Full paper
Sitagliptin alleviated myocardial remodeling of the left ventricle and
improved cardiac diastolic dysfunction in diabetic rats
Yu-Sheng Liu
a,b, Zhi-Wei Huang
a,1, Lin Wang
a, Xin-Xin Liu
a, Yong-Mei Wang
b,
Yun Zhang
a, Mei Zhang
a,*aDepartment of Cardiology, The Qilu Hospital of Shandong University, Jinan, Shandong, 250033, PR China bDepartment of Cardiology, The Second Hospital of Shandong University, Jinan, Shandong, 250033, PR China
a r t i c l e i n f o
Article history:Received 9 April 2014 Received in revised form 11 December 2014 Accepted 12 December 2014 Available online 23 December 2014
Keywords: Diabetes mellitus Sitagliptin Myocardial remodeling Cardiac function
a b s t r a c t
Objective: Sitagliptin, a dipeptidyl peptidase IV (DPP-Ⅳ) inhibitor, has a biological role in improving the serum levels of glucagon-like peptide 1 (GLP-1). Hence, we sought to determine the effect of sitagliptin on myocardial inflammation, collagen metabolism, lipid content and myocardial apoptosis in diabetic rats.
Materials and methods: The type 2 diabetic rat model was induced by low-dose streptozotocin and a high-fat diet. Characteristics of diabetic rats were evaluated by electrocardiography, echocardiography and blood analysis. Cardiac inflammation, fibrosis, cardiomyocyte density, lipid accumulation, and receptor-interacting protein kinase 3 (RIP3) level, related to apoptosis, were detected by histopathologic
analysis, RT-PCR and western blot analysis to evaluate the effects of sitagliptin on myocardial remodeling of the left ventricle.
Results: Diabetic rats showed myocardial hypertrophy or apoptosis, inflammation, lipid accumulation, myocardial fibrosis, elevated collagen content, RIP3 overexpression, and left-ventricular dysfunction.
Sitagliptin could reverse the overexpression of RIP3and alleviate cellular apoptosis in myocardial tissues.
It could significantly improve left-ventricular systolic pressure and þdp/dt max, reduce the E/E0ratio, left
ventricular end diastolic pressure,dp/dt max and Tau in diabetic rats.
Conclusions: Sitagliptin might have a myocardial protective effect by inhibiting apoptosis, inflammation, lipid accumulation and myocardial fibrosis in diabetic rats, for a potential role in improving left-ventricular function in diabetes.
© 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
Accumulating evidence indicates that diabetic cardiomyopathy
(1,2)is a disorder of myocardial lesions induced by high glucose. It features myocardial hypertrophy or apoptosis(3), inflammation, lipid accumulation, and myocardialfibrosis(4). As well, it might feature systolic and/or diastolic dysfunction of the heart and
increased mortality(5). Sari et al.(6)declared that cardiac endo-plasmic reticulum stress (ERS) and ERS-initiated apoptosis were involved in diabetes. Receptor-interacting protein 3 (RIP3), an
important signal molecule, is involved in tumor necrosis factor
a
(TNF-a
)-mediated apoptosis and related to mitochondrial energy metabolism(7,8), but its expression in the diabetic heart is not known.Sitagliptin is a dipeptidyl peptidase IV (DPP-Ⅳ) inhibitor and has a role in improving the activity of glucagon-like peptide-1 (GLP-1)(9), an incretin hormone secreted from the distal intes-tine L cells with glucose-dependent release that promotes the secretion of postprandial insulin(10). GLP-1 receptors (GLP-1Rs) exist widely in islets, myocardium and brain tissue. Relatively low GLP-1 concentrations as compared with in vivo blood con-centrations promoted insulin secretion independent of the cAMP-protein kinase A pathway(11). With decreased activity of * Corresponding author. Wenhua Xi Street, No. 107, Jinan, Shandong Province,
250012, China. Tel./fax:þ86 531 8587 5464.
E-mail addresses: lyssyl188@sina.com (Y.-S. Liu), huangzhiwei88@tom.com
(Z.-W. Huang),wanglin2004@yahoo.com.cn(L. Wang),liuxx@tom.com(X.-X. Liu),
xinzang2012@126.com(Y.-M. Wang),daixh@vip.sina.com(Y. Zhang),daixh@vip. sina.com(M. Zhang).
Peer review under responsibility of Japanese Pharmacological Society.
