 7B); it also resulted in levelsclose to those seen in linagliptin-treated wild-type mice.However, linagliptin did not have any significant effectson glucose tolerance, insulin sensitivity, or plasma insulin

Table 1—Effects of DPP-4 inhibitors on metabolic parameters at 14 weeks of treatment

NC HF HF+Lina HF+Sita

Body weight (g) 27.6 6 0.7 43.9 6 1.3** 39.3 6 2.2** 41.7 6 2.0**

Food intake (kcal/day/kg body weight) 430.2 6 3.7 443.5 6 43.3 489.8 6 16.0 443.3 6 52.9

Liver weight (g) 1.11 6 0.03 1.41 6 0.10* 1.18 6 0.06 1.34 6 0.17

eWAT weight (g) 0.47 6 0.07 2.24 6 0.17** 2.06 6 0.14** 2.14 6 0.16**

Plasma TG (mg/dL) 26.3 6 2.2 45.9 6 5.3* 35.7 6 1.2**† 38.6 6 1.4**

Plasma TC (mg/dL) 72.6 6 2.8 144.8 6 7.6** 104.9 6 11.9*† 114.8 6 9.8**†

Plasma NEFA (mEq/L) 0.16 6 0.02 0.41 6 0.02** 0.34 6 0.02**† 0.36 6 0.02**

Plasma AST (IU/L) 14.5 6 0.6 19.1 6 0.4** 15.2 6 1.0†† 16.8 6 1.08

Plasma ALT (IU/L) 5.24 6 0.19 6.96 6 0.48* 4.25 6 0.33†† 5.15 6 0.47†

Data were obtained from 22-week-old mice on different diets. Data are presented as mean 6 SEM. n = 6–8. *P , 0.05, **P , 0.01 vs.mice fed the NC diet; †P , 0.05, ††P , 0.01 vs. mice fed the HF diet.

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levels in obese MIP-1a2/2 mice (Fig. 7C–E). The total num-ber of ATMs and the percentages of M1 and M2 macro-phages in the eWAT and liver were unaffected by linagliptin,which was in accordance with the pattern of F4/80

immunostaining (Fig. 7F and G). The effects of DPP-4 andlinagliptin on the regulation of M1/M2 polarization werenot observed in peritoneal macrophages obtained fromMIP-1a2/2 mice (Supplementary Fig. 9).

Figure 5—Effects of linagliptin and sitagliptin on insulin resistance, hepatic steatosis, and DPP-4 activity in DIO mice. A: Weight gain ofmice from age 8 to 22 weeks. B: H&E-stained liver sections and hepatic TG content. Scale bars: 100 mm. C: GTT. D: ITT. E: Plasma insulinlevels, HOMA of insulin resistance (HOMA-IR), and active GLP-1 levels. F: DPP-4 activity levels in plasma, eWAT, and liver. n = 6–8. *P <0.05, **P < 0.01 vs. NC group; †P< 0.05, ††P < 0.01 vs. HF group; ‡P< 0.05, ‡‡P< 0.01 vs. HF+Lina group. G: DPP-4 activity in plasma(before, 2 h after, or 24 h after last gavage), eWAT, and liver from mice gavaged with vehicle, linagliptin (Lina), or sitagliptin (Sita). n = 6. *P<0.05, **P < 0.01 vs. vehicle; ††P < 0.01 vs. linagliptin-treated group. H: Urine 8-OHdG levels and TBARS content in eWAT and liver. n = 6–8.*P < 0.05, **P < 0.01 vs. NC group; †P < 0.05, ††P < 0.01 vs. HF group; ‡P < 0.05 vs. HF+Lina group.

