Sitagliptin Reduces Cardiac Apoptosis, Hypertrophy and Fibrosis Primarily by Insulin-Dependent Mechanisms in Experimental type-II Diabetes. Potential Roles of GLP-1 Isoforms

Background Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1) enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart. Methods Goto-Kakizaki (GK) rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin) or vehicle (n=10, each). After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays. Results Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36), alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells. Conclusions Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36) promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actions.


Introduction
The global prevalence of type-II diabetes mellitus (T2DM) has increased in such a way that has achieved epidemic proportions [1]. Experimental and clinical studies have shown an association between T2DM and cardiomyopathy, being defined by functional and structural changes at the level of myocardium, independent of any vascular or cardiac diseases. Diabetic cardiomyopathy (DCM) is characterized by myocardial apoptosis, hypertrophy and subsequent fibrosis, as well as cardiac dysfunction [2]. The intervention to prevent the development of fibrosis has been proposed for the treatment of DCM [3,4]. An excess of plasma free fatty-acid (FFA) and glucose triggers pro-fibrotic factors and extracellular matrix (ECM) deposition from fibroblasts and myocytes [2,3]. However, the molecular underlying mechanisms of these responses are poorly known, leading to a lack of treatment. In this regard, metformin is the only anti-diabetic conclusively probed to avoid cardiac complications in diabetes. Unfortunately, this insulin-sensitizer can produce unwanted side effects [1]. Dipeptidyl peptidase-IV (DPP-IV) inhibitors, as sitagliptin, are a new class of anti-diabetics that prevent the degradation of insulinotropic incretins, without producing hypoglycemia [5]. The most active incretin is glucagon-like peptide-1- , usually termed GLP-1. GLP-1 is released from intestinal L-cells to the circulation in response to ingested nutrient. GLP-1 regulates blood glucose mainly by enhancing pancreatic insulin secretion, and confers cardioprotection after myocardial infarction, congestive heart failure and ischemia [6,7]. However, the expression of DPP-IV and GLP-1 receptors (GLP-1R) has been also described in different tissues including liver, vessel and heart, suggesting extra-pancreatic actions [6][7][8][9]. Via RISK (cAMPPKAPI3KAkt) pathway GLP-1R induce transcription factor activation [10,11]. In this sense, peroxisome proliferator-activated receptors (PPAR) are FFA-binding nuclear receptors that act as transcription factors to regulate cardiac metabolic and inflammatory genes [12]. In particular, PPARδ may control pro-fibrotic genes to prevent cardiac fibrosis and heart failure [13]. However, due to the plasma DPP-IV activity, GLP-1 is degraded within minutes to the insulinotropic-inactive GLP-1(9-36) [5]. The aim of this study was to investigate whether sitagliptin could induce cardioprotection for T2DM hearts by GLP-1/PPARδ direct actions on cardiac cells.

Ethics Statement
These investigations adhered to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996)

Animal Model
A polygenic non-obese non-hypertensive model of T2DM was used for the study. Male Goto-Kakizaki (GK) rats were purchased from Taconic, Denmark and were kept on an artificial 12-hour light-dark cycle (7 a.m.-7 p.m.) at 25°C. GK exhibit similar metabolic, hormonal and vascular disorders that the human T2DM, offering a convenient model for the study of T2DM per se, without the confounding effects of obesity or hypertension [14]. Once GK became diabetic (at 16th week), some were treated with sitagliptin [Merck Sharp & Dohme (Spain), 10 mg/Kg/day] or metformin clorhidrato [Acofarma (Spain), 200 mg/Kg/day]. Both drugs were dissolved in water and daily administrated (10 a.m.) by a gavage. Vehicle-treated GK and wistar were also examined. N=10, each group. Body weight, diet consumption and systolic blood pressure (measured by tail-cuff method) were weekly evaluated. After 10 weeks of treatment, plasma (collected from cava vein) and hearts were isolated (3-7 p.m.) under isoflurane (1.5% in O 2 ) anaesthesia. Plasma lipid profile, glucose, hepatic enzymes and renal parameters were enzymatically measured in the clinical department of the Hospital. Hearts were rinsed, dried and weighted. After atria excision, a ventricular slice was included in p-formaldehyde and paraffin for histology. Then, left ventricles were frozen in liquid-N2 for biochemical assays.