1 Contributed equally to this work.
H O S T E D BY Contents lists available atScienceDirect
Journal of Pharmacological Sciences
j o u r n a l h o me p a g e :w w w . e l s e v i e r . c o m / l o ca t e / j p h s
http://dx.doi.org/10.1016/j.jphs.2014.12.007
We purchased 70 male, 8-week-old Wistar rats (120e140 g) from the experimental animal center of Shandong University (Jinan, China). All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee. Rats were housed at 22C with 12-h light/dark cycles. All mice were fed a high-fat diet (34.5% fat, 17.5% protein, 48% carbohydrate; Beijing HFK Bio-Technology). Four weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed and blood was sampled by the jugular vein. Fasting insulin and fasting blood glucose (FBG) were measured, and the insulin sensitivity index [ISI¼ ln (FBG fasting insulin)1] was calculated. Control rats received citrate buffer (intraperitoneally) alone and rats with insulin resistance received a single intraperitoneal injection of STZ (Sigma, St. Louis, MO; 30 mg/kg intraperitoneally in 0.1 mol/L cit-rate buffer, pH 4.5) as described (16). At one week after STZ administration, rats with FBG> 11.1 mmol/L in 2 consecutive ana-lyses were considered the diabetic model. Animals were then randomized to one of 2 groups for treatment: control rats (n¼ 10), normal chow; or diabetes rats, induced with DPP-Ⅳ inhibitor (low-dosage sitagliptin, 30 mg/kg/d) (n¼ 20) [DPP-IVi (Low)] and (high-dosage sitagliptin, 50 mg/kg/d) (n¼ 20) [DPP-IVi (High)]. Treat-ment with sitagliptin started 4 weeks after STZ injection. The rats were killed after 17 weeks of diabetes.
2.2. Blood analyses
Blood was collected from the jugular vein after rats fasted for 8 h. FBG and circulating levels of cholesterol, triglycerides, low-density lipoprotein (LDL) and high-low-density lipoprotein (HDL) were analyzed with use of the Bayer blood chemistry analyzer (Bayer, Tarrytown, NY). Free fatty acid (FFA) concentrations were measured by use of an enzymatic test kit (CSB-E08770r; HuaMei BIO-TECH, Wuhan, China). ISI was calculated. Plasma insulin levels were measured by use of the rat insulin ELISA kit based on the direct Sandwich ELISA technique (Mercodia AB, Sweden). Hemo-globin A1C (HbA1c) level was measured by the Nycocard Reader
(Ranbaxy, India).
2.3. Electrocardiography and echocardiography
ECG was recorded with the limb leads (I, II, III, aVR, aVL, aVF) to evaluate cardiac electrical activity. Echocardiography involved use of the Philips IE33 imaging system (S12-4). Images were obtained from 2D, M-mode, pulsed-wave Doppler and tissue Doppler im-aging (TDI). All measurements were performed by the same researcher and were averaged from 6 consecutive cardiac cycles. Wall thickness and LV dimensions, including LV end systolic
max), blood pressure, and heart rate with a microtip pressure transducer (Millar Instruments) and LVED pressure (LVEDP) was measured. Tau was calculated with the LV time constant Tau¼ P/ (dp/dt).
2.5. Morphometric analysis
Heart tissues werefixed in 10% formalin, embedded in paraffin, and sectioned at 5-mm thick. A single myocyte was measured with images captured from H&E-stained sections. The myocyte cross-sectional area was assessed by400 magnification within the LV, and a mean was obtained by quantitative morphometry with automated image analysis (Image-Pro Plus 5.0; Media Cybernatics, Houston, TX).
Dark greenestained collagen fibers were quantified to measure fibrosis in Masson trichrome-stained sections. The collagen volume fraction (CVF) and perivascular collagen area to luminal area (PVCA/ LA) were analyzed by quantitative morphometry with automated image analysis (Image-Pro Plus 5.0). CVF was calculated as reported previously(18). Perivascular collagen was excluded from the CVF measurement. To normalize the PVCA around vessels with different sizes, perivascular collagen content was represented as the PVCA/ LA ratio. Interstitial and perivascular fibrosis was evaluated by Picrosirius red staining. Sections were stained with 0.5% sirius red (Sigma) in saturated picric acid for 25 min. Collagen was stained red. Myocardial frozen sections (5 mm) were stained with Oil-red O (Sigma) for 10 min, washed, then counterstained with hematoxylin for 30 s. A Nikon microscope (Nikon, Melville, NY) was used to capture images.
An in situ cell death detection kit (Roche GmBH, Germany) was used for TUNEL assay. Briefly, slides were deparaffinized, rehy-drated with xylene; underwent a graded ethanol series; and were permeablized with hot 0.1 M citrate buffer, pH 6.0; incubated with reaction mixture containing TdT; and labeled with dUTP for 1 h at 37C. Images were captured by confocal laser scanning microscopy (Zeiss LSM510). For a negative control, TdT was omitted from the reaction mixture.
2.6. Immunohistochemical staining
Paraffin sections underwent immunohistochemistry by a microwave-based antigen retrieval method. Sections were incu-bated with primary antibodies for rabbit polyclonal collagen I and III, tumor necrosis factor TNF-
a
, and interleukin 6 (IL-6) (Abcam, Cambridge, MA) overnight, then with biotinylated secondary anti-body for 30 min at 37C. Negative controls were omission of the primary antibody. Stained sections were developed with dia-minobenzidine and counterstained with hematoxylin. Sectionswere viewed under a confocal FV 1000 SPD laser scanning micro-scope (Olympus, Japan).
