Dipeptidyl peptidase-4 (DPP-4) inhibition with linagliptin reduces western diet-induced myocardial TRAF3IP2 expression, inflammation and fibrosis in female mice.

BACKGROUND: Diastolic dysfunction (DD), a hallmark of obesity and primary defect in heart failure with preserved ejection fraction, is a predictor of future cardiovascular events. We previously reported that linagliptin, a dipeptidyl peptidase-4 inhibitor, improved DD in Zucker Obese rats, a genetic model of obesity and hypertension. Here we investigated the cardioprotective effects of linagliptin on development of DD in western diet (WD)-fed mice, a clinically relevant model of overnutrition and activation of the renin-angiotensin-aldosterone system.
METHODS: Female C56Bl/6 J mice were fed an obesogenic WD high in fat and simple sugars, and supplemented or not with linagliptin for 16 weeks.
RESULTS: WD induced oxidative stress, inflammation, upregulation of Angiotensin II type 1 receptor and mineralocorticoid receptor (MR) expression, interstitial fibrosis, ultrastructural abnormalities and DD. Linagliptin inhibited cardiac DPP-4 activity and prevented molecular impairments and associated functional and structural abnormalities. Further, WD upregulated the expression of TRAF3IP2, a cytoplasmic adapter molecule and a regulator of multiple inflammatory mediators. Linagliptin inhibited its expression, activation of its downstream signaling intermediates NF-κB, AP-1 and p38-MAPK, and induction of multiple inflammatory mediators and growth factors that are known to contribute to development and progression of hypertrophy, fibrosis and contractile dysfunction. Linagliptin also inhibited WD-induced collagens I and III expression. Supporting these in vivo observations, linagliptin inhibited aldosterone-mediated MR-dependent oxidative stress, upregulation of TRAF3IP2, proinflammatory cytokine, and growth factor expression, and collagen induction in cultured primary cardiac fibroblasts. More importantly, linagliptin inhibited aldosterone-induced fibroblast activation and migration.
CONCLUSIONS: Together, these in vivo and in vitro results suggest that inhibition of DPP-4 activity by linagliptin reverses WD-induced DD, possibly by targeting TRAF3IP2 expression and its downstream inflammatory signaling.

Background

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in obese, insulin resistant, and type 2 diabetic individuals. Diastolic dysfunction (DD) is an early manifestation of diabetes and obesity related CVD, and a strong predictor of future CV events and progression to systolic dysfunction [1]. Impaired diastolic relaxation is associated with oxidative stress, inflammation, insulin resistance, left ventricular hypertrophy (LVH), and myocardial fibrosis [2]. In general, premenopausal women are at lower risk for CVD than men. However, obesity offsets this advantage. Indeed, young overweight [3], obese [4] or obese and diabetic [5] women exhibit subclinical DD accompanied by LVH, and are at a higher risk of developing heart failure compared to their male counterparts [6, 7]. Thus, DD and the eventual progression to heart failure are major health care concerns associated with the ongoing epidemics of obesity and diabetes, especially in premenopausal women [8, 9]. Given the increased propensity of developing cardiac stiffness in females with insulin-resistance, investigating the molecular mechanisms underlying the development of DD in females is of paramount importance.

Ideally, therapeutic strategies for diabetes would improve glycemia and have neutral or favorable effects on CVD outcomes, including DD. In this regard, inhibitors of dipeptidyl peptidase-4 (DPP-4) have shown promising results [10, 11]. DPP-4 inhibitors were developed largely to prevent the degradation of the incretin hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP) that are secreted by enteroendocrine cells in response to postprandial hyperglycemia and account for as much as 70% of postprandial insulin secretion (the incretin effect) [11]. In addition to prolonging the half-life of GLP-1 and GIP to improve glycemia, DPP-4 inhibitors also inactivate various substrate hormones, chemokines, neuropeptides and growth factors, and these actions can occur independent of effects on glycemia and have positive implications for CV health [11]. In this regard, linagliptin is a potent, long acting, and highly specific DPP-4 inhibitor [12]. Because of its favorable disposal kinetics, renal safety profile and potent direct effects on vasculature [11, 13], it is being used to treat T2D patients. In these patients, linagliptin lowered blood glucose and the risk of hypoglycemia related to glycemic treatment [14]. Importantly, in a double-blind, randomized, controlled trial, linagliptin did not further increase CV risk in T2D patients [15]. Moreover, in various preclinical models, it also improved cardiac and vascular dysfunction, fibrosis and stiffness [16-21].

Utilizing an overnutrition model, we have recently reported that female C57Bl/6 J mice fed a western diet (WD) high in fat and simple sugars for 16 weeks developed insulin resistance, oxidative stress, inflammation, cardiac fibrosis and DD [22]. In addition, the WD-fed mice demonstrated heightened mineralocorticoid receptor (MR) activation, as evidenced by abrogation of the abnormal cardiac phenotype by genetic ablation of MR or co-treatment with the MR antagonist spironolactone [22, 23]. Whether linagliptin exerts cardioprotective effects in WD-induced obesity in females is not known [22-26].