Glucose tolerance test
Glucose tolerance was evaluated as published elsewhere [15]. Blood samples were collected (from tail vein) at the day before sacrifice after overnight fasting (N=10, each group). Then, rats received the corresponding dose of vehicle or sitagliptin and plasma was immediately obtained, after which glucose solution (0.5 g/kg) was administrated by i.p. Fifteen and sixty minutes after glucose loading, plasma was obtained again. Plasma glucose and insulin were measured by ELISA kits (Mercodia AB; Sweden). Plasma GLP-1 was determined by modification of Orskov method [16]. Samples [collected in glass tubes with DPP-IV inhibitors (Vacutainer P700, BD; USA)] were mixed with 0.5M EDTA, 10,000 UIC/ml aprotinin and absolute ethanol for 1h at 4°C, and centrifuged (3,000 rpm, 15 min at 4°C). Supernatants were frozen in liquid-N 2 , lyophilized and dissolved in 0.2M glycin-0.5% human serum albumin-500U/ml aprotinin solution (pH 8,8). Then, 100 µl were used for GLP-1 quantification by ELISA (Epitope Diagnostic Inc.; USA).

Cardiac structure and function measurement
Cardiac echocardiography was performed under 1.5% isoflurane-O 2 anaesthesia in all rats before (not shown) and after the treatments. Both M-mode and two-dimensional (2D) echocardiograms were obtained using a 12 MHz ultra-band sector transducer (En Visor-C-HD, Philips). Images were obtained from the left and right parasternal window in a supine decubitus position. The following parameters were measured and calculated from M-mode tracing: left ventricular (LV) enddiastolic diameter (LVDD), LV end-systolic diameter (LVSD) and ejection fraction (EF; by Teichzol method). Wall thickness of four segments [anterior, inter-ventricular-septum (IVS), lateral, and posterior (LVPW) walls] was evaluated on short axis 2D images. LV mass index (LVMI) was calculated according to Devereux method using LVPW, IVS and LVDD parameters, and normalized to body weight as previously described [17]. For histological quantification of the LVPW and IVS thicknesses, serial paraffin sections (4 µm) of half-height sliced myocardium were fixed on slides and stained with Haematoxylin/Eosin (H/E). LVPW and IVS thicknesses were evaluated as mean of 4-5 measurements in the same heart region of all rats by using Metamorph software. A representative photograph taken with an optical microscopy, and the score for each rat are shown. Cell size of LVPW cardiomyocytes was quantified in 50 transversally oriented H/E-stained cells of twenty randomly fields by average of the cross-sectional areas (at nucleus level), using Metamorph.

Cardiac fibrosis and cell-death examination
Masson trichrome was used to detect extra-cellular matrix (ECM) deposition by sequent addition of Bouin's, Weigert's and Biebrich solutions (Bio-Optica, Milan, Italy) on paraffin sections (4 µm) of all myocardia. Interstitial, perivascular and replacement fibrosis were quantified together on five fields of each myocardium using the Metamorph software. Photographs with a scale bar were taken at 40x magnification under optical microscopy. Apoptosis was detected by a TUNEL-based apoptosis detection kit, following manufacture's instructions (ApopTag®, Invitrogen). The percentage of TUNEL-positive nuclei relative to total nuclei was determined in a blinded manner by counting 200-300 cells on ten randomly chosen fields per coverslip for each myocardium. In vitro, cells were cultured in chamber slides (Nunc; Naperville, IL), stimulated, fixed with methanol:acetone (1:1) and nuclear-stained with DAPI. Condensed, pyknotic and/or fragmented nuclei of death cells were identified by confocal microscopy and were counted by using Metamorph. Necrosis was evaluated in the hearts by loss of cytoskeleton vinculin in paraffin-fixed slides by incubations with an anti-vinculin antibody followed by a biotinylated secondary antibody (Cell Signalling, USA), as previously described [32]. Representative photographs were taken under confocal microscopy. In cultured cardiomyocytes necrosis was measured by the release of the cytolytic glucose 6-phosphate dehydrogenase (G6PD) from damage and dying cells into the media, using the Vybranto Cytotoxicity Assay Kit (Invitrogen).

