# A Possible Mechanism: Vildagliptin Prevents Aortic Dysfunction through Paraoxonase and Angiopoietin-Like 3

Authors: Qian Zhang; Xinhua Xiao; Jia Zheng; Ming Li; Miao Yu; Fan Ping; Tong Wang; Xiaojing Wang
Journal: BioMed Research International
 (2018)

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## Abstract

The collected data have revealed the beneficial effects of dipeptidyl peptidase-4 (DPP-4) inhibitors on the vascular endothelium, including vildagliptin. However, the involved mechanisms are not yet clear. In this study, Sprague-Dawley rats were randomly divided into the following four groups: control, diabetic, diabetic + low-dose vildagliptin (10 mg/kg/d), and diabetic + high-dose vildagliptin (20 mg/kg/d). The diabetic model was created by feeding a high-fat diet for four weeks and injection of streptozotocin. Then, vildagliptin groups were given oral vildagliptin for twelve weeks, and the control and diabetic groups were given the same volume of saline. The metabolic parameters, endothelial function, and whole genome expression in the aorta were examined. After 12 weeks of treatment, vildagliptin groups showed significantly reduced blood glucose, blood total cholesterol, and attenuated endothelial dysfunction. Notably, vildagliptin may inhibit angiopoietin-like 3(Angptl3) and betaine-homocysteine S-methyltransferase(Bhmt) expression and activated paraoxonase-1(Pon1) in the aorta of diabetic rats. These findings may demonstrate the vasoprotective pathway of vildagliptinin vivo.


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## Body

## 1. Introduction

The incidence and prevalence of diabetes mellitus are dramatically increasing worldwide [1]. Epidemiological research shows that diabetic patients have a higher risk of cardiovascular diseases [2]. The main cause of morbidity and mortality in diabetic patients is cardiovascular diseases.Glucagon-like peptide-1 (GLP-1) is produced in gut L-cells. It contains 30 amino acids. The main physiological function of GLP-1 is stimulation of insulin secretion from pancreaticβ cells when glucose is orally taken up in the human body. GLP-1 can also inhibit glucose production and appetite, activate adipose and muscle glucose uptake and storage, and thus moderate insulin sensitivity. However, GLP-1 is quickly hydrolyzed by dipeptidyl peptidase-4 (DPP-4).DPP-4 inhibitors are a new class of GLP-1 based antidiabetic drugs. As one type of DPP-4 inhibitors, vildagliptin controls blood glucose by inhibiting the enzymatic activity of DPP-4. DPP-4 is also found on endothelial cells in the cardiovascular system, and increasing research has focused on the benefit of DPP-4 inhibitors on cardiovascular function. Sitagliptin (one type of DPP-4 inhibitor) has been proven to significantly attenuate heart failure-related left ventricular (LV) end-diastolic pressure, systolic performance, and chamber stiffness in an ablation-induced cardiac dysfunction rat model [3]. In a clinical trial, sitagliptin enhanced global and regional LV function in type 2 diabetes mellitus (T2DM) patients with coronary artery [4]. Vildagliptin exerts cardioprotective effects in obesity-based insulin resistance [5, 6], myocardial infarction (MI) [7], and ischemia-reperfusion (I/R) injury rat models [8]. However, the exact mechanism of the beneficial effect of vildagliptin on the aorta in diabetic rats remains to be elucidated.In this study, we hypothesized that vildagliptin improved aorta function through multiple pathways. We employed a whole genomic expression array and bioinformatics method to explore the pathway involved in aorta vascular function moderation in diabetic rats.


## 2. Materials and Methods

### 2.1. Animal Treatments and Diets

Five-week-old male Sprague-Dawley rats were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College (Beijing, China, SCXK-2014-0013). The animal protocol was approved by the Animal Care Committee of the Peking Union Medical Hospital Animal Ethics Committee (Project XHDW-2015-0051, 15 Feb 2015), and all efforts were made to minimize suffering. All the rats were fed in a light/dark cycle (12 hours : 12 hours) environment and were free to drink water. Three days after arrival, rats were randomly divided into four groups (n=6 per group): normal control group, diabetic group, low-dose vildagliptin (vil-low), and high-dose vildagliptin (vil-high). The normal control group was fed a standard rodent diet (kcal%: 10% fat, 20% protein, and 70% carbohydrate; 3.85 kcal/gm). Other groups were fed a high-fat diet (kcal%: 45% fat, 20% protein, and 35% carbohydrate; 4.73 kcal/gm, Research Diet, New Brunswick, NJ, USA). After 4 weeks, diabetic, low-dose vildagliptin, and high-dose vildagliptin groups were given a single injection of streptozotocin (STZ, 30 mg/kg body weight, i.p., Sigma-Aldrich, St. Louis, MO, USA). Fasting blood glucose > 11.1 mmol/L was the standard for the diabetic model. Then, vil-low and vil-high groups were treated with 10 mg or 20 mg vildagliptin (Novartis Pharma AG, Basel, Switzerland)/kg of body weight by daily gavage for 12 weeks. Normal control and diabetic groups were given normal saline. After 12 weeks of treatment, the rats were anesthetized using ketamine (100 mg/kg i.p., Pharmacia and Upjohn Ltd., Crawley, UK), followed by withdrawal of food overnight. Blood samples were obtained from the abdominal aorta. Then, the rats were sacrificed by decapitation. The thoracic aorta was quickly removed. Some aortas were placed in Krebs solution (120 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.18 mmol/L of KH2PO4, 2.25 mmol/L of CaCl2, 24.5 mmol/L of NaHCO3, 1.2 mmol/L of MgSO4·7H2O, 11.1 mmol/L of glucose, and 0.03 mmol/L of EDTA) and aerated with 95% O2 and 5% CO2. Other aortas were frozen in liquid nitrogen and stored at −80°C for a gene microarray experiment.