WD enhances oxidative stress and inflammation, and many of the inflammatory mediators are transcriptionally upregulated by NF-κB and AP-1, two ubiquitously expressed oxidative stress-responsive dimeric nuclear transcriptional factors. Recently, we demonstrated that transgenic overexpression of TRAF3IP2, a cytoplasmic adapter molecule and an upstream regulator of NF-κB and AP-1, results in spontaneous development of myocardial hypertrophy, fibrosis and dysfunction [27]. However, it is not known whether WD upregulates TRAF3IP2 expression in mouse hearts, and whether linagliptin inhibits its expression and downstream signaling intermediates. Our results show that linagliptin exerts cardioprotective effects, including the suppression of WD-induced myocardial oxidative stress, TRAF3IP2 overexpression, inflammation, interstitial fibrosis, and DD in female mice. Supporting these in vivo results, linagliptin suppressed aldosterone-induced oxidative stress, TRAF3IP2 expression, and multiple inflammatory mediators in isolated adult mouse cardiac fibroblasts, resulting in reduced activation and migration.

Methods

Animals

This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Three week-old female C57Bl6/J mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were cared for according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Missouri-Columbia. Animals were housed in groups of four under a 12-h/day illumination regimen. Water was provided ad libitum. Three month-old wild type C57Bl/6 J mice were used for isolation of cardiac fibroblasts (CF) as previously described [28].

Linagliptin treatment

At 4 weeks of age, mice were divided into three groups; Group 1 were fed a control diet (CD; Test Diet 58Y2, Richmond, Indiana), group 2 were fed a WD (WD), and group 3 were fed the WD supplemented with linagliptin (WDL). The WD (Test Diet 58Y1) consisted of high fat (46%) and high carbohydrate as sucrose (17.5%) and high fructose corn syrup (17.5%). Linagliptin (BI 1356; (R)-8-(3-aminopiperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3, 7-dihydro-purine-2, 6-dione) [29] was added to WD so that the final concentration was 83 mg linagliptin kg−1. At this dose, the plasma levels reach approximately 8 mg kg−1/day or approximately 50–100 nM [13]. The animals remained on these diets for 4 months.

Baseline data

Body weights were recorded prior to euthanasia. Plasma DPP-4 activity was analyzed by an established fluorometric assay using the substrate H-Ala-Pro-AFC as reported by us [18, 30]. Myocardial DPP-4 activity was determined as previously described [17].

Echocardiography

Echocardiography was performed on isoflurane (2%) anesthetized mice using a GE Vivid i system with an 11.5-MHz phased-array pediatric probe, as previously described [22, 24].

Assessment of cardiac hypertrophy and fibrosis

Cardiac hypertrophy was analyzed by four different, but complimentary, methods: heart weight to tibia length, cardiomyocyte cross-sectional area, fetal gene (ANP) re-expression, and echocardiography. For cardiomyocyte cross-sectional area, tissue sections were stained with Alexa Fluor® 488-tagged wheat germ agglutinin (WGA, 1:100; #W11261, Thermo Fisher Scientific), and 10 cardiomyocytes from each section were used for analysis by MetaVue. ANP expression was analyzed by RT-qPCR and western blotting. Cardiac fibrosis was analyzed by picrosirius red staining and interstitial fibrosis was quantified by NIH image J software.

Immunohistochemistry (IHC) and immunofluorescence (IF)

A 1 mm-thick slice from the midsection of the heart was fixed in 4% paraformaldehyde overnight, embedded in paraffin, sectioned at 4 μm and used for histological analysis. 3-nitrotyrosine (AB5411; 1:150 dilution; Millipore, Billerica, MA) was localized by IHC. TRAF3IP2 was localized by IF using anti-TRAF3IP2 antibody (1:25; #sc-100647, Santa Cruz Biotechnology, Inc.) and Alexa Fluor® 488-tagged donkey anti-mouse secondary antibody (1:400; # A-21202; Thermo Fisher Scientific). Endothelial cells were detected using anti-CD31 antibody (1:50, #ab28364, abcam) and donkey anti-goat Alexa 488 secondary antibody 1:400 (Life Technologies; A11055). Macrophages were identified using anti-CD68 antibody (1:50, #SC-17832, SC) and Alexa Fluor®-647-tagged donkey anti-mouse antibody. Hoechst (1:400; #H-3570, Thermo Fisher Scientific) was used to visualize nuclei. Photomicrographs were obtained using a Nikon Eclipse 80i microscope and a Spot RT digital camera, and analyzed by SPOT Advanced Software (Sterling Heights, MI).

Lipid peroxidation assay

Myocardial extracts were analyzed for lipid peroxidation products MDA/4-HNE (malondialdehyde/4-hydroxyalkenals) using a Lipid Peroxidation Assay kit (Calbiochem) as previously described [31].