2.7. RT-PCR
Total RNA was prepared with the TRIzol reagent (Gibco/Invi-trogen, Carlsbad, CA). RT-PCR involved the primer sequences for
RIP3, forward 50-ACC ACTGAGCGAGCATCCTTCC-30and reverse 50
-CCCGGAACACGGCTCCGAAC-30; and
b
-actin, forward 50-AGA CCT TCA ACA CCC CAG-30and reverse 50-CAC GAT TTC CCT CTC AGC-30. Reactions were carried out on a real-time PCR thermocycler (IQ5 Real-Time PCR cycler; Bio-Rad), with SYBR green as fluo-rescence dye. Relative expression analysis involved the 2△△CT method.Fig. 1. (continued).
Fig. 1. Blood analysis demonstrating metabolic disturbance. A. FBG: fasting blood glucose, BG at 10:00 am; HbA1c: Glycated hemoglobin, FINS: Fasting insulin, ISI: insulin sensitivity
index; B. TC: Total cholesterol levels, TG: triglyceride levels, HDL-C: high density lipoprote in-cholesterol, LDL-C: low density lipoprotein-cholesterol, FFAs: free fatty acids; C. Fasting serum levels of GLP-1 and serum levels of GLP-1 at 30 min after oral glucose testing. D and E. intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)findings. (Data are mean ± SEM; n ¼ 7e10 per group. Compared with the control group, *P ＜ 0.05, **P ＜ 0.01; Compared with DPP-Ⅳi (High), #P ＜ 0.05, ##P ＜ 0.01; Compared with DPP-Ⅳi (Low), xP ＜ 0.05, xxP ＜ 0.01.).
2.8. Western blot analysis
Western blot analysis was performed as described previously
(19)with antibodies for RIP3(Calbiochem, La Jolla, CA), collagen I
and III, TNF-
a
and IL-6 (Abcam), followed by anti-IgG horseradish peroxidaseeconjugated secondary antibody. Protein levels were normalized to that ofb
-actin as an internal control and phospho-specific proteins to that of total protein.2.9. Statistical analysis
Data are presented as mean± SD for continuous variables or proportions. After testing for normal distribution of variables, data
were analyzed by Student's two-tail t test and one-way ANOVA followed by post-hoc least significant difference test as appropriate. Correlation was assessed by Pearson or Spearman rank order cor-relation test, as appropriate. Statistical analyses involved use of SPSS 16.0 (SPSS, Chicago, IL, USA). A two-tailed P < 0.05 was considered statistically significant.
3. Results
3.1. Baseline characteristics of diabetic rats
Diabetic rats showed polydipsia, polyphagia, and polyuria as compared with other rats (P< 0.01). As compared with the control
Fig. 2. Electrocardiography and cardiac function measured by echocardiography and Millar catheter. 2.1 Electrocardiogram A: control group, B: diabetic rats with DPP-Ⅳi (Low), and C: diabetic rats with DPP-Ⅳi (High); 2.2 A1: 4D echocardiograms, A2: 2D echocardiograms by short-axis side, A3: M-mode, A4: pulse Doppler echocardiograms of mitral inflow, and A5: tissue Doppler echocardiograms. BeG: Sequential evaluations of left ventricular end diastolic diameter (LVEDD) (B), FS (C), LVEF (D), E/A (E), E0/A0(F) and E/E0(G); 2.3 AeC: LVSP
and LVEDP, DeF: Representative þdp/dt and dp/dt, and G: Tau. (Data are mean ± SEM; n ¼ 7e10 per group. Compared with control, *P ＜ 0.05, **P ＜ 0.01; Compared with DPP-Ⅳi (High), #P＜ 0.05, ##P ＜ 0.01; Compared with DPP-Ⅳi (Low), xP ＜ 0.05, xxP ＜ 0.01.).
group, diabetic rats showed decreased body weight (P < 0.01e0.05) and heart rate and increased blood pressure (P< 0.01e0.05); however, with sitagliptin treatment, body weight increased and heart rate and blood pressure decreased (P< 0.01e0.05).
3.2. Sitagliptin improved blood glucose and lipid metabolism Insulin resistance appeared after 4 weeks with a high-fat diet. One week after STZ injection, FBG increased markedly in
diabetic rats. Diabetic rats showed insulin resistance, moderate hyperglycemia, hyperlipidemia, and decreased ISI (P < 0.05) (Fig. 1A). Serum levels of total cholesterol, total triglycerides, LDL-C, and FFA were higher for diabetic rats than controls (P < 0.05) and decreased with sitagliptin treatment (P < 0.01e0.05) (Fig. 1B). Plasma GLP-1 decreased under both fasting and 30 min after oral glucose (P < 0.01e0.05) and increased with sitagliptin treatment (P< 0.01e0.05) in diabetic rats (Fig. 1C). Sitagliptin had no effect on IPGTT and IPITT results (Fig. 1D and E).