### 2.2. Body Weight and Fasting Blood Glucose Measurements

The rats were weighed every 4 weeks. Fasting blood glucose levels were measured by Bayer Contour TS glucometer (Hamburg, Germany).


### 2.3. Oral Glucose Tolerance Test (OGTT)

An OGTT was performed after 12 weeks of treatment. Blood glucose levels were measured at 30, 60, and 120 min after an oral administration of 20% glucose at a dose of 2 g/kg. The area under the curve (AUC) was calculated by the linear trapezoid method [9].


### 2.4. Serum Insulin and Lipid Panel Measurements

Serum fasting insulin was analyzed using an ELISA kit (Millipore, Billerica, MA, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by the following formula: FBG (mmol/L) × fasting insulin (μIU/mL)/22.5. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured using an enzyme end-point kit (Roche Diagnostics GmbH, Mannheim, Germany).


### 2.5. Isometric Contractile Tension Assay

Isometric contractile tension was determined as follows and described previously [10]. The aortic ring segments (3 mm width) were incubated with Krebs solution (120 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.18 mmol/L of KH2PO4, 2.25 mmol/L of CaCl2, 24.5 mmol/L of NaHCO3, 1.2 mmol/L of MgSO4·7H2O, 11.1 mmol/L of glucose, and 0.03 mmol/L of EDTA), aerated with 95% O2 and 5% CO2, and then preconstricted with phenylephrine (Phe, 10−7 mmol/L). Isometric contractile tension was measured by a BL-410 Biological function system (Chengdu Tai Meng Science and Technology Co., Ltd., Chengdu, China). Once a basal contraction was obtained, 10−10 to 10−4 mol/L of acetylcholine (Ach) was cumulatively added to the solution.


### 2.6. RNA Extraction, Amplification, Labeling, and Hybridization

To look for the differentially expressed genes under vildagliptin treatment in diabetic rats, we performed gene whole transcript-based array in vil-high group and diabetic group (n=3 in each group). Total RNA was extracted from aortas using a mirVana™ RNA Isolation Kit (Ambion, San Paulo, SP, Brazil). The RNA was purified using an RNeasy Kit (Qiagen, Hilden, Germany), quantified by NanoDrop ND-2000 spectrophotometry (Nanodrop Tech, Rockland, Del, Wilmington, DE, USA), and qualified by agarose gel electrophoresis. Total RNA (100 ng) was used for cDNA synthesis. cRNA was synthesized followed by two-strand cDNA. Biotin-labeled cRNA was hybridized to an Affymetrix GeneChip Rat Gene 2.0 ST whole transcript-based array (Affymetrix Technologies, Santa Clara, CA, USA). After hybridization, the microarrays were washed, stained, and scanned with an Affymetrix Scanner 3000 7G (Santa Clara, CA, USA).The microarray signals were analyzed using Expression Console software (version 1.4.1, Affymetrix, Santa Clara, CA, USA). The significance of the difference in genes was determined by one-way ANOVA. Differentially expressed genes were defined as genes with a fold change > 2.0 andP < 0.05. The microarray raw data were submitted to the Gene Expression Omnibus (GEO) repository (GSE102196).


### 2.7. Bioinformatics Analysis for Microarray

DAVID (Database for Annotation, Visualization and Integrated Discovery) software (https://david.abcc.ncifcrf.gov/) [11] was used to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The Search Tool for the Retrieval of Interacting Genes (STRING) database (https://string-db.org/) [12] was used to analyze the interaction network for differentially expressed genes.


### 2.8. Validation of Differentially Expressed Genes by Quantitative PCR

The expression levels of three differentially expressed genes were measured by qPCR to validate the results of the microarray. All primers are listed in Table1.Actin was used as an internal control.Table 1
Oligonucleotide sequences for qPCR analysis.
Gene symbol GenBank ID Forward primer Reverse Primer Product size Angptl3 NM_001025065 AAAGGGTTTTGGGAGGCTTGA CCCAAAAGCGCTATGGTCTC 117 Bhmt NM_030850 GATGCTTGGGGAGTGACGAA TGTGGCTACTGTGCGGATTT 119 Pon1 NM_032077 AAGCTGGCTACACCCACATC CAACATTCGTTGGTGAGCGG 103 Angptl3: angiopoietin-like 3; Bhmt: betaine-homocysteine S-methyltransferase; Pon1: paraoxonase 1.


### 2.9. Statistical Analyses

Data are presented as the means ± standard deviation (SD). One-way ANOVA analysis followed by Student’st-test was used to compare differences among groups. P<0.05 was considered significant.