Immunoblotting

Preparation of LV homogenates, electrophoresis and western blotting were described previously [27]. The following antibodies were used: TRAF3IP2 (1:600; #bs-6202R, Bioss), p65 (1:1000; #8242, CST), phospho-p65 (Ser536; 1:1000; #3031, cell signaling technology, Inc or CST), c-Jun (1:1000; #9165, CST), phospho-c-Jun (Ser63, 1:1000; #9261, CST), p38 MAPK (1:1000; #9212, CST), phospho-p38 MAPK (Thr180/Tyr182, 1:1000; #9211, CST), S6K1 (1:1000, #9202, CST), phospho-S6 K (Thr389, 1:1000, #9205, CST), ANP (1:200, #sc20158, Santa Cruz Biotechnology), IL-10 (1:200, #sc-365858, Santa Cruz Biotechnology, Inc) and GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Inc.).

Plasma cytokine concentrations

Plasma concentrations of IL-17A, IL-6 and IL-18 were analyzed by respective ELISAs (IL-17A, #BMS6001, eBioscience; IL-6, #BMS603/2, eBioscience; IL-18, #7625, R&D Systems).

mRNA expression

Total RNA was isolated from frozen LV tissue using Trizol reagent (Sigma) and 0.5 μg of RNA was reverse transcribed into cDNA using a reverse transcription kit (Agilent Technologies). mRNA expression was quantified by RT-qPCR using the following Applied Biosystems™ TaqMan™ probes: ANP (Assay ID: Mm01255748), TRAF3IP2 (Assay ID: Mm00506094_m1), IL-18 (Assay ID: Mm00434226), IL-6 (Assay ID: Mm00446191), IL-17A (Assay ID: Mm00439618-m1), IL-17F (Assay ID: Mm00521423-m1), Ccl2/MCP-1 (Assay ID: Mm00441242-m1), CD68 (Assay ID: Mm03047343-m1), AGTR1a/AT1 (Assay ID: Mm01957722-s1), ColIα1 (Assay ID: Mm00801666), ColIIIα1 (Assay ID: Mm1254476), CTGF (Assay ID: Mm01192932_g1), and LOX (Assay ID: Mm00495386). 18S rRNA (Assay ID: Hs99999901) served as a house keeping gene. All data were normalized to corresponding 18S levels and analyzed using 2−ΔΔ method.

Ultrastructure analysis with transmission electron microscopy

Details of myocardial tissue preparation, sectioning, staining and viewing are all previously described [25]. Briefly, a JOEL 1400-EX transmission electron microscope (Joel Ltd. Tokyo, Japan) was utilized to review three fields chosen randomly per mouse to obtain three 2000× images/heart.

In vitro studies

Cardiac fibroblasts (CF) isolated from 3 month-old wild type C57Bl/6 J mice were used between passages 2 and 3. CF were treated with aldosterone (Aldo; 0.1 μM) for indicated periods as previously described [28, 32]. MR was targeted by spironolactone (5 μM for 15 min) or silenced using lentiviral shRNA (CCGGCCAAGGTACTTCCAGGAT TTACTCGAGTAAATCCTGGAAGTACCTTGGTTTTT, Sigma-Aldrich; multiplicity of infection: 0.5 for 48 h). We have chosen Aldo because myocardial MR activation was shown to be an important contributor to WD-induced oxidative stress, inflammation, cardiac fibrosis and DD [22, 23]. Hydrogen peroxide generation was quantified by Amplex Red assay [28]. TRAF3IP2 expression was targeted by lentiviral shRNA (CCGGAGAACCATTCCCGAGTCAATTC TCGAGAATTGACTCGGGAATGGTTCTTTTTTG, Sigma-Aldrich; moi 0.5 for 48 h) [28]. shRNA against eGFP served as a control. Alpha-smooth muscle actin (α-SMA; #F3777, Fitzgerald Industries International, 2 μg/ml) and vimentin (#sc-373717, Santa Cruz Biotechnology, Inc.; 1 μg/ml) served as markers of CF activation, and were evaluated by western blotting. CF migration was analyzed by BioCoat Matrigel migration assay [28]. CF were treated with linagliptin (30 nM for 1 h) prior to the addition of Aldo. These in vitro experiments were performed at least three times, and representative immunoblots are shown in the Figure.

Statistical analysis

Results are reported as the mean ± SEM. One way ANOVA and post hoc t tests (Bonferroni) were performed to examine differences in outcomes between CD fed mice and WD fed mice with or without linagliptin (Sigma Plot 12.0, Systat Software). All differences were considered significant when p < 0.05.

Results

Linagliptin suppresses cardiac DPP-4 activity in WD-fed mice. Compared to CD fed mice, cardiac DPP-4 activity did not differ statistically from that of WD-fed mice (CD 4764 ± 337 vs. WD 5180 ± 385 relative light units (RLU); p > 0.05). Linagliptin suppressed DPP-4 activity in WD fed mice by 62% (1946 ± 118 RLU vs. WD; p < 0.05).