3.3. Sitagliptin improved heart features
Control rats showed P waves, T waves and amplitude consis-tency of QRS waves (Fig. 2.1). For diabetic rats, 66.7% showed abnormal ECG results, and QRS“electrical alternans” phenomena were slightly more common than in controls rats, with super-ventricular tachycardia, super-ventricular tachycardia, slow heart rate and other complex arrhythmia in diabetic rats. However, with sita-gliptin treatment, for diabetic rats, 42.4% showed abnormal ECG results, with no complex arrhythmia.
3.4. Sitagliptin improved LV structure and function
At the beginning, the size of LV chambers, LVIDs and LVIDd did not differ in control and diabetic rats (P> 0.05); however, at the
end, compared with controls, diabetic rats showed increased LVIDs and LVIDd (P < 0.05) (Fig. 2.2). Sitagliptin treatment decreased LVIDs and LVIDd (P< 0.05).
At the beginning, control and diabetic rats showed no diastolic or systolic dysfunction (P> 0.05). However, at the end, compared with controls, diabetic rats showed decreased LVEF and FS (P< 0.01e0.05), which was increased with sitagliptin treatment (P< 0.01e0.05) (Fig. 2.2). Compared with controls, from 13 to 21 weeks, E/A, E0/A0 decreased (P < 0.01e0.05) and E/E0 increased
(P< 0.01e0.05) in diabetic rats, which was reversed with sitagliptin treatment (Fig. 2.2).
Compared with controls, diabetic rats showed decreased LVSP (P < 0.01) and ±dp/dt max (P < 0.01e0.05) and increased LVEDP (P < 0.01); however, sitagliptin reversed these findings (Fig. 2.3).
Fig. 3. Pathology features of the diabetic hearts. A1: Heart size; A2: cross sections of hearts at the papillary muscle level; A3: Longitudinal sections of LV stained with hematoxylin and eosin (H&E). A4: Transverse sections of LV stained with H&E. Quantitative data of B: heart weight to body weight (HW/BW) ratio and C: myocyte size. (Data are mean ± SEM; Compared with control, *P＜ 0.05, **P ＜ 0.01; Compared with DPP-Ⅳi (High), #P ＜ 0.05, ##P ＜ 0.01; Compared with DPP-Ⅳi (Low), xP ＜ 0.05, xxP ＜ 0.01.).
Fig. 4. Expression of glucagon-like peptide 1 (GLP-1) and receptors in the heart. Expression of A: GLP-1 and B: GLP-1 receptor. (Data are mean± SEM; Compared with control, *P＜ 0.05, **P ＜ 0.01; Compared with DPP-Ⅳi (High), #P ＜ 0.05, ##P ＜ 0.01; Compared with DPP-Ⅳi (Low), xP ＜ 0.05, xxP ＜ 0.01.).
Fig. 5. DPP-Ⅳ inhibitor sitagliptin alleviated cardiac fibrosis in diabetic rats. A1: Masson trichrome staining (collagen is blue and myocardium red) and Picrosirius red staining-collagenfibers stained bright red; bright-field (A2), dark-field (A3) show myocardial interstitial fibrosis. A4eA5: Picrosirius red staining-bright-field (A4), dark-field (A5) show perivascularfibrosis. Quantitative analysis of B: CVF, C: collagen content by hydroxyproline assay, and D: perivascular collagen area to luminal area (PVCA/LA). (Data are mean± SEM; Compared with control, *P ＜ 0.05, **P ＜ 0.01; Compared with DPP-Ⅳi (High), #P ＜ 0.05, ##P ＜ 0.01; Compared with DPP-Ⅳi (Low), xP ＜ 0.05, xxP ＜ 0.01.).
3.5. Sitagliptin reversed the size of both the heart and cardiomyocytes
Compared to controls, diabetic rats showed larger heart sizes, higher ratio of heart weight to body weight (HW/BW) (P< 0.05) (Fig. 3A), and increased myocardial hypertrophy and cellular cross-sectional area (498.37± 14.31 vs 347.84 ± 12.27
m
M3, P< 0.01) (Fig. 3AeC). Diabetic rats also showed myocardial cell hypertro-phy, muscle fiber derangement or fracture, irregular shape, and uneven size of nuclei. Sitagliptin treatment reversed these findings.3.6. Sitagliptin increased GLP-1 content and downregulated the density of GLP-1 receptors in diabetic hearts
As compared with controls, diabetic myocardial tissue showed decreased GLP-1 content (P< 0.01) (Fig. 4A) and expression of
GLP-1 receptor (GLP-GLP-1R) (P< 0.01) (Fig. 4B), which were reversed with sitagliptin treatment (P< 0.01).