## 2.1. Animal Treatments and Diets

Five-week-old male Sprague-Dawley rats were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College (Beijing, China, SCXK-2014-0013). The animal protocol was approved by the Animal Care Committee of the Peking Union Medical Hospital Animal Ethics Committee (Project XHDW-2015-0051, 15 Feb 2015), and all efforts were made to minimize suffering. All the rats were fed in a light/dark cycle (12 hours : 12 hours) environment and were free to drink water. Three days after arrival, rats were randomly divided into four groups (n=6 per group): normal control group, diabetic group, low-dose vildagliptin (vil-low), and high-dose vildagliptin (vil-high). The normal control group was fed a standard rodent diet (kcal%: 10% fat, 20% protein, and 70% carbohydrate; 3.85 kcal/gm). Other groups were fed a high-fat diet (kcal%: 45% fat, 20% protein, and 35% carbohydrate; 4.73 kcal/gm, Research Diet, New Brunswick, NJ, USA). After 4 weeks, diabetic, low-dose vildagliptin, and high-dose vildagliptin groups were given a single injection of streptozotocin (STZ, 30 mg/kg body weight, i.p., Sigma-Aldrich, St. Louis, MO, USA). Fasting blood glucose > 11.1 mmol/L was the standard for the diabetic model. Then, vil-low and vil-high groups were treated with 10 mg or 20 mg vildagliptin (Novartis Pharma AG, Basel, Switzerland)/kg of body weight by daily gavage for 12 weeks. Normal control and diabetic groups were given normal saline. After 12 weeks of treatment, the rats were anesthetized using ketamine (100 mg/kg i.p., Pharmacia and Upjohn Ltd., Crawley, UK), followed by withdrawal of food overnight. Blood samples were obtained from the abdominal aorta. Then, the rats were sacrificed by decapitation. The thoracic aorta was quickly removed. Some aortas were placed in Krebs solution (120 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.18 mmol/L of KH2PO4, 2.25 mmol/L of CaCl2, 24.5 mmol/L of NaHCO3, 1.2 mmol/L of MgSO4·7H2O, 11.1 mmol/L of glucose, and 0.03 mmol/L of EDTA) and aerated with 95% O2 and 5% CO2. Other aortas were frozen in liquid nitrogen and stored at −80°C for a gene microarray experiment.


## 2.2. Body Weight and Fasting Blood Glucose Measurements

The rats were weighed every 4 weeks. Fasting blood glucose levels were measured by Bayer Contour TS glucometer (Hamburg, Germany).


## 2.3. Oral Glucose Tolerance Test (OGTT)

An OGTT was performed after 12 weeks of treatment. Blood glucose levels were measured at 30, 60, and 120 min after an oral administration of 20% glucose at a dose of 2 g/kg. The area under the curve (AUC) was calculated by the linear trapezoid method [9].


## 2.4. Serum Insulin and Lipid Panel Measurements

Serum fasting insulin was analyzed using an ELISA kit (Millipore, Billerica, MA, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by the following formula: FBG (mmol/L) × fasting insulin (μIU/mL)/22.5. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured using an enzyme end-point kit (Roche Diagnostics GmbH, Mannheim, Germany).


## 2.5. Isometric Contractile Tension Assay

Isometric contractile tension was determined as follows and described previously [10]. The aortic ring segments (3 mm width) were incubated with Krebs solution (120 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.18 mmol/L of KH2PO4, 2.25 mmol/L of CaCl2, 24.5 mmol/L of NaHCO3, 1.2 mmol/L of MgSO4·7H2O, 11.1 mmol/L of glucose, and 0.03 mmol/L of EDTA), aerated with 95% O2 and 5% CO2, and then preconstricted with phenylephrine (Phe, 10−7 mmol/L). Isometric contractile tension was measured by a BL-410 Biological function system (Chengdu Tai Meng Science and Technology Co., Ltd., Chengdu, China). Once a basal contraction was obtained, 10−10 to 10−4 mol/L of acetylcholine (Ach) was cumulatively added to the solution.


## 2.6. RNA Extraction, Amplification, Labeling, and Hybridization

To look for the differentially expressed genes under vildagliptin treatment in diabetic rats, we performed gene whole transcript-based array in vil-high group and diabetic group (n=3 in each group). Total RNA was extracted from aortas using a mirVana™ RNA Isolation Kit (Ambion, San Paulo, SP, Brazil). The RNA was purified using an RNeasy Kit (Qiagen, Hilden, Germany), quantified by NanoDrop ND-2000 spectrophotometry (Nanodrop Tech, Rockland, Del, Wilmington, DE, USA), and qualified by agarose gel electrophoresis. Total RNA (100 ng) was used for cDNA synthesis. cRNA was synthesized followed by two-strand cDNA. Biotin-labeled cRNA was hybridized to an Affymetrix GeneChip Rat Gene 2.0 ST whole transcript-based array (Affymetrix Technologies, Santa Clara, CA, USA). After hybridization, the microarrays were washed, stained, and scanned with an Affymetrix Scanner 3000 7G (Santa Clara, CA, USA).The microarray signals were analyzed using Expression Console software (version 1.4.1, Affymetrix, Santa Clara, CA, USA). The significance of the difference in genes was determined by one-way ANOVA. Differentially expressed genes were defined as genes with a fold change > 2.0 andP < 0.05. The microarray raw data were submitted to the Gene Expression Omnibus (GEO) repository (GSE102196).