Linagliptin improves diastolic dysfunction

Compared to CD, WD induced DD (Fig. 1, Table 1). Early mitral inflow velocity (E) did not differ among the three groups of mice (Table 1). The velocity of the septal annulus early in diastole (E’), assessed by tissue doppler imaging, tended to slow in WD mice (p > 0.05), followed by more rapid wall movement later in diastole (A′) (p < 0.05) compared to CD (Table 1). The E′/A′ ratio was significantly lower in the WD fed animals, indicating that diastolic filling relies to a greater extent on movement of the septal wall later in diastole (Fig. 1a). Mitral inflow propagation velocity (Vp) was also slower in the WD group (p < 0.05) (Fig. 1b). Moreover, the ratio of E/Vp, an index of LV filling pressure, significantly increased in the WD group (p < 0.05) (Fig. 1c). The myocardial performance index (MPI or the Tei index of global cardiac function) was elevated in WD mice (p < 0.05) suggesting impaired global cardiac function (Fig. 1d). Isovolumic relaxation time (IVRT) was prolonged in WD mice compared to the CD group (p < 0.05) (Fig. 1e), possibly resulting in elevated MPI in WD mice. Isovolumic contraction time was also prolonged in WD mice (Fig. 1f). Importantly, linagliptin prevented these functional abnormalities. Of note, these abnormalities occurred in the absence of significant changes in heart rate (Table 1).

Fig. 1

Linagliptin prevents WD-induced diastolic dysfunction. Echocardiographic assessment of cardiac function indicates that WD induces impairments in a E’/A’ tissue Doppler index of diastolic function, b Vp, flow propagation velocity of early mitral inflow; c E/Vp, an index of LV filling pressure; d MPI myocardial performance index of global cardiac function; e IVRT isovolumic relaxation time; and f IVCT isovolumic contraction time. These functional impairments are prevented by administration of linagliptin. *p < 0.05 CD vs WD;†p < 0.05 WD vs WDL. N = 10/group

Table 1

Summary of parameters obtained during cardiac ultrasound not shown in Fig. 1

4 months of WD
Ultrasound parameters	CD (10)	WD (10)	WDL (10)
HR, bpm	441 ± 9	441 ± 12	419 ± 9
E, cm s−1	81 ± 3	85 ± 3	80 ± 4
E’, cm s−1	4.1 ± 0.1	3.7 ± 0.2	4.1 ± 0.2
A’, cm s−1	2.0 ± 0.0	2.5* ± 0.2	2.1 ± 0.1

Values are mean ± SE. Numbers in parentheses are sample sizes

HR heart rate, E velocity of early mitral inflow, E’ peak early velocity of septal annulus, A’ peak late velocity of mitral inflow, Post hoc comparisons

P < 0.05 * CD vs WD

Picrosirius red staining demonstrated a twofold increase in interstitial fibrosis in the WD group (p < 0.001 vs. CD; Fig. 2a–b), and linagliptin inhibited this effect (p < 0.01 vs. WD). Compared to CD, gene expression of collagen Iα1 and collagen IIIα1 was increased in the WD group, and suppressed by linagliptin (Fig. 2c). Moreover, expression of CTGF, a key regulator of collagen expression [33], was markedly increased in WD hearts, and linagliptin inhibited its expression. Myocardial stiffening is caused by increased expression of collagens and their crosslinking. Lysyl oxidase (LOX) plays a role in collagen cross-linking and the deposition of insoluble collagen [34]. WD increased LOX expression, and linagliptin reduced its expression (p < 0.05 vs. CD).

Fig. 2

Linagliptin prevents WD-induced cardiac fibrosis. a WD induces profibrotic mineralocorticoid receptor (MR) protein expression and this effect is blunted by linagliptin. b Representative micrographs (4× with 40× inset) show interstitial fibrosis by picrosirius red (PSR) staining in the heart and semi-quantitative assessment of PSR staining. Scale bars 50 μm. c Bar graphs show quantitative analysis of RT-qPCR- generated mRNA results for Col Iα, Col IIIα1, CTGF and LOX expressed as fold change from baseline in the CD group. *p < 0.05 vs CD and †p < 0.05 vs WD. N = 5–8/group

Linagliptin inhibits WD-induced cardiac hypertrophy

Compared to CD, WD-fed mice developed cardiac hypertrophy, as evidenced by increased heart weight to tibia length (p < 0.05; Fig. 3a), cardiomyocyte cross-sectional area (Fig. 3b–c), and ANP protein and mRNA expression (p < 0.05; Fig. 3c). p70 S6 kinase 1 (S6K1) is a threonine/serine kinase and plays a role in protein synthesis. While WD increased S6K1 activation, as evidenced by increased levels of phospho-S6K1 (Thr389) (p = 0.06), linagliptin tended to inhibit its activation (Fig. 3d).