Western Blot (WB)
A piece (50 mg) of homogenized ventricle (by Bullet Blender, Cultek) or cell extract were dissolved in protein lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 2% SDS + 1/250 mammalian protease inhibitors), and equal amounts (20-30 μg) of protein extracts were separated on polyacrylamide gels, transferred to membranes and probed with specific primary antibodies [anti-fibronectin (Millipore, Darmstadt, Germany),collagen type-I precursor (Millipore), -PPARδ (Aviva System Biology; USA), -caspase-3, -AMPK or -APMK-P (Cell Signalling)]. Anti-GAPDH (Sigma) was used as loading control. Then, secondary antibodies (GE Healthcare) were used for chemo-luminescence development. A representative gel of at least three independent experiments with the semiquantification score (n-fold) is shown in the figures. For quantification of soluble fibronectin, cultured cells were starved in 1% FBS (to prevent proteases activity) before stimulation, after which, cell media were collected, centrifuged (12,000 rpm at 4°C) to remove cell debris, and loaded (20 μg) on polyacrylamide gels. Since cells do not secrete GAPDH, Ponceau staining was used as a loading control.

Immunofluorescence (IF)
Fibronectin was localized in HL-1. After stimulation, cells were washed and fixed with 4% p-formaldehyde. Antifibronectin (Millipore) was added overnight at 4°C and developed with anti-rabbit FITC-linked goat antibody (Sigma). 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was used for nuclear labeling. Fibronectin staining was quantified by Metamorph on ten randomly fields in at least three independent experiments. Cardiomyocyte size was quantified as previously described [24] as surface area from ten randomly chosen fields of actin-stained cells [with an anti-F-actin phalloidin antibody (Sigma)], in at least three independent experiments. Representative photographs with a bar scale were taken at 40x magnification under confocal microscopy.

Statistical analysis
Data are expressed as mean±standard deviation. Multiple comparisons were performed by non-parametric Kruskal-Wallis test followed by a Mann-Whitney test. A two-tailed p<0.05 was considered significant.

Characterization of the GK associated T2DM model
After 10 weeks of treatments, the patho/physiological parameters of the experimental model are shown in Figure 1a. GK rats showed elevated circulating levels of glucose, lipid profile [cholesterol (Ch), triglycerides (TG), non-HDL Ch, nonesterified fatty acids (NEFA) and high-density lipoproteins (HDL)], and proteinuria, compared to control hearts. GK also exhibited a modest but significant increase in heart-to-femur length ratio (HW/FL). Interestingly, both sitagliptin and metformin attenuated hyperglycemia, hyperlipidemia and proteinuria, and restored HW/FL (Figure 1a). Plasma ions (Na + , Cland K + ), markers of severe renal (urea, blood urea nitrogen, creatin and albumin) and liver (ASAT and ALAT) injury (not shown), and systolic blood pressure remained within the normal ranges in all groups of rats.