## 2.7. Bioinformatics Analysis for Microarray

DAVID (Database for Annotation, Visualization and Integrated Discovery) software (https://david.abcc.ncifcrf.gov/) [11] was used to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The Search Tool for the Retrieval of Interacting Genes (STRING) database (https://string-db.org/) [12] was used to analyze the interaction network for differentially expressed genes.


## 2.8. Validation of Differentially Expressed Genes by Quantitative PCR

The expression levels of three differentially expressed genes were measured by qPCR to validate the results of the microarray. All primers are listed in Table1.Actin was used as an internal control.Table 1
Oligonucleotide sequences for qPCR analysis.
Gene symbol GenBank ID Forward primer Reverse Primer Product size Angptl3 NM_001025065 AAAGGGTTTTGGGAGGCTTGA CCCAAAAGCGCTATGGTCTC 117 Bhmt NM_030850 GATGCTTGGGGAGTGACGAA TGTGGCTACTGTGCGGATTT 119 Pon1 NM_032077 AAGCTGGCTACACCCACATC CAACATTCGTTGGTGAGCGG 103 Angptl3: angiopoietin-like 3; Bhmt: betaine-homocysteine S-methyltransferase; Pon1: paraoxonase 1.


## 2.9. Statistical Analyses

Data are presented as the means ± standard deviation (SD). One-way ANOVA analysis followed by Student’st-test was used to compare differences among groups. P<0.05 was considered significant.


## 3. Results

### 3.1. Vildagliptin Moderated Blood Glucose and Insulin Resistance

After 12 weeks of treatment, vildagliptin significantly reduced fasting blood glucose AUC in OGTT (P<0.05, Figures 1(b) and 1(f)). Additionally, fasting serum insulin and HOMA-IR index in the vildagliptin-treated group were lower than in the diabetic group (P<0.05, Figures 1(c) and 1(d)).Figure 1
Effect of vildagliptin on metabolic parameters in rats. (a) Body weight, (b) fasting blood glucose, (c) insulin, (d) homeostasis model assessment (HOMA-IR), (e) blood glucose in oral glucose tolerance test (OGTT), (f) area under the curve (AUC) in OGTT, (g) total cholesterol, (h) triglyceride, (i) high-density lipoprotein cholesterol (HDL-C), (j) low-density lipoprotein cholesterol (LDL-C), and (k) relaxation responses to Ach in aortic rings. Values are mean ± SD (n=6), P∗<0.05, P∗∗<0.01, versus control group; P#<0.05, P##<0.01 versus diabetic group. vil-low: low dose of vildagliptin; vil-high: high dose of vildagliptin.
(a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)


### 3.2. Vildagliptin Decreased Blood Lipid Levels

Vildagliptin decreased TC and LDL-C levels in diabetic rats (P<0.01, Figures 1(g) and 1(j)).


### 3.3. Vildagliptin Depressed Vasorelaxation Responses of Aortic Rings to Ach

As shown in Figure1(k), endothelium-dependent vasodilation was impaired in diabetic rats compared with normal rats. Treatment with vildagliptin ameliorated this impairment (P<0.01).


### 3.4. Identification of Differentially Expressed Genes

After filtering genes that had a cutoff of a 1.5-fold change or greater, we found that 150 genes were upregulated and 120 genes were downregulated in the vil-high group compared with the diabetic group (P<0.05).