Fig. 3

Linagliptin inhibits WD-induced cardiac hypertrophy and induction of prohypertrophic mediators. a The ratio of heart weight to tibia length (HW/TL). N = 10/group. b Micrographs show wheat germ agglutinin (WGA) staining in the myocardium and accompanying bar graph shows semi-quantitative assessment of cardiomyocyte cross-sectional area (CSA). Scale bars 50 μm. c Western blot shows myocardial ANP expression and accompanying bar graphs show quantitative analysis of protein and mRNA expression as fold change from baseline in the CD group. N = 3/group. d Western blot shows myocardial phospho-S6K1 (Thr389) and pan-actin expression and accompanying bar graph shows fold change in expression from CD. *p < 0.5 vs CD and †p < 0.05 vs WD. N = 4–5/group

Linagliptin attenuates WD-induced cardiac nitrative and oxidative stress

3-nitrotyrosine (3-NTY) staining revealed low levels of 3-NTY in hearts from CD-fed mice, but increased in the WD-fed group (p < 0.05 vs. CD), and linagliptin inhibited this increase (p < 0.05 vs. WD) (Fig. 4a–b). Compared to CD, WD enhanced lipid peroxidation by fourfold in heart tissue, as evidenced by increased MDA/4HNE levels, and this effect was inhibited by linagliptin (p < 0.05; Fig. 4c).

Fig. 4

Linagliptin abrogates WD-induced oxidative stress. a Representative micrographs show myocardial 3-nitrotyrosine immunostaining at low (4×) and high (40×) magnification (inset). L indicates lumen of the left ventricle. b Bar graph shows semi-quantitative analysis of 3-nitrotyrosine staining expressed as average gray scale intensities (AGSI). c Bar graph shows myocardial levels of malondialdehyde MDA and its degradation product, 4-hydroxynonenal (4HNE). *p < 0.05 vs CD and †p < 0.05 vs WD. N = 3–6/group

Linagliptin suppresses WD-induced TRAF3IP2 protein and gene expression

TRAF3IP2 is an oxidative stress-responsive cytoplasmic adapter molecule. Given the robust increase in oxidative stress in the heart of female WD-fed mice and suppression by linagliptin, we next investigated whether WD induces TRAF3IP2 expression and its inhibition by linagliptin. While TRAF3IP2 protein and mRNA expression were detected at low levels in CD hearts, a marked increase in its expression was detected in the WD-fed mice (Fig. 5a). Notably, linagliptin inhibited their increased expression. Confirming the Western blot data, immunofluorescence revealed increased TRAF3IP2 expression in WD hearts and its reduced levels in linagliptin co-treated animals (Fig. 5b). Dual staining with cardiomyocyte- and endothelial cell-specific markers, phalloidin and CD31, respectively, revealed localization of TRAF3IP2 in cardiomyocytes, fibroblasts and endothelial cells. TRAF3IP2 expression was also identified in the nuclei of some of these cells.

Fig. 5

Linagliptin prevents WD-induced increases in TRAF3IP2 expression. a Western blot shows myocardial TRAF3IP2 expression and accompanying bar graphs b show quantitative analysis of protein and mRNA expression as fold change from baseline in the CD group. c Immunofluorescent localization of TRAF3IP2 in the myocardium. The left panels show co-localization of phalloidin (cardiomyocytes, green) and TRAF3IP2 (red). The right panels show colocalization of CD31 (endothelial cells,) and TRAF3IP2 (yellow). d Bar graphs show quantitative analysis of TRAF3IP2 immunofluorescence in the myocardium (top bar graph) and in the coronary endothelium (bottom bar graph) expressed relative to CD. *p < 0.05 vs CD and †p < 0.05 vs WD. N = 5–6/group

Linagliptin suppresses WD-induced NF-κB, AP-1 and p-38 MAPK activation, and proinflammatory and fibrotic gene expression

WD induced activation of NF-κB, AP-1 and p38 MAPK in the heart, as evidenced by increased levels of phosphorylated p65, c-Jun and p38 MAPK (Fig. 6). Linagliptin inhibited their activation. Recent studies indicated that DPP-4 inhibition attenuates obesity-associated inflammation [30, 35]. Since linagliptin suppressed WD-induced NF-κB, AP-1, and p38-MAPK activation, we next investigated whether linagliptin would inhibit induction of proinflammatory mediators. The results showed increased cytokine expression in the WD group, including IL-18, a pro-inflammatory and pro-hypertrophic cytokine, and linagliptin inhibited their upregulation (Fig. 7a). Similarly, linagliptin inhibited the WD-induced increases in mRNA expression of multiple pro-inflammatory and pro-fibrotic cytokines, including IL-18, IL-6, IL-17A, IL-17F, and MCP-1 in the heart (Fig. 7c). Furthermore, linagliptin inhibited WD-induced increases in angiotensin II type 1 receptor (AGTR1) gene expression in the heart (Fig. 7c). In addition to their increased local expression, systemic levels of IL-6, IL-17A, and IL-18 were also increased in the WD fed mice, and linagliptin suppressed their increase (Fig. 7d). Compared to CD fed mice, expression of CD68, a marker of macrophages, was increased in WD hearts and this increase was reduced by linagliptin (p > 0.05; Fig. 7e–f). Linagliptin also inhibited WD-induced increases in CD68 mRNA expression in the hearts (p > 0.05; Fig. 7g). IL-10 is an antiinflammatory cytokine and an inhibitor of oxidative stress [36]. Although IL-10 protein expression was readily detected in the CD group, its levels were not modulated by WD. However, linagliptin markedly increased its expression in the hearts of WD fed mice (Fig. 7h).