3.7. Sitagliptin alleviated cardiacfibrosis in diabetic rats
The diabetic heart showed cardiac fibrosis, with a diffuse, small, patchy, and nonuniform pattern, as well as destroyed and disorganized collagen network structure in the interstitial areas (Fig. 5A1eA3) and perivascular areas (Fig. 5A4eA5). CVF (4.8± 0.49 vs 0.47 ± 0.06, P < 0.01e0.05) and collagen content (15.8 6 ± 0.43 vs 7.06 ± 0.31
m
g/mg, P< 0.01) were higher in diabetic rats than controls (Fig. 5B and C), and perivascular collagen area/lumen area ratio (PVCA/LA) was increased (2.74± 0.37 vs 0.5 ± 0.02, P < 0.01e0.05) (Fig. 5D). Compared with controls, diabetic rats showed increased expression of collagen I and III (Fig. 6.1). These findings were reversed with sitagliptin treatment.Fig. 6. Sitagliptin reversed collagen, collagen degrading enzyme, inflammation and the fatty content in diabetic heart. Sitagliptin reversed 6.1 collagen Ⅰ and Ⅲ; 6.2: collagen degrading enzyme MMP-1 and -9; 6.3: TNF-aand IL-6; and 6.4: fatty content in myocardial and epicardial tissues. (Data are mean± SEM; Compared with control, *P ＜ 0.05, **P＜ 0.01; Compared with DPP-Ⅳi (High), #P ＜ 0.05, ##P ＜ 0.01; Compared with DPP-Ⅳi (Low), xP ＜ 0.05, xxP ＜ 0.01.).
3.8. Sitagliptin downregulated the expression of MMP-1 and 9, TNF-
a
and IL-6 in diabetic heartsCompared with controls, diabetic rats showed increased expression of MMP-1 and -9 and TNF-
a
and IL-6 (Fig. 6.2 and 6.3), which was decreased with sitagliptin treatment.3.9. Sitagliptin reduced lipid accumulation in myocardial and epicardial tissue in diabetic hearts
The accumulation of triglycerides caused myocardial fatty degeneration in diabetic rats, with increased staining in myocardial tissue (7.03± 0.31 vs 0.32 ± 0.01, P < 0.01) (Fig. 6.4) and epicardial adipose tissue (12.76± 0.48 vs 0.32 ± 0.01, P < 0.01), which was reduced with sitagliptin treatment (P< 0.01e0.05).
3.10. Sitagliptin downregulated the expression of RIP3in diabetic
hearts
Compared with controls, diabetic myocardial tissues showed increased number of RIP3-positive particles distributed around the
nucleus, which were decreased in number with sitagliptin treat-ment (Fig. 7.1). The mRNA and protein levels of RIP3were increased
in diabetic myocardial tissues (P< 0.01e0.05) and were decreased with sitagliptin treatment (P< 0.01e0.05).
3.11. Sitagliptin inhibits myocardial apoptosis
Diabetic rats showed irregular myocardial nuclei, nuclear pyk-nosis, chromatin aggregation, early presentation of apoptotic cells, with clear myocardial cellular nucleolus, smooth and complete nuclear membrane in controls and no obvious aggregation of Fig. 6. (continued).
chromatin, no nuclear pyknosis and nuclear membrane still intact; sitagliptin treatment smoothed the nuclear membrane in diabetic rats (Fig. 7.2). Compared with controls, diabetic rats showed increased proportion of apoptotic cells (P < 0.01), which was decreased with sitagliptin treatment (P< 0.01). With the correla-tion between the RIP3mRNA and cellular apoptotic rate in diabetic
myocardial tissues (Pearson r ¼ 0.795, P < 0.001), the positive percentage of apoptosis was related to RIP3 mRNA in diabetic
myocardial tissues. 4. Discussion
Sitagliptin, a DPP-Ⅳ inhibitor, has a biological role in improving the serum levels of GLP-1. GLP-1 reduces hypergly-cemia but may also trigger direct effects on the heart(20). We sought to determine the effect of sitagliptin on myocardial
inflammation, collagen metabolism, lipid content and myocardial apoptosis in diabetic rats. Sitagliptin improved metabolic disturbance, reversed myocardial remodeling and lipid accumu-lation, inhibited cardiac inflammation, reduced collagen synthe-sis, and minimized apoptotic cells in STZ-induced diabetic rat hearts. RIP3 was overexpressed and positively correlated with
number of apoptotic cells in diabetic myocardial tissues; sita-gliptin downregulated the expression and minimized the num-ber of apoptotic cells. Sitagliptin could improve the systolic and diastolic functions of the left ventricle in diabetic rats by reducing the E/E0 ratio, promoting LVSP and decreasing LVEDP, ±dp/dt max and Tau. It reversed the altered blood glucose, HbA1
and lipid levels, and plasma GLP-1 and insulin levels, and reduced fat deposition in diabetic myocardial and epicardial tissues, so sitagliptin may play an essential role in improving diabetic cardiac dysfunction and cardiomyopathy.