Sitagliptin attenuated the ventricular thickness, cell hypertrophy and cell-death of GK hearts
By Echo-Doppler (Figure 1b), GK showed a significant increase of LV posterior wall (LVPW) thickness and LV mass index (LVMI), resulting in a decreased LV diastolic (LVDD) and systolic (LVSD) diameters. Deceleration time was also prolonged in GK, suggesting diastolic dysfunction, and the ejection fraction (EF) was elevated. Sitagliptin treatment significantly reduced the LVPW, LVMI and deceleration time (Figure 1b). Metformin exhibited a decreased deceleration time and a non-significant trend to restore LVPW and LVMI. Cardiac wall thickness was also analysed in sitagliptin-treated hearts stained with H/E (Figure 1c). We confirmed the increase of LVPW in the GK myocardium and its diminution by sitagliptin. Moreover, we measured the cardiomyocyte diameters in this LVPW area. GK showed a significant increase of the crosssectional area (1.41±0.04-fold vs. 1.0±0.03 wistar, p<0.05), which was reduced after sitagliptin (1.14±0.09-fold vs. wistar, p<0.05).

Sitagliptin reduced myocardial fibrosis and pro-fibrotic factors
Left ventricular myocardium in the wistar rats showed a normal architecture with regular interstitial space (Figure 2c). In contrast, abnormal myocardial architecture (cardiomyocyte disarray and increased interstitial space) was observed in the GK group. Masson trichrome staining detected a deposition of ECM within the interstitial and mainly peri-vascular areas (green-blue staining, Figure 2c). Interestingly, in both sitagliptin and metformin-treated rats, ECM accumulation was markedly reduced. Thus, we next focused on the fibrotic component by examining the expression of key cardiac ECM proteins. Protype-I collagen and fibronectin were found elevated in GK rats (Figure 2d, left). However, only fibronectin was reverted with both sitagliptin and metformin. Furthermore, the mRNA expression of pro-fibrotic inducers, such as transforming growth factor-ß 1 (TGFß 1 ) and its cofactor, connective tissue growth factor (CTGF), were also up-regulated in the GK myocardium and restored with both treatments (Figure 2d, right).

Sitagliptin increased GLP-1 secretion in fasting and glucose-overload states in GK rats
Previous data had indicated that sitagliptin could increase plasma GLP-1 in a glucose-dependent manner [24,25]. By a glucose tolerance test, we observed in the fasting state (0 min) that GLP-1 levels were diminished in GK rats and moderately reverted after sitagliptin (Figure 3a), which may correlate with the increase of insulin (Figure 3b) and ameliorated glucose (Figure 3c) in these rats. Then, mainly after 60 min of glucose loading, GK showed decreased levels of GLP-1 ( Figure 3a) and a delayed insulin response (Figure 3b), which could be responsible for the increasing concentration of plasma glucose (Figure 3c). However, also after 60 min, sitagliptin-treated rats kept higher levels of GLP-1 ( Figure 3a) and insulin (Figure 3b), which may achieve for the slight but significant glycemic reduction (Figure 3c).

Expression of cardiac GLP-1 receptors
The existence of GLP-1 receptor (GLP-1R) has been described in the heart, specifically in cardiomyocytes but not in fibroblasts [8]. We indeed detected GLP-1R mRNA expression in the rat hearts (Figure 4a, left). GK exhibited higher GLP-1R expression than wistar, and sitagliptin did not modify this value. As expected, in contrast to cardiac fibroblast and TFB (not