Fig. 6

Linagliptin prevents WD-induced increases in p-65, c-jun and p38-MAPK activation. a Western blots show myocardial phospho- and total p-65, c-jun and p-38 MAPK. Accompanying bar graphs show results of densitometric analysis b of protein expression as fold change from baseline in the CD group. WD induces activation of these proinflammatory proteins which is largely prevented by linagliptin. *p < 0.05 vs CD and †p < 0.05 vs WD. N = 5–6/group

Fig. 7

Linagliptin inhibits the cardiac and systemic inflammatory responses to western diet. a WD increases myocardial IL-18 protein and mRNA expression, b pro-inflammatory cytokine gene expression (1L-6, IL-17A, IL-17F and MCP-1), c plasma pro-inflammatory cytokine levels (IL-6, IL-17A, and IL-18), and d myocardial angiotensin type 1 receptor (AGTR1) gene expression. These WD-induced inflammatory responses are prevented by linagliptin e, f WD increases CD68 positive immunofluorescence in the heart, indicating enhanced macrophage infiltration. g WD also increased CD68 mRNA expression. Linagliptin tended to reduce CD68 protein and mRNA levels. h Linagliptin induces anti-inflammatory IL-10 expression. *p < 0.05 vs CD and †p < 0.05 vs WD. N = 5–6/group. AVGI average grey scale intensities

Linagliptin prevents WD-induced abnormalities in myocardial ultrastructure

We recently documented ultrastructural remodeling in cardiomyocytes of mice fed a WD [22, 24, 26]. Herein, we investigated whether linagliptin suppresses these structural abnormalities. Transmission electron microscopy revealed normal organized appearance of a row of cardiomyocyte (CMC) sarcomere(s) (S) alternating with a row of intermyofibrillar (IMF) mitochondria (Mt) in the CD hearts (Fig. 8 A, D, F). There are normally only one or two layers of IMF Mt and they typically exhibit an electron dense Mt matrix and intact Mt crista. Each sarcomere is bound by prominent electron dense Z lines as depicted by the arrows in Fig. 8D. Confirming our earlier results, WD feeding resulted in abnormal remodeling in the heart as evidenced by Mt accumulation and enlargement (arrow), loss of Mt matrix electron density, and Mt fragmentation with loss of crista (Fig. 8B, E, H). Notably, linagliptin treatment prevented these ultrastructural abnormalities in the heart (Fig. 8C, F, I). In addition to more electron dense Mt matrix, linagliptin treatment is associated with improvement in Mt crista structure (see inserts) and sarcomere organization (Fig. 8C, F, I).

Fig. 8

Linagliptin inhibits ultrastructural abnormalities in WD-fed hearts. Panels A, D and G depict the normal organized appearance of sarcomeres (S) alternating with a row of intermyofibrillar mitochondria (Mt). WD induces abnormal remodeling of mitochondria and sarcomeres as depicted in Panels B, E and H. Inset in panel E shows Mt swelling and loss of cristae structure. Linagliptin prevented these WD-induced ultrastructural abnormalities (Panels C, F and I). Panels A–C ×800: scale bar 2 μm. Panels B–I ×2000; scale bar 0.5 or 1 μm

Linagliptin inhibits Aldo-induced cardiac fibroblast (CF) activation and migration

Consistent with previous reports [28, 32], Aldo induced a dose-dependent (0.001–1 μM) increase in TRAF3IP2 expression (Fig. 9a), with peak levels detected at ~0.1 μM concentration. Therefore, in all subsequent experiments, Aldo was used at 0.1 μM concentration. Aldo-induced TRAF3IP2 expression was inhibited by the MR antagonist spironolactone or linagliptin (Fig. 9b). Confirming the inhibitory effects of spironolactone, silencing MR also attenuated Aldo-induced TRAF3IP2 expression (Fig. 9c).