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Figure 6—Decreased M1 and increased M2 macrophages in eWAT and liver of DIO mice due to linagliptin administration. A and B: FACSanalysis of the stromal vascular cells of epididymal fat pads of mice fed the NC, HF, HF+Lina, or HF+Sita diet for 14 weeks. A: Repre-sentative plot of total macrophages (top) and expression of M1 and M2 macrophages (bottom) of mice. B: Quantification of ATMs, M1ATMs, and M2 ATMs. Data are total ATM counts, percentages of M1 ATMs, percentages of M2 ATMs, and M1/M2 ratios. C and D: FACSanalysis of the hepatic nonparenchymal cell fractions. C: Representative plot of total macrophages (top) and expression of M1 and M2 LMs(bottom). D: Quantification of total macrophage counts, percentages of M1 LMs, percentages of M2 LMs, and M1/M2 ratios. n = 6–8. *P <0.05, **P < 0.01 vs. NC group; †P < 0.05, ††P < 0.01 vs. HF group; ‡P < 0.05 vs. HF+Lina group.

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DISCUSSION

Our study provides firm evidence for DPP-4 playing acrucial role in regulating the macrophage-mediated in-flammatory response to obesity and the development ofinsulin resistance. We demonstrated how DPP-4 is pre-dominantly expressed in macrophages, particularly M1macrophages, rather than in adipocytes in WAT. We alsofound that administration of the DPP-4 inhibitor linaglip-tin attenuates oxidative stress, inflammation, and insulinresistance, at least in part, through reduction of macro-phage accumulation and alternative macrophage activation

in both WAT and liver in DIO mice. Moreover, despiteequivalent reductions in blood glucose, we showed thatlinagliptin reduces markers of oxidative stress to a greaterextent than sitagliptin. Interestingly, the apparently protectiveeffects of linagliptin are abrogated in MIP-1a–deficient mice,suggesting that linagliptin may alleviate obesity-associated in-flammation partly due to its actions on a chemokine MIP-1a–dependent mechanism.

Recent studies show that DPP-4 expression and releasefrom visceral fat is augmented in obese and insulin-resistant subjects (12–14). Furthermore, plasma DPP-4

Figure 7—Loss of protection by linagliptin against obesity-related inflammation and insulin resistance in MIP-1a2/2 mice. A: Weight gain ofMIP-1a2/2 mice on an HF diet with or without linagliptin. B: DPP-4 activities in plasma, eWAT, and liver. C: GTT. D: ITT. E: Plasma insulinlevels. F and G: F4/80 immunostaining (left) and quantification of total macrophage counts and percentages of M1 macrophages and M2macrophages (right) in eWAT (F ) and liver (G) by FACS. n = 8. Scale bars: 100 mm. **P < 0.01 vs. HF group.

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activity is positively correlated with HbA1c levels in patientswith type 2 diabetes (27,28). However, the cellular sourcesresponsible for DPP-4 in WAT remain unclear. Our findingssuggest that dominant DPP-4 expression in SVF of WAT(Fig. 1A and B) may derive from several types of stromalvascular cells, including preadipocytes, infiltrated macro-phages, and other hematopoietic cells. However, consistentwith previous findings (14), our results show that DPP-4 isexpressed mainly on F4/80+ macrophages rather than ad-ipocytes (Fig. 1B and C). FACS analysis also revealed thatDPP-4+ ATMs increase in obese mice compared with leanmice (Fig. 1D). Similar to a previous report (14), high levelsof DPP-4 were detected on adipose macrophages (Fig. 1Cand D) but not on T cells (data not shown) in obese mice.This observation suggests that DPP-4+ macrophages are amajor source of circulating DPP-4 in the obese state.