### 3.5. GO Term Enrichment Analysis of Differentially Expressed Genes

In general, 91 GO terms were significantly enriched (P<0.05, Table 2). The top 20 most significant biological process (BP) terms are shown in Figure 2 (P<0.01). They were negative regulation of endopeptidase activity, epoxygenase P450 pathway, acute-phase response, blood coagulation, oxidation-reduction processes, cytoskeleton organization, negative regulation of fibrinolysis, microtubule-based processes, phosphatidylcholine metabolic processes, fibrinolysis, organ regeneration, complement activation classical pathway, inflammatory response, positive regulation of lipid catabolic process, triglyceride homeostasis, aging, cellular response to interferon gamma, cholesterol metabolic process, cholesterol homeostasis, and tissue regeneration.Table 2
The enriched GO terms with differentially expressed genes(P<0.05).
Term ID Term name Count P-value Fold Enrichment catalogue Genes GO:0010951 negative regulation of endopeptidase activity 16 7.22 × 10−10 8.425 Biology Process KNG1, MUG2, MUG1, SERPINA10, PZP, C5, AHSG, AMBP, SERPINA3N, SERPINF2, SERPINA4, SERPINC1, ITIH4, HRG, ITIH2, SERPIND1 GO:0019373 epoxygenase P450 pathway 7 5.88 × 10−7 21.821 Biology Process CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2C13, CYP2A1, CYP2A2 GO:0006953 acute-phase response 7 7.97 × 10−6 14.356 Biology Process KNG1, MUG1, CRP, ITIH4, SAA4, SERPINA1, AHSG GO:0007596 blood coagulation 8 2.08 × 10−5 9.446 Biology Process F13B, F12, SERPINA10, C9, F9, SERPIND1, CPB2, PLG GO:0055114 oxidation-reduction process 23 3.29 × 10−5 2.753 Biology Process ALDH8A1, CYP2B3, CYP2J4, HSD3B5, DECR2, RGD1304810, CYP2D3, EGLN1, KMO, P4HTM, VAT1, IYD, CYP2C6V1, TDO2, CYP3A23/3A1, HIF1AN, CYP4F4, CYP2C23, CYP2C13, SH3BGRL3, RDH16, CYP2A1, CYP2A2 GO:0007010 cytoskeleton organization 10 3.95 × 10−5 6.089 Biology Process CCL24, TNIK, CAP2, TUBB2A, SVIL, CAMSAP1, STRIP2, TUBB6, TUBA1A, TUBB4B GO:0051918 negative regulation of fibrinolysis 4 6.93 × 10−5 44.533 Biology Process SERPINF2, HRG, CPB2, PLG GO:0007017 microtubule-based process 6 1.68 × 10−4 11.405 Biology Process TUBB2A, TUBB5, TUBB6, TUBA1A, DCTN1, TUBB4B GO:0046470 phosphatidylcholine metabolic process 4 6.75 × 10−4 22.267 Biology Process APOA5, PON1, GPLD1, LIPC GO:0042730 fibrinolysis 4 8.35 × 10−4 20.782 Biology Process F12, HRG, CPB2, PLG GO:0031100 organ regeneration 7 0.00110 5.995 Biology Process APOA2, BAAT, ADH1, ALDOC, APOA5, PRPS2, AHSG GO:0006958 complement activation, classical pathway 5 0.00123 10.532 Biology Process C9, C5, CRP, C4BPB, C4BPA GO:0006954 inflammatory response 12 0.00158 3.149 Biology Process CCL24, KNG1, TLR10, SERPINA3N, TNFRSF10B, MUG1, CCL21, C5, HRH4, CCL9, SERPINA1, CXCR3 GO:0050996 positive regulation of lipid catabolic process 3 0.00236 38.967 Biology Process APOA2, APOA5, ANGPTL3 GO:0070328 triglyceride homeostasis 4 0.00431 11.990 Biology Process APOC4, APOA5, LIPC, ANGPTL3 GO:0007568 aging 11 0.00745 2.721 Biology Process ADRB3, CYP3A23/3A1, CRYAB, ENDOG, ALDOC, CRP, SPINK1, IGFBP1, ATP5G3, SREBF2, HTR2A GO:0071346 cellular response to interferon-gamma 5 0.00766 6.388 Biology Process CCL24, SERPINA3N, CCL21, CCL9, CFH GO:0008203 cholesterol metabolic process 5 0.00811 6.285 Biology Process APOA2, PON1, LIPC, ANGPTL3, SREBF2 GO:0042632 cholesterol homeostasis 5 0.00906 6.089 Biology Process CAV3, APOA2, APOA5, LIPC, ANGPTL3 GO:0042246 tissue regeneration 4 0.00996 8.907 Biology Process SERPINA10, APOA5, IGFBP1, PLG GO:0046330 positive regulation of JNK cascade 5 0.0112 5.730 Biology Process DIXDC1, CCL21, SERPINF2, MAP3K10, TPD52L1 GO:0050921 positive regulation of chemotaxis 3 0.0134 16.700 Biology Process CCL21, C5, CXCR3 GO:0006956 complement activation 3 0.0153 15.587 Biology Process C8A, C5, CFH GO:2000649 regulation of sodium ion transmembrane transporter activity 3 0.0153 15.587 Biology Process CAV3, SCN1B, SCN2B GO:0042573 retinoic acid metabolic process 3 0.0173 14.613 Biology Process ALDH8A1, ADH1, RDH16 GO:0009395 phospholipid catabolic process 3 0.0217 12.989 Biology Process APOA2, ENPP2, ANGPTL3 GO:0001666 response to hypoxia 9 0.0230 2.598 Biology Process CRYAB, ANG, CAMK2G, ALDOC, CRP, SERPINA1, EGLN1, PAK1, LIPC GO:0055117 regulation of cardiac muscle contraction 3 0.0241 12.305 Biology Process CAV3, P2RX4, CALM3 GO:0055090 acylglycerol