GLP-1 regulated HF-and/or HG-induced pro-fibrosis, hypertrophy and cell-death in cardiomyocytes
An excess of FFA and glucose are main components of the hyperlipidemic and hyperglycemic milieu in DCM [2,22]. We stimulated HL-1 cardiomyocytes with both compounds and focused on the pro-fibrotic response. After 24h, highconcentration of FFA (HF) or glucose (HG) triggered fibronectin expression (Figure 4b, by WB and IF) and secretion to the cultured media (Figure 4c). When both HF and HG were added together, fibronectin was not significantly further stimulated (not shown). In concordance, the mRNA expression of pro-fibrotic cytokines, such as TGFß 1 and CTGF, were also up-regulated after 6h of HF or HG (Figure 4d). Then, since sitagliptin increased GLP-1 plasma levels ( Figure 3a) and reduced myocardial fibrosis in GK rats (Figure 2a), we assayed whether GLP-1 may modulate the pro-fibrotic response to HF and HG in cardiomyocytes. GLP-1 pre-treatment (1 nM) attenuated fibronectin expression (Figure 4b, by WB and IF) and secretion (Figure 4c) in HF or HG-induced cells. GLP-1 alone did not significantly alter fibronectin content in control cells (Figure 4bc). Moreover, GLP-1 decreased pro-fibrotic TGFß 1 transcripts after HG, and CTGF after HF or HG incubation (Figure 4d).
In addition, although fibroblasts did not express GLP-1R (our data and [8]), we tested whether they could respond to GLP-1 isoforms by different receptors, as previously suggested [27]. HF and HG also triggered fibronectin expression in cardiac fibroblasts, and both GLP-1 and GLP-1(9-36) reduced also these levels (Figure 5c

PPARδ mediated the anti-fibrotic actions of GLP-1 stimulation
A proposed GLP-1 downstream mediator could be the peroxisome proliferator activating receptor-δ (PPARδ) [28,29]. In this regard, a significant decrease of PPARδ levels was noted in the GK myocardium, and this effect was normalized by sitagliptin (Figure 6a), but not metformin (Figure 6b). Since PPARδ had demonstrated anti-fibrotic proprieties in the heart [12,13], but its role on the diabetic scenario was unknown, we first tested whether PPARδ activation may affect fibronectin upregulation induced by HF or HG. Intriguingly, a PPARδ agonist (GW0742) pre-treatment attenuated fibronectin levels only in HG-stimulated cardiomyocytes (Figure 6c, 6 th lane). Thus, in concordance, the anti-fibrotic effect of GLP-1, but not GLP-1(9-36), was significantly reverted by a specific PPARδ antagonist (G5797) in HF-or HG-stimulated cardiomyocytes (Figure 5a, 4 th lanes). Similar data were also observed in cardiac fibroblasts (Figure 5c, 4 th lanes).

Direct protective effects of metformin on cardiac cells
Since both sitagliptin and metformin similarly reduced fibrosis in the GK heart, and GLP-1 triggered anti-fibrotic actions on cultured cardiomyocytes and cardiac fibroblasts, we tested whether metformin may also induce direct anti-fibrotic influence on cardiac cells. Indeed, we observed a reduction of fibronectin up-regulation in HG-stimulated cardiomyocytes (not shown) and TFB fibroblasts (Figure 6d) pre-treated with metformin. In addition, a known metformin-mediator playing key roles in DCM [30], AMP-activated protein kinase (AMPK), was also activated (phosphorylated) in HG+metformin cells. However, metformin did not diminish fibronectin levels after HF, nor increased phosphorylated-AMPK despite AMPK was over-expressed ( Figure 6d). In this regard, AMPK could be used for deacetylation processes in mitochondria biogenesis [30]. Of note, metformin also reduced the expression of caspase-3, and the release of G6PD, but not BNP/α-SMA mRNA overexpression, in HF-and/or HG-stimulated cells (Figure S1 a, c).