Dysregulation of M1/M2 polarization in macrophages isemerging as a central mechanism underlying the pathogen-esis of obesity and comorbidities such as insulin resistanceand nonalcoholic fatty liver disease (29,30). Deletion of M1macrophages normalizes sensitivity to insulin in obese mice(3,31), whereas reducing the number of M2 macrophagespredisposes lean mice to insulin resistance (4). M2-typeKupffer cells in the liver serve to protect against nonalco-holic fatty liver disease (32). Thus, strategies restrainingM1 polarization and/or driving alternative M2 activationof macrophages may have the potential to protect againstexacerbated inflammation and insulin resistance and atten-uate the progression to steatohepatitis. Our in vitro find-ings (Fig. 2) and FACS data (Fig. 6) indicate that DPP-4inhibition causes an anti-inflammatory macrophage polari-zation in ATMs and LMs, which contributes to the atten-uation of whole-body insulin resistance. Furthermore,DPP-4 per se induced M1 polarization but suppressed M2polarization of macrophages (Fig. 2A and C), whereas thedysregulation of M1/M2 status was reversed by linagliptin(Fig. 2B and D). These data imply that there is a direct linkbetween DPP-4 activation and M1 polarization in the mac-rophage. Elevated levels of plasma GLP-1 could representthe mechanism underlying this process, and this wasreflected in the inhibition of DPP-4 that accompaniedchronic inflammation. However, linagliptin increasedplasma GLP-1 in DIO mice (Fig. 5E), but only to a smallextent compared with exogenous GLP-1 administration(10). Several lines of evidence suggest that the conse-quences of DPP-4 inhibition are far more complex thanpreviously thought and may involve both GLP-1–dependentand –independent effects (8,33–35). Considering its pat-tern of expression and the multiplicity of functions andtargets of DPP-4, DPP-4 may play a distinct role in regu-lating macrophage polarity in addition to its effect on theincretin axis.

The infiltration of Th1 and CD8+ T cells precedes M1-polarized macrophage recruitment, and interactions betweenT cells and macrophages constitute a maladaptive feed-forward loop, which leads to adipose inflammation and insu-lin resistance (36,37). Therefore, DPP-4 inhibition may reduce

the accumulation of T cells as well as M1 activation of mac-rophage to alleviate insulin resistance and inflammation inobesity (Supplementary Fig. 6). In humans with type 2 di-abetes, treatment with DPP-4 inhibitors decreases proinflam-matory markers in the blood and inhibits NF-kB activationin mononuclear cells (38). However, DPP-4 inhibitor treat-ment might be a double-edged sword (39), as it may increasethe risk of infection in patients with diabetes (40). Currently,the long-term effects of DPP-4 inhibitors on T cell matura-tion and activation remain unknown, and further researchis needed to clarify their long-term immunological effects.

An important question is whether DPP-4 inhibitors canregulate the recruitment of monocytes and affect M1/M2status in bone marrow or peripheral blood given the linkbetween Ly6Chi/Ly6C2 monocyte subtypes and their fateas M1/M2 macrophages (41–43). In the current study, HFfeeding caused a significant increase in recruited inflamma-tory Ly6Chi monocytes in the peripheral blood, whereasDPP-4 inhibitors did not affect Ly6Chi or Ly6C2 monocyteseither in bone marrow or peripheral blood (SupplementaryFig. 7). According to the FACS analysis, although the num-ber of crown-like structures revealed by F4/80 immuno-staining was decreased by linagliptin, the number of totalATMs was unaffected by DPP-4 inhibitors. This inconsis-tency can be explained by differences in the antibodiesused for the immunostaining and flow cytometry proce-dures. Thus, inhibiting DPP-4 caused a dynamic M2 shiftof macrophages within WAT and liver, which contributedto the attenuation of local and systemic insulin resistance.In contrast, LMs, which consist of resident Kupffer cellsand recruited bone marrow–derived macrophages, de-creased in HF-fed mice following treatment with linagliptin(Fig. 6C and D), which suggests that the reduction of LMswas mainly due to the decreased activation of residentKupffer cells.

Increased oxidative stress causes MCP-1 productionfrom accumulated fat, which, in obese subjects, leads toinfiltration of ATMs (2,44). A major contributor to oxidativestress in fat and vasculature (44,45), NADPH oxidase canactivate both NF-kB and MAPK subfamilies, thus increasinginflammatory response. In this study, linagliptin decreasedROS generation and suppressed the expression of NADPHoxidase subunits in both WAT and liver (Fig. 5H and Sup-plementary Fig. 5). Furthermore, DPP-4 activity, ROS pro-duction, and M1/M2 polarity were associated (Fig. 2 andSupplementary Fig. 2). Thus, the anti-inflammatory effectsof DPP-4 inhibition are due to the attenuation of oxidativestress from accumulated fat or infiltrated macrophages inWAT and/or liver.