homeostasis 2 0.0254 77.933 Biology Process APOA5, ANGPTL3 GO:0034370 triglyceride-rich lipoprotein particle remodeling 2 0.0254 77.933 Biology Process APOA2, APOA5 GO:0031116 positive regulation of microtubule polymerization 3 0.0265 11.690 Biology Process CAV3, MAP1B, DCTN1 GO:0032956 regulation of actin cytoskeleton organization 4 0.0287 5.995 Biology Process DIXDC1, HRG, PAK1, SH3BGRL3 GO:0048247 lymphocyte chemotaxis 3 0.0317 10.627 Biology Process CCL24, CCL21, CCL9 GO:0070098 chemokine-mediated signaling pathway 4 0.0363 5.469 Biology Process CCL24, CCL21, CCL9, CXCR3 GO:0045959 negative regulation of complement activation, classical pathway 2 0.0378 51.956 Biology Process C4BPB, C4BPA GO:0002542 Factor XII activation 2 0.0378 51.956 Biology Process KNG1, F12 GO:0009725 response to hormone 5 0.0381 3.936 Biology Process ANG, APOA5, LIPC, ANGPTL3, SREBF2 GO:0042311 vasodilation 3 0.0402 9.352 Biology Process KNG1, P2RX4, ALB GO:0006631 fatty acid metabolic process 4 0.0413 5.196 Biology Process BAAT, DECR2, LIPC, ANGPTL3 GO:0007399 nervous system development 7 0.0428 2.741 Biology Process PLXNA4, SCN2B, CAMK2G, KREMEN1, MAP1B, DPYSL3, NUMBL GO:0048675 axon extension 3 0.0431 8.992 Biology Process SEMA7A, MAP1B, POU4F3 GO:0072659 protein localization to plasma membrane 4 0.0485 4.871 Biology Process CAV3, TNIK, FGF13, CDH1 GO:0006935 chemotaxis 4 0.0485 4.871 Biology Process CCL24, ENPP2, C5, CXCR3 GO:0006810 transport 6 0.0489 3.017 Biology Process P2RX4, DYNC1LI1, AFM, LOC360919, ALB, LOC500473 GO:0072562 blood microparticle 22 1.84 × 10−18 14.631 Cellular Components KNG1, GC, C9, MUG1, APCS, C4BPA, PLG, AHSG, C8A, AMBP, APOA2, AFM, SERPINF2, ALB, HPX, APOA5, PON1, CFH, SERPINC1, ITIH4, HRG, ITIH2 GO:0005615 extracellular space 49 2.19 × 10−11 2.922 Cellular Components MUG2, MUG1, PZP, CRP, GPLD1, SPINK1, ANPEP, AZGP1, APOA2, ANG, SEMA7A, SERPINA4, APOA5, CFH, SERPINA1, SEMA3B, KNG1, F12, APCS, LOC360919, F9, C8A, AMBP, SERPINA3N, SERPINF2, SCGB2A2, GC, SERPINA10, ENPP2, C5, CCL9, AHSG, CCL24, ALB, CCL21, SERPINC1, ANGPTL3, C4BPB, DPYSL3, C4BPA, PLG, AFM, HPX, PON1, METRNL, SERPIND1, IGFBP1, LIPC, CPB2 GO:0034364 high-density lipoprotein particle 7 4.31 × 10−8 32.313 Cellular Components APOA2, APOC4, APOA5, PON1, GPLD1, SAA4, LIPC GO:0070062 extracellular exosome 66 6.06 × 10−8 1.957 Cellular Components ALDH8A1, CYP2J4, MUG1, TUBB2A, CRP, GPLD1, ANPEP, CKB, AZGP1, APOA2, DES, PACSIN3, ANG, SERPINA4, ITIH4, TUBB5, CFH, TUBB6, ITIH2, SEMA3B, SERPINA1, TUBA1A, KNG1, F12, TNIK, APCS, CRYAB, F9, METTL7A, VAT1, AMBP, C8A, RPS16, SERPINF2, BHMT, PRNP, SLC27A2, PRPS2, GC, C9, SERPINA10, ALDOC, C5, CDH1, KMO, AHSG, ALCAM, ALB, SERPINC1, DOPEY2, HRG, TUBB4B, COTL1, PLG, P2RX4, AFM, HPX, ARF3, PON1, CALM3, PAPPA2, METRNL, SERPIND1, SH3BGRL3, MYH14, CPB2 GO:0031090 organelle membrane 11 2.10 × 10−7 9.591 Cellular Components CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, CYP4F4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0030018 Z disc 9 1.24 × 10−4 6.037 Cellular Components CAV3, JPH2, DES, CRYAB, BAG3, PDLIM3, PAK1, HOMER1, FLNC GO:0014704 intercalated disc 6 4.60 × 10−4 9.232 Cellular Components CAV3, SCN1B, DES, ATP2A2, FGF13, PAK1 GO:0030426 growth cone 9 6.13 × 10−4 4.772 Cellular Components ANG, CRP, MAP1B, CALM3, DPYSL3, FGF13, MYH14, PAK1, HAP1 GO:0005874 microtubule 11 8.97 × 10−4 3.658 Cellular Components DYNC1LI1, TUBB2A, CAMSAP1, MAP1B, TUBB5, TUBB6, FGF13, TUBA1A, DCTN1, TUBB4B, GLYATL2 GO:0043034 costamere 4 0.00196 15.695 Cellular Components SVIL, SYNM, HOMER1, FLNC GO:0005576 extracellular region 19 0.00456 2.088 Cellular Components APCS, C9, CRP, NTN4, GPLD1, SAA4, C4BPB, FGF13, PTH2, C4BPA, PLG, LOC500473, CCL24, C8A, SERPINA3N, ALB, APOA5, HRG, LIPC GO:0005789 endoplasmic reticulum membrane 16 0.00504 2.258 Cellular Components CYP2B3, CDIPT, HSD3B5, CYP2D3, SREBF2, CYP2C6V1, UGT2B37, UGT2B17, CYP3A23/3A1, ATP2A2, CYP4F4, CYP2C23, CYP2C13, CYP2A1, CYP2A2, SLC27A2 GO:0030424 axon 12 0.00657 2.609 Cellular Components GC, ALCAM, P2RX4, CRYAB, ALDOC, MAP1B, FGF13, CDH1, MYH14, PAK1, HOMER1, HTR2A GO:0034361 very-low-density lipoprotein particle 3 0.0192 13.848 Cellular Components APOA2, APOC4, APOA5 GO:0043231 intracellular membrane-bounded organelle 16 0.0302 1.825 Cellular Components KNG1, CYP2B3, CAV3, CYP2J4, HSD3B5, GPLD1, PLG, SREBF2, AMBP, UGT2B17, CYP3A23/3A1, PON1, CYP2C23, UGT2A3, CYP2A1, SLC27A2 