Inflammation is a key pathophysiologic factor in diabetic car-diomyopathy(1,2,16,21), and is closely related to fat deposition. Ti et al. (16)reported that TNF-
a
and IL-6 were overexpressed in diabetic rats. Biomarkers of inflammation including TNF-a
and IL-6 levels are associated with risk of developing type 2 diabetes. Consistent with these reports, our results showed an increase in TNF-a
and IL-6 levels in diabetic myocardial tissues as well as fat deposition in diabetic myocardial and epicardial tissues. However, sitagliptin could reduce the expression of TNF-a
and IL-6 and fat deposition in the diabetic heart, so sitagliptin might lower the morbidity and mortality of diabetic complications associated with inflammation(22,23).Previous studies(1,2,16) have demonstrated that myocardial fibrosis is a typical characteristic of diabetic cardiomyopathy. Both the deposition and disordered arrangement of type I and/or Ⅲ collagen and abnormal expression of MMPs/TIMPs, and cyto-kines TGF-
b
1 or insulin-like growth factor 1 contribute to thedevelopment of myocardial fibrosis and cardiac diastolic dysfunction. However, some reports(24,25)demonstrated that a GLP-1R agonist can inhibit the expression of TGF-
b
1. However,this study found that sitagliptin reduced aberrant interstitial and perivascular collagen accumulation, as well as total collagen content. Echocardiography and catheterization results revealed that the LV diastolic dysfunction improved, which was attributed Fig. 6. (continued).
to decreased cardiacfibrosis, reduced collagen Ⅰ and Ⅲ content and MMP-1 and -9 expression. Thus, sitagliptin improved LV diastolic dysfunction in diabetic rats by attenuating myocardial fibrosis.
In addition to the above changes, diabetic myocardial tissues show apoptosis and necrosis of myocardiocytes. Recent reports
(26e29) demonstrated that GLP-1 could play a key role in the progress of inhibiting apoptosis during I/R injury. In contrast, gli-benclamide exacerbates I/R injury and deteriorates cardiac func-tion(30,31). A GLP-1R agonist enhanced
b
-cell proliferation and neogenesis in STZ-treated rats and reducedb
-cell apoptosis in part via endoplasmic reticulum stress (32,33). DeNicola et al. (34)found that GLP-1R served as a novel approach to eliciting car-dioprotection and mitigating oxidative stress-induced injury. Consistent with these reports, our results show that sitagliptin minimized the number of apoptotic cells and RIP3 was
overexpressed and correlated positively with the number of apoptotic cells in the diabetic rat heart; furthermore, sitagliptin downregulated the expression of RIP3and minimized the number
of apoptotic cells. Attenuating cardiac apoptosis might be due to downregulated RIP3after administration of sitagliptin. Thereafter,
sitagliptin improved LV systolic dysfunction. An GLP-1 analog protected cardiomyocytes against high glucose-induced apoptosis by activating the Epac-1/Akt pathway (35). However, the exact mechanism of sitagliptin reversing these changes and the signal pathway remains unknown.
In summary, our study is afirst step in unifying many factors in the diabetogenic processes in a relevant in vivo system and in-vestigates sitagliptin as a DPP-Ⅳ inhibitor for the diabetic heart to help move us closer tofinding effective treatment or prevention strategies. Future study could investigate difference doses of sitagliptin.
Fig. 7. Sitagliptin reduced the expression of RIP3and inhibited myocardial apoptosis. Sitagliptin reduced the expression of 7.1(AeC): RIP3; 7.2(AeB): inhibited myocardial apoptosis;
and 7.2(C): proportion of myocardial apoptotic cells positively correlated with relative levels of RIP3mRNA (Data are mean± SEM; Compared with control, *P ＜ 0.05, **P ＜ 0.01;
Conflict of interest
The authors declare that they have no competing interests. Acknowledgment
Yu-Sheng Liu, wrote the manuscript and researched data. Zhi-Wei Huang, is studying for her doctorate (her tuter Prof. Xiu-Li Ju) in the Shandong University and also is working in the Department of Hematology, The Qilu Childrens' Hospital of Shandong Univer-sity, performed the animal model and molecular analysis. Lin Wang and Xin-Xin Liu reviewed/edited the manuscript, and Yong-Mei Wang performed echocardiology. Yun Zhang and Mei Zhang, researched data and reviewed/edited manuscript. The study was supported by the National Basic Research Program of China (973 Program, 2011CB503906) and the National Science Foundation of China (No. 81270404,81470559) and by the Seed Fund (grant S2013).
References
(1) Cas AD, Spigoni V, Ridolfi V, Metra M. Diabetes and chronic heart failure: from diabetic cardiomyopathy to therapeutic approach. Endocr Metab Immune Disord Drug Targets. 2013;15(1):220e223.
(2) Hamby RI, Zoneraich S, Sherman L. Diabetic cardiomyopathy. JAMA. 1974;229(13):1749e1754.
(3) Sharifi AM, Mousavi SH, Farhadi M, Larijani B. Study of high glucose-induced apoptosis in PC12 cells: role of bax protein. J Pharmacol Sci. 2007;104(3): 258e262.