Fig. 9

Linagliptin inhibits aldosterone (Aldo)-induced cardiac fibroblast activation and migration. The MR agonist, Aldo upregulated TRAF3IP2 expression in a dose-dependent manner (a) and pretreatment with the MR antagonist spironolactone and silencing MR each attenuated Aldo-induced TRAF3IP2 expression (b and c). Further, linagliptin inhibited Aldo-induced oxidative stress as evidenced by reduced H2O2 generation (d), and the induction of CTGF, MCP-1, and IL-18 (e). Moreover, linagliptin inhibited upregulation in extracellular matrix proteins collagens Iα1 and IIIα1, and AT1R (f). These results were recapitulated by TRAF3IP2 knockdown (e and f). Importantly, linagliptin inhibited CF activation and migration (g), the hallmarks of cardiac fibrosis. These in vitro experiments were performed at least three times, and a representative immunoblot is shown

Linagliptin inhibits Aldo-induced oxidative stress and inflammation

Further investigations revealed that Aldo induced oxidative stress as evidenced by increased generation of hydrogen peroxide, and its inhibition by linagliptin. Furthermore, linagliptin treatment and silencing TRAF3IP2 each attenuated Aldo-induced pro-inflammatory cytokine (CTGF, MCP-1 and IL-18) (Fig. 9e) and pro-fibrotic Col1α1, ColIIIα1 and AT1R expression (Fig. 9f).

Linagliptin inhibits Aldo-induced CF activation and migration

Because CF activation and migration play critical roles in cardiac fibrosis, we next investigated whether linagliptin inhibits these critical phenomena. Indeed, the results in Fig. 9g show that while Aldo induced CF activation (increased expression of α-SMA and vimentin), linagliptin inhibited their expression. In addition, linagliptin inhibited Aldo-induced CF migration (Fig. 9g). Collectively, these in vitro studies suggest that linagliptin exerts anti-fibrotic effects possibly through inhibition of oxidative responsive TRAF3IP2 expression, inflammatory cytokine and collagen expression, and importantly, CF activation and migration (Fig. 10).

Fig. 10

Schematic illustrates a possible causal role of TRAF3IP2 in western diet induced oxidative stress, inflammation, fibrosis and diastolic dysfunction, and the efficacy of linagliptin in reducing these cardiac impairments. Area within the dotted grey box summarizes novel data presented in this investigation

Discussion

In this investigation we report prevention of diastolic dysfunction (DD) and cardiac fibrosis by linagliptin, a DPP-4 inhibitor, in western diet (WD) fed obese female mice. Even though most previous investigations into the anti-fibrotic effects of linagliptin in the heart were performed in male animals [16, 21, 37], here we focused on female mice, in large part, because, females develop diastolic dysfunction earlier than males, though both sexes gain weight and develop insulin resistance when fed the WD [24]. Moreover, DD is more pronounced in the WD-fed females than males [22, 25]. Thus, this model better recapitulates the loss of CV protection in premenopausal women [18, 24]. This feeding paradigm also mimics the loss of CV protection that occurs in insulin resistant/obese premenopausal women who have a higher propensity to develop diastolic dysfunction compared to age-matched men [3, 4].

Here we provide the first evidence of TRAF3IP2 upregulation in an overnutrition model and its inhibition by linagliptin. Importantly, suppressed TRAF3IP2 is accompanied by reduced cardiac nitrative/oxidative stress, RAAS activation (decreased AT1 and MR expression), inflammation, cardiac fibrosis, and DD. Supporting these in vivo observations, our in vitro studies using isolated CF also demonstrated that linagliptin inhibits Aldo-induced TRAF3IP2 expression, oxidative stress, inflammatory cytokine expression, and CF activation and migration.

Cardiac fibrosis is one of the major determinants of impaired diastolic relaxation. Chamber stiffness results, in part, from increased accumulation of collagens [38]. Linagliptin significantly suppressed WD-induced interstitial fibrosis. This was associated with decreased expression of multiple profibrotic factors, including IL-6, IL-17 and IL-18, CTGF, and collagens Iα1 and IIIα1 in the heart [39]. In addition, systemic levels of these inflammatory mediators were also decreased by linagliptin. In fact, both cardiac fibrosis and DD in obesity and diabetes are associated with a state of chronic sub-acute systemic and tissue inflammation [40, 41], and low grade inflammation serves as a proximate trigger, as well as an ultimate modulator of various markers of cardiac dysfunction in obesity, such as insulin resistance, diabetes and CVD [41]. In this context, inappropriate renin-angiotensin-aldosterone system (RAAS) activation is implicated in inflammation and immune cell recruitment in multiple organs, including heart and vasculature, in animal models of obesity, diabetes and hypertension, and blockade of RAAS is known to ameliorate inflammation associated with obesity [42, 43].

TRAF3IP2 is a cytoplasmic adapter molecule and an upstream regulator of NF-κB, AP-1 and p38 MAPK activation [44, 45], whose persistent activation mediates cardiac immune and inflammatory responses, as well as fibrosis [46-48]. It is emerging as a convergence point in immune and inflammatory responses elicited by cytokines, such as IL-17, and hormones, such as angiotensin II (AngII) and aldosterone. The causal role of IL-17, which signals exclusively via TRAF3IP2, is well recognized in myocardial fibrosis and DD [49-51]. Interestingly, DPP-4 is abundantly expressed in the Th17 lymphocytes that secrete IL-17, and inhibition of DPP-4 suppresses Th17-mediated immune responses [11, 52, 53]. In addition, TRAF3IP2 also plays a role in AngII- and aldosterone-induced NF-κB activation in cardiomyocytes [54] and cardiac fibroblasts [32] respectively. TRAF3IP2 also plays a role in aldosterone-induced AT1R expression [54], suggesting a critical role for TRAF3IP2 in aldosterone and AngII crosstalk. Recently, linagliptin has been shown to suppress AT1R expression and AngII-induced cardiac fibrosis [20]. It has also been shown to inhibit AngII-induced NF-κB activation and collagen synthesis in cultured cardiac fibroblasts [55]. Therefore, it is highly likely that linagliptin might have exerted anti-fibrotic effects by inhibiting TRAF3IP2 expression and activation of downstream signaling intermediates NF-κB and AP-1.