GO:0005856 cytoskeleton 8 0.0345 2.605 Cellular Components TNIK, DES, FILIP1, MAP1B, FLNC, COTL1, TPM2, HAP1 GO:0044216 other organism cell 2 0.0376 52.316 Cellular Components C4BPB, C4BPA GO:0033017 sarcoplasmic reticulum membrane 3 0.0457 8.719 Cellular Components JPH2, ATP2A2, CAMK2G GO:0004867 serine-type endopeptidase inhibitor activity 15 6.34 × 10−11 11.167 Molecular Function MUG2, PZP, MUG1, SERPINA10, SPINK1, AMBP, SERPINA3N, SERPINF2, SERPINA4, SERPINC1, ITIH4, HRG, ITIH2, SERPINA1, SERPIND1 GO:0070330 aromatase activity 9 2.65 × 10−8 18.039 Molecular Function CYP2C6V1, CYP2B3, CYP3A23/3A1, CYP4F4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0008392 arachidonic acid epoxygenase activity 8 1.97 × 10−7 18.393 Molecular Function CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0008395 steroid hydroxylase activity 8 5.35 × 10−7 16.035 Molecular Function CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0020037 heme binding 13 1.46 × 10−6 6.122 Molecular Function CYP2B3, CYP2J4, CYP2D3, AMBP, CYP2C6V1, TDO2, CYP3A23/3A1, CYP4F4, CYP2C23, HRG, CYP2C13, CYP2A1, CYP2A2 GO:0008092 cytoskeletal protein binding 8 1.07 × 10−5 10.423 Molecular Function DES, PACSIN3, CRYAB, ALDOC, PDLIM3, CDH1, FLNC, ABCG2 GO:0005506 iron ion binding 13 1.11 × 10−5 5.031 Molecular Function CYP2B3, CYP2J4, CYP2D3, EGLN1, P4HTM, CYP2C6V1, CYP3A23/3A1, HIF1AN, CYP4F4, CYP2C23, CYP2C13, CYP2A1, CYP2A2 GO:0004866 endopeptidase inhibitor activity 5 5.40 × 10−4 13.028 Molecular Function MUG2, MUG1, C5, SERPINA1, AHSG GO:0017080 sodium channel regulator activity 5 6.95 × 10−4 12.214 Molecular Function CAV3, SCN1B, SCN2B, GPLD1, FGF13 GO:0016712 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen 5 8.78 × 10−4 11.495 Molecular Function CYP2B3, CYP2J4, CYP3A23/3A1, CYP2D3, CYP2A1 GO:0005200 structural constituent of cytoskeleton 6 0.00223 6.514 Molecular Function TUBB2A, TUBB5, TUBB6, SYNM, TUBA1A, TUBB4B GO:0016706 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors 4 0.00262 14.213 Molecular Function HIF1AN, RGD1304810, EGLN1, P4HTM GO:0008201 heparin binding 8 0.00268 4.283 Molecular Function SERPINA10, ANG, APOA5, SERPINC1, CFH, HRG, SERPIND1, LIPC GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 5 0.00297 8.316 Molecular Function CYP2C6V1, CYP2B3, CYP2C23, EGLN1, CYP2C13 GO:0005102 receptor binding 12 0.00666 2.598 Molecular Function KNG1, HAO1, P2RX4, BAAT, ANG, LRRC4B, DECR2, HRG, HOMER1, SLC27A2, HAP1, PLG GO:0004497 monooxygenase activity 5 0.00944 6.013 Molecular Function CYP2C6V1, CYP2B3, CYP2C23, CYP2D3, CYP2C13 GO:0004622 lysophospholipase activity 3 0.01328 16.751 Molecular Function ENPP2, LIPC, GDPD1 GO:0042803 protein homodimerization activity 19 0.01474 1.845 Molecular Function RBPMS2, CRYAB, GIMAP7, CAMK2G, CRP, NR4A1, TPD52L1, ABCG2, AMBP, ADRB3, APOA2, HIF1AN, ANG, SERPINF2, ADH1, TENM3, PON1, RDH16, PRPS2 GO:0005543 phospholipid binding 5 0.0198 4.825 Molecular Function APOA2, APOA5, MAP1B, PON1, SYTL3 GO:0044325 ion channel binding 6 0.0233 3.693 Molecular Function CAV3, CALM3, FGF13, PRNP, HOMER1, HAP1 GO:0048020 CCR chemokine receptor binding 3 0.0239 12.342 Molecular Function CCL24, CCL21, CCL9 GO:0043395 heparan sulfate proteoglycan binding 3 0.0289 11.167 Molecular Function CFH, HRG, LIPC GO:0030215 semaphorin receptor binding 3 0.0289 11.167 Molecular Function SEMA7A, SEMA3B, SH3BGRL3 GO:0005509 calcium ion binding 16 0.0330 1.797 Molecular Function MATN2, F12, APCS, CALR4, TBC1D9, ENPP2, CRP, DECR2, F9, CDH1, P4HTM, ATP2A2, PON1, ITIH4, CALM3, SYTL3 GO:0008307 structural constituent of muscle 3 0.0371 9.771 Molecular Function PDLIM3, SYNM, TPM2 GO:0003779 actin binding 8 0.0406 2.511 Molecular Function GC, DIXDC1, CAP2, ANG, DIAPH3, MAP1B, COTL1, TPM2 GO:0015020 glucuronosyltransferase activity 3 0.0460 8.685 Molecular Function UGT2B37, UGT2B17, UGT2A3 GO:0005507 copper ion binding 4 0.0463 4.963 Molecular Function P2RX4, ANG, PRNP, ATP7B GO:0005515 protein binding 29 0.0484 1.427 Molecular Function CAV3, GC, SCN1B, ALDOC, CRP, CDH1, RHOV, CKB, A1CF, TUBB5, SERPINC1, SERPINA1, PAK1, TUBA1A, HAP1, TUBB4B, SCN2B, CRYAB, MAP1B, HRK, NR4A1, HOMER1, NUMBL, P2RX4, ATP2A2, CALM3, SLC27A2, PRPS2, HTR2AFigure 2
Enriched biological process for the differentially expressed genes between vil-high group and diabetic group.