Discussion
T2DM per se can damage the heart. Non-hypertensive nonobese GK rats exhibited an accumulation of ECM in the myocardium, and up-regulation of pro-fibrotic TGFß 1 , CTGF and main ECM components such as type-I collagen and fibronectin. These data are in consonance with previous results in type-I diabetic rodents [31,32]. Importantly, cardiac fibrosis may be induced by direct stimulation of released cytokines and/or indirectly by a mechanism of replacing died cells, thus preserving the structural integrity of the myocardium. However, fibrosis may increase hypertrophy and cardiac dysfunction [2,4,33]. In this regard, GK showed cardiac and myocellular hypertrophy that along with data suggest diastolic dysfunction. Unexpectedly, EF was elevated in GK rats. This could be a consequence of the reduction of cavity volume and nearcomplete emptying of the ventricle in order to maintain cardiac output, as occurs in hypertrophied hearts [34]. In this sense, human and experimental diabetic-associated heart failure has been also described without a reduction of EF [35,36].
To date, there is not a specific treatment for DCM-associated fibrosis. DPP-IV inhibitors, as sitagliptin, suppress the DPP-IV activity and consequently, prolong GLP-1 half-life. GLP-1 accounts for at least 50-70% of postprandial insulin secretion, improving the glycemic control by a glucose-dependent mechanism [5]. In our data, sitagliptin reduced hyperglycemia and glucose intolerance. Moreover, we described a lipidlowering influence. Other DPP-IV inhibitors have also showed hypolipidemic effects on T2DM patients [37,38]. The mechanisms for this lipidemic control may be related to the GLP-1 effect on lipid absorption [39], metabolism [40] and/or PPARs activation (see later). More interestingly, sitagliptin attenuated cardiac apoptosis/necrosis, hypertrophy and fibrosis in experimental T2DM. These effects may respond to an increased insulin response by plasma GLP-1 stabilization. In fact, in these rats metformin induced similar anti-apoptotic/ necrotic, -hypertrophic and -fibrotic actions, and improved cardiac function. However, other sitagliptin-associated cardioprotective actions have been reported in non-diabetic injuries. Sitagliptin reduced the infarct size in mice with ischemia-reperfusion [41], and diminished post-ischemic stunning in patients with coronary artery disease and preserved LV function [42]. Also, a GLP-1-analogue treatment improved cardiac function in non-diabetic infarcted patients. Thus, besides its insulin-dependent glycemic and lipidemic control, sitagliptin might play salutary roles by direct actions of GLP-1 on cardiac cells [6]. In this regard, although GLP-1 is expressed in brain, pancreas or intestine, but not heart [26], the presence of GLP-1R have been demonstrated in cardiomyocytes [8], and GLP-1R knockout mice displayed impaired LV contractibility and diastolic function [45]. We have confirmed GLP-1R expression in rat hearts and HL-1 cardiomyocytes, and also, we have detected alleviation of the pro-apoptotic/necrotic, hypertrophic and fibrotic expression in GLP-1 pre-treated cardiomyocytes exposed to HF and/or HG. Previous data indicated also anti-apoptotic actions of GLP-1 in HL-1 cells [18]. However, these effects may respond also to the action of its metabolite GLP-1 . In fact, we found similar effects on HF-and/or HG-stimulated cardiac cells for GLP-1(9-36), and sitagliptin reversed at least the anti-fibrotic action of GLP-1. GLP-1(9-36) may also promote cardioprotection in T2DM. In this regard, GLP-1(9-36) exerted anti-oxidant effects on cardiac and vascular cells [28,46]. Thus, since metformin and GLP-1(9-36) induce similar antiapoptotic/necrotic, -hypertrophic and -fibrotic actions than GLP-1, the cardioproptective effects observed after sitagliptin administration should be firstly explained by its insulinotropic proprieties. Moreover, we have also described direct antiapoptotic/necrotic and anti-fibrotic effects of metformin on HFor HG-stimulated cardiac cells, likely involving AMPK activation. Similar results were found in H 2 O 2 -and TGFßincubated cardiomyocytes and fibroblasts, respectively [47] [48]. However, these in vitro approaches may not accurately reproduce in vivo GLP-1 secretion and DPP-IV inhibition (or metformin cardiac bioavailability), and the large population of cardiac non-myocytes/fibroblasts that express pro-fibrotic factors could differentially respond to GLP-1 isoforms (or metformin). Further investigations studying the potential interactions between GLP-1R and metformin-linked mediators (i.e. AMPK) could be of high interest (Figure 7). In this sense, GLP-1 may also directly counteract the pro-oxidative, inflammatory and apoptotic activities induced by angiotensin-II [43,44].
Moreover, the cellular mechanisms activated by GLP-1 isoforms are not elucidated. GLP-1 and GLP-1  can signal by GLP-1R or different receptors [28,46]. Indeed, we observed that although cardiac fibroblasts did not express GLP-1R, both GLP-1 and GLP-1(9-36) reduced the pro-fibrotic expression after HF or HG. Downstream, cardiac cAMP-dependent RISK kinases have been involve in the GLP-1R signalling to activate different transcription factors, such as PPARδ [49][50][51][52]. PPARδ, a highly expressed nuclear receptor in cardiac cells, promotes healthy activities in the diabetic heart by up-regulation of FFAoxidation enzymes [53]. Additionally, PPARδmay also control the pro-fibrotic expression [54]. We observed a lessening of HG-induced fibronectin expression after PPARδ-agonist administration. In HF-stimulated cells a PPARδ-agonist did not change fibronectin levels possibly because PPARδ can be also a mediator of FFA signaling. Wagner et al. described a reduction of myocardial collagen in PPARδ-agonist treated mice [52]. In addition, we noted that sitagliptin, but not metformin, returned PPARδ levels in GK hearts. Also, in HF-/HG-stimulated cardiomyocytes, GLP-1, but not GLP-1(9-36), reduced pro-fibrotic factors in a PPARδdependent way. In this regard, a GLP-1-analogue increased myocardial PPARδ and reduced apoptosis in infarcted mice [29]. Altogether, at least for the anti-fibrotic effect, GLP-1 could show more affinity for GLP-1R than GLP-1 , and this receptor may be linked to PPARδ activation ( Figure 7). However, further experiments are required to establish the precise assembly of this phenomenon.