(4) Lee TI, Kao YH, Chen YC, Huang JH, Hsiao FC, Chen YJ. Peroxisome proliferator-activated receptors modulate cardiac dysfunction in diabetic cardiomyopathy. Diabetes Res Clin Pract. 2013;8227(13):9e10.
(5) Bai Y, Cui W, Xin Y, Miao X, Barati MT, Zhang C, et al. Prevention by sulforaphane of diabetic cardiomyopathy is associated with up-regulation of Nrf2 expression and transcription activation. J Mol Cell Cardiol. 2013;S0022e2828(13):00011e00014. (6) Sari FR, Watanabe K, Thandavarayan RA, Harima M, Zhang S, Muslin AJ, et al. 14-3-3 protein protects against cardiac endoplasmic reticulum stress (ERS) and ERS-initiated apoptosis in experimental diabetes. J Pharmacol Sci. 2010;113(4):325e334.
(7) Zhang DW, Zheng M, Zhao J, Li YY, Huang Z, Li Z, Han J. Multiple death pathways in TNF-treatedfibroblasts: RIP3- and RIP1-dependent and inde-pendent routes. Cell Res. 2011;21(2):368e371.
(8) Vanlangenakker N, Vanden Berghe T, Bogaert P, Laukens B, Zobel K, Deshayes K, et al. Bertrand MJ.cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species pro-duction. Cell Death Differ. 2011;18(4):656e665.
(9) Akarte AS, Srinivasan BP, Gandhi S. A novel long acting DPP-IV inhibitor PKF-275-055 stimulatesb-cell proliferation resulting in improved glucose ho-meostasis in diabetic rats. Biochem Pharmacol. 2012;83(2):241e252. (10) Kim MK, Chae YN, Kim HD, Yang EK, Cho EJ, Choi SH, et al. DA-1229, a novel
and potent DPP4 inhibitor, improves insulin resistance and delays the onset of diabetes. Life Sci. 2012;90(1e2):21e29.
(11) Shigeto M, Katsura M, Matsuda M, Ohkuma S, Kaku K. Low, but physio-logical, concentration of GLP-1 stimulates insulin secretion independent of the cAMP-dependent protein kinase pathway. J Pharmacol Sci. 2008;108(3): 274e279.
(12) Yin M, Sillje HH, Meissner M, van Gilst WH, de Boer RA. Early and late effects
of the DPP-4 inhibitor vildagliptin in a rat model of post-myocardial infarction heart failure. Cardiovasc Diabetol. 2011;10(5):85e87.
(13) Cuthbertson DJ, Irwin A, Gardner CJ, Daousi C, Purewal T, Furlong N, et al. Improved glycaemia correlates with liver fat reduction in obese, type 2 dia-betes, patients given glucagon-like peptide-1 (GLP-1) receptor agonists. PLoS One. 2012;7(12):111e113.
(14) Yamazaki K, Inoue T, Yasuda N, Sato Y, Nagakura T, Takenaka O, et al. Com-parison of efficacies of a dipeptidyl peptidase IV inhibitor and alpha-glucosidase inhibitors in oral carbohydrate and meal tolerance tests and the effects of their combination in mice. J Pharmacol Sci. 2007;104(1):29e38. (15) Ku HC, Chen WP, Su MJ. DPP-4 deficiency preserves cardiac function via GLP-1
signaling in rats subjected to myocardial ischemia/reperfusion. Naunyn Schmiedebergs Arch Pharmacol. 2011;384(2):197e207.
(16) Ti Y, Xie GL, Wang ZH, Bi XL, Ding WY, Wang J, et al. TRB3 gene silencing alleviates diabetic cardiomyopathy in a type 2 diabetic rat model. Diabetes. 2011;60(11):2963e2974.
(17) Sahn DJ, DeMaria A, Kisslo J, Weyman A. Recommendations regarding quan-titation in M-mode echocardiography: results of a survey of echo-cardiographic measurements. Circulation. 1978;58:1072e1083.
(18) Sun M, Dawood F, Wen WH, Chen M, Dixon I, Kirshenbaum LA, et al. Excessive tumor necrosis factor activation after infarction contributes to susceptibility of myocardial rupture and left ventricular dysfunction. Circulation. 2004;110: 3221e3228.
(19) Bi XP, Tan HW, Xing SS, Wang ZH, Tang MX, Zhang Y, et al. Overexpression of TRB3 gene in adipose tissue of rats with high fructose-induced metabolic syndrome. Endocr J. 2008;55:747e752.
(20) B1 Picatoste, Ramírez E, Caro-Vadillo A, Iborra C, Ares-Carrasco S, Egido J, et al. Sitagliptin reduces cardiac apoptosis, hypertrophy andfibrosis primarily by insulin-dependent mechanisms in experimental type-II diabetes. Potential roles of GLP-1 isoforms. PLoS One. 2013;21:e78330.