It is possible that the anti-inflammatory effects oflinagliptin are attributable to its impact on glucose con-trol mediated by DPP-4 inhibition rather than any othermolecule-specific properties (46,47). When comparing lina-gliptin and sitagliptin, although we find that both DPP-4inhibitors lower glucose levels to an equivalent extent inDIO mice, linagliptin elicited a greater DPP-4 inhibitionand may also have had stronger antioxidative (Fig. 5) and

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anti-inflammatory actions (Fig. 6). The diversity of the drugeffects may result from differences in their tissue availabil-ity and/or inhibitory efficiencies against DPP-4. Linagliptinis highly tissue penetrative, whereas sitagliptin is onlyweakly tissue penetrative (47,48). Linagliptin also has alonger duration of DPP-4 inhibition, something we haveshown in our own work (Fig. 5G) and that has beenobserved previously (46).

DPP-4 is a 766-amino acid serine protease thatpreferentially cleaves N-terminal dipeptides from varioussubstrates (26). Among them, MIP-1a, also known asCCL3, is converted into an efficient monocyte/macrophageattractant after cleavage by DPP-4 (25,26). MIP-1a isrobustly upregulated in WAT of obese mice, and infiltratedATMs or preadipocytes can secrete MIP-1a in inflamed fat(49). The present results showed that the loss of MIP-1aresulted in improved glucose homeostasis and the reduc-tion of macrophage accumulation as well as in a polariza-tion toward an anti-inflammatory phenotype in the WATand liver (Supplementary Fig. 8). Linagliptin did not conferthe protection against obesity-induced inflammation or in-sulin resistance in MIP-1a2/2 mice despite DPP-4 activitybeing markedly inhibited by linagliptin (Fig. 7). Furthermore,DPP-4 unaffected M1/M2 marker mRNA expression or ROSproduction in peritoneal macrophages from MIP-1a2/2

mice (Supplementary Fig. 9), suggesting that MIP-1amay be a substrate for DPP-4 that, at least in part, con-tributes to the regulation of macrophage polarization un-der conditions of obesity.

In conclusion, our findings suggest that DPP-4 plays acritical role in obesity-induced inflammation and insulinresistance by regulating the M1/M2 status of macro-phages. DPP-4+ macrophages accumulate in WAT of obesemice, and, importantly, inhibition of DPP-4 with linaglip-tin results in macrophage polarization toward an anti-in-flammatory phenotype in adipose tissue and liver, therebyattenuating obesity-induced inflammation and insulin re-sistance. Overall, the current investigation highlights apotential clinical utility for DPP-4 inhibition in the atten-uation of macrophage-mediated inflammation and pre-vention of insulin resistance and type 2 diabetes.

Acknowledgments. The authors thank M. Nakayama and K. Hara(Kanazawa University, Kanazawa, Japan) for technical assistance and animalcare and Tim Hardman of Niche Science & Technology for help in the preparationof the manuscript.Funding. This work was supported by Grant-in-Aid for Scientific Research (B)(25282017) and Challenging Exploratory Research (15K12698) from the Ministryof Education, Culture, Sports, Science, and Technology of Japan and BoehringerIngelheim Pharma GmbH & Co. KG grant (to T.O.).Duality of Interest. T.O. received research support from BoehringerIngelheim Pharma GmbH & Co. KG. No other potential conflicts of interest rele-vant to this article were reported.Author Contributions. F.Z., Y.N., M.N., N.N., and L.X. performedexperiments and acquired data. N.M. and S.K. contributed to discussion andedited the manuscript. T.O. contributed to the study concept and design andwrote the manuscript. T.O. is the guarantor of this work and, as such, had full

access to all the data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Prior Presentation. Parts of this study were presented in abstract form atthe 75th Scientific Sessions of the American Diabetes Association, Boston, MA,5–9 June 2015.

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