Study limitations
Another incretin, termed glucose-dependent insulinotropic polypeptide (GIP), will be increased after DPP-IV inhibition. However, GIP is much less active and its receptor is profoundly decreased under hyperglycemia [55]. Also, we cannot exclude that other potential DPP-IV targets, such as stromal cellderived factor-1 chemokine [5] [28], might affect some cardiovascular responses.

Conclusions
In chronic experimental DCM, the apoptotic and fibrotic responses may promote myocardial hypertrophy and remodelling. However, DPP-IV inhibitors as sitagliptin, through plasma GLP-1 stabilization and insulin control of hyperglycemia/lipidemia, reduced the cardiac pro-apoptotic/ necrotic, hypertrophic and fibrotic expression in a similar way to metformin. However, in the presence of high concentrations of palmitate or glucose, cultured cardiac cells demonstrated a direct effect of GLP-1PPARδ, GLP-1(9-36) and metformin on related factors through GLP-1R or distinct receptors. Furthermore, the cardioprotective actions of GLP-1  suggests additional benefits of GLP-1 analogues, which do not interfere with the physiological GLP-1 degradation, over the ones from DPP-IV inhibitors in diabetic and, interestingly, nondiabetic cardiomyopathies. Figure S1. GLP-1 isoforms alleviated the pro-hypertrophic and lethal responses after HF-and/or HG-stimulation in cardiac cells. (a, left) HF-/HG-incubated cardiomyocytes were pre-treated with GLP-1, and F-actin (red) was detected by IF for cell-size quantification. (a, right) Some cells were used for BNP and α-SMA mRNA detection. (b) Dying cardiomyocytes (left) or cardiac fibroblasts (right) were observed under microscopy after HF/HG-stimulation and GLP-1 pre-treatment. (c) Caspase-3 expression and the release of G6PD to the cultured media was measured in stimulated cardiomyocytes. *p<0.05 and **p<0.01 vs. control. †p<0.05 and † †p<0.01 vs. HF or HG. (AI)