(21) Yamasaki Y, Katakami N, Kaneto H, Nakajima K, Kusuoka H, Kashiwagi A, et al. Improved lipid profiles are associated with reduced incidence of coronary vascular events in asymptomatic patients with type 2 diabetes and impaired myocardial perfusion. J Atheroscler Thromb. 2012;18(11):1203e1206. (22) Akarte AS, Srinivasan BP, Gandhi S. Vildagliptin selectively ameliorates GLP-1,
GLUT4, SREBP-1c mRNA levels and stimulatesb-cell proliferation resulting in improved glucose homeostasis in rats with streptozotocin-induced diabetes. J Diabetes Complications. 2012;26(4):266e274.
(23)Satoh-Asahara N, Sasaki Y, Wada H, Tochiya M, Iguchi A, Nakagawachi R, et al. A dipeptidyl peptidase-4 inhibitor, sitagliptin, exerts anti-inflammatory ef-fects in type 2 diabetic patients. Metabolism. 2012;10(11):135e137. (24)Noyan-Ashraf MH, Shikatani EA, Schuiki I, Mukovozov I, Wu J, Li RK, et al.
A glucagon-like peptide-1 analog reverses the molecular pathology and car-diac dysfunction of a mouse model of obesity. Circulation. 2013;127(1): 74e85.
(25)Chaykovska L, von Websky K, Rahnenführer J, Alter M, Heiden S, Fuchs H, et al. Effects of DPP-4 inhibitors on the heart in a rat model of uremic cardiomy-opathy. PLoS One. 2011;6(11):e27861.
(26)Parlevliet ET, Wang Y, Geerling JJ, Schr€oder-Van der Elst JP, Picha K, O'Neil K, et al. GLP-1 receptor activation inhibits VLDL production and reverses hepatic steatosis by decreasing hepatic lipogenesis in high-fat-fed APOE-/-mice. PLoS One. 2012;7(11):e49152.
(27)Terasaki M, Nagashima M, Watanabe T, Nohtomi K, Mori Y, Miyazaki A, et al. Effects of PKF275-055, a dipeptidyl peptidase-4 inhibitor, on the development of atherosclerotic lesions in apolipoprotein E-null mice. Metabolism. 2012;61(7):974e977.
(28)Giannocco G, Oliveira KC, Crajoinas RO, Venturini G, Salles TA, Fonseca-Alaniz MH, et al. Dipeptidyl peptidase IV inhibition upregulates GLUT4 translocation and expression in heart and skeletal muscle of spontaneously hypertensive rats. Eur J Pharmacol. 2013;698(1e3):74e86.
(29)Palaniyappan A, Uwiera RR, Idikio H, Menon V, Jugdutt C, Jugdutt BI. Atten-uation of increased secretory leukocyte protease inhibitor, matricellular proteins and angiotensin II and left ventricular remodeling by candesartan and omapatrilat during healing after reperfused myocardial infarction. Mol Cell Biochem. 2013;30(1):223e225.
(30)Zhu BM, Miyamoto S, Nagasawa Y, Wajima T, Hashimoto K. Effect of the sarcolemmal K (ATP) channel blocker HMR1098 on arrhythmias induced by programmed electrical stimulation in canine old myocardial infarction model: comparison with glibenclamide. J Pharmacol Sci. 2003;93(1):106e113. (31)Schramm TK, Gislason GH, Vaag A, Rasmussen JN, Folke F, Hansen ML,
Fosbøl EL, et al. Mortality and cardiovascular risk associated with different insulin secretagogues compared with metformin in type 2 diabetes, with or without a previous myocardial infarction: a nationwide study. Eur Heart J. 2011;32(15):19008.
(32)Kwon DY, Kim YS, Ahn IS, Kim da S, Kang S, Hong SM, et al. Exendin-4 po-tentiates insulinotropic action partly via increasing beta-cell proliferation and neogenesis and decreasing apoptosis in association with the attenuation of endoplasmic reticulum stress in islets of diabetic rats. J Pharmacol Sci. 2009;111(4):361e371.
(33)Kim JY, Lim DM, Park HS, Moon CI, Choi KJ, Lee SK, et al. Exendin-4 protects against sulfonylurea-inducedb-cell apoptosis. J Pharmacol Sci. 2012;118(1): 65e74.
(34)DeNicola M, Du J, Wang Z, Yano N, Zhang L, Wang Y, et al. Stimulation of glucagon-like peptide-1 receptor through exendin-4 preserves myocardial performance and prevents cardiac remodeling in infarcted myocardium. Am J Physiol Endocrinol Metab. 2014;307(8):E630eE643.
(35)Wu XM, Ou QY, Zhao W, Liu J, Zhang H. The GLP-1 analogue Liraglutide protects cardiomyocytes from high glucose-induced apoptosis by activating the Epac-1/Akt pathway. Exp Clin Endocrinol Diabetes. 2014;11(2):20e26.