Linagliptin also prevented WD-induced LOX expression. LOX plays a role in collagen crosslinking and myocardial stiffness [51]. We have identified LOX as one of the important downstream targets of TRAF3IP2 in vitro and in vivo [27, 32]. Therefore, multiple mechanisms might have contributed to the anti-fibrotic effects of linagliptin, including TRAF3IP2-dependent LOX expression, and collagen expression and crosslinking.

In addition to cardiac fibroblasts, macrophages also play a critical role in the development of cardiac fibrosis. MCP-1 is a macrophage chemoattractant, and its gene deletion suppresses cardiac macrophage infiltration, inflammatory response, and fibrosis [56]. In this regard, we observed an increase in MCP-1 expression in WD-fed mice and its inhibition by linagliptin. These data are consistent with previous reports that demonstrated TRAF3IP2-dependent MCP-1 expression and macrophage accumulation in the heart [27]. Of note, linagliptin is also known to reduce macrophage accumulation in adipose tissue of female C57BL/6 N mice fed an obesogenic diet [57]. In the present study, we have demonstrated an increase in macrophage accumulation in the heart, as evidenced by increased expression of CD68, a macrophage marker. Linagliptin moderately suppressed its expression, suggesting that linagliptin might have suppressed WD-induced cardiac fibrosis, possibly by targeting macrophage accumulation.

In addition to enhanced pro-inflammatory cytokine expression, inappropriate RAAS activation in obesity induces oxidative stress in the heart [58, 59], resulting in myocardial injury and adverse remodeling [60]. In fact, we recently reported that AngII and Aldo induce oxidative stress, in part via TRAF3IP2 upregulation [32, 54]. We have also reported that linagliptin decreases WD-induced oxidative stress in vasculature [18]. In this study, linagliptin effectively prevented WD-induced nitrative/oxidative stress in the heart, as seen by reduced levels of 3-NTY and MDA/4-HNE levels. Therefore, it is plausible that linagliptin might have suppressed WD-induced adverse myocardial remodeling by suppressing nitrative and oxidative stress. Oxidative stress is also known to contribute to ultrastructural abnormalities in the heart. While WD promoted disorganized mitochondria along with mitochondrial enlargement and fragmentation, these abnormalities were prevented by linagliptin. Suppression of oxidative stress and improvement in mitochondrial structural remodeling by linagliptin might have contributed to improvement in diastolic dysfunction in the WD-fed mice [22, 61].

We have demonstrated that linagliptin suppresses systemic [18], as well as cardiac DPP-4 activity in WD-fed mice. DPP-4 has multiple substrates, including GLP-1. Prolonging the half-life of GLP-1 in order to extend its insulinotropic effect is the principle rationale for use of DPP-4 inhibitors for treatment of hyperglycemia in diabetes. Administration of native GLP-1 or GLP-1 agonists have been shown to be cardioprotective [11, 62]. The extent to which DPP-4 inhibitors induce GLP-1-dependent responses may be more limited given the modest increases in circulating active GLP-1 levels induced by DPP-4 inhibitors, relative to those induced by GLP-1-based therapies. Given the numerous and varied substrates enzymatically cleaved or bound by DPP-4, DPP-4 inhibitors may have the potential to exhibit a broader range of salutary pleiotropic effects in the heart and vasculature, including reduction in oxidative and nitrative stress and inflammation, as well as, improvement in nitric oxide dependent vasodilation [13, 17, 18], and these salutary effects may be independent of GLP-1 and its receptor.

Conclusions

We have demonstrated that linagliptin, a DPP-4 inhibitor, improves DD in WD-fed female mice, a clinically relevant model of obesity-associated CVD. Improvement in DD occured in association with suppressed oxidative stress, TRAF3IP2 expression, inflammation and fibrosis. Importantly, its cardioprotective effects appeared to be independent of changes in body weight [18], heart rate (Table 1) and blood pressure [22]. Our in vitro studies in cardiac fibroblasts recapitulated these in vivo observations. Although these data suggest that the efficacy of linagliptin in the heart is mediated by suppression of TRAF3IP2 expression and downstream inflammatory signaling, we have not demonstrated a direct role for TRAF3IP2 in WD-induced DD. Future studies utilizing TRAF3IP2-null mice are necessary to determine its direct causal role in WD-induced DD and adverse remodeling.