### 3.6. Pathway Enrichment Analysis of Differentially Expressed Genes

In the pathway enrichment analysis, 12 pathway terms were significantly enriched (P<0.05, Table 3). The top 10 most significant pathway terms were complement and coagulation cascades, retinol metabolism, steroid hormone biosynthesis, chemical carcinogenesis, linoleic acid metabolism, inflammatory mediator regulation of TRP channels, Gap junction, prion diseases, metabolic pathways, and arachidonic acid metabolism.Table 3
The enriched Kegg pathway with differentially expressed genes(P<0.05).
Pathway ID Pathway name Count P-value Fold Enrichment Genes rno04610 Complement and coagulation cascades 16 7.92 × 10−14 14.904 KNG1, F12, C9, C5, F9, C4BPB, C4BPA, PLG, F13B, C8A, SERPINF2, CFH, SERPINC1, SERPINA1, SERPIND1, CPB2 rno00830 Retinol metabolism 12 3.00 × 10−8 9.697 CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, ADH1, CYP2C23, CYP2C13, UGT2A3, CYP2A1, RDH16, CYP2A2 rno00140 Steroid hormone biosynthesis 10 2.63 × 10−6 8.280 CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, HSD3B5, CYP2C23, CYP2D3, CYP2C13, UGT2A3 rno05204 Chemical carcinogenesis 9 5.47 × 10−5 6.633 CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, ADH1, CYP2C23, CYP2C13, UGT2A3 rno00591 Linoleic acid metabolism 5 3.01 × 10−3 8.179 CYP2C6V1, CYP2J4, CYP3A23/3A1, CYP2C23, CYP2C13 rno04750 Inflammatory mediator regulation of TRP channels 7 6.98 × 10−3 4.082 CYP2C6V1, CYP2J4, CAMK2G, CYP2C23, CALM3, CYP2C13, HTR2A rno04540 Gap junction 6 9.57 × 10−3 4.573 TUBB2A, TUBB5, TUBB6, TUBA1A, TUBB4B, HTR2A rno05020 Prion diseases 4 1.14 × 10−2 8.384 C8A, C9, C5, PRNP rno01100 Metabolic pathways 30 1.17 × 10−2 1.556 CYP2B3, CYP2J4, PGS1, CDIPT, TUSC3, ALDOC, HSD3B5, KMO, ANPEP, ATP5G3, CKB, TDO2, ADH1, CYP2C13, UGT2A3, CYP2C6V1, MAN2A2, HAO1, UGT2B37, UGT2B17, BAAT, CYP3A23/3A1, BHMT, PON1, CYP2C23, LIPC, RDH16, CYP2A1, CYP2A2, PRPS2 rno00590 Arachidonic acid metabolism 5 3.16 × 10−2 4.140 CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2C13 rno04726 Serotonergic synapse 6 3.63 × 10−2 3.245 CYP2C6V1, CYP2J4, CYP2C23, CYP2D3, CYP2C13, HTR2A rno04360 Axon guidance 6 4.08 × 10−2 3.144 PLXNA4, SEMA7A, NTN4, SEMA3B, PAK1, UNC5C


### 3.7. Gene Interaction Network

Based on 270 differentially expressed genes, a gene interaction network was analyzed by the String software. We found that 257 nodes and 338 edges were constructed (Figure3). Twenty-five genes had more than 5 edges. The top 10 genes by degree are listed in Table 4.Table 4
The top 10 genes from gene interaction analysis.
Gene Accession Gene Symbol gene name degree NM_019369 Itih4 inter-alpha-trypsin inhibitor heavy chain family, member 4 17 NM_053491 Plg plasminogen 16 NM_001014006 F12 coagulation factor XII (Hageman factor) 12 NM_001012027 Serpinc1 serpin peptidase inhibitor, clade C (antithrombin), member 1 11 NM_138514 Cyp2c13 cytochrome P450, family 2, subfamily c, polypeptide 13 9 NM_019287 Apob apolipoprotein B 8 ENSRNOT00000074103 Cyp2c6 cytochrome P450, family 2, subfamily C, polypeptide 6, variant 1 8 NM_053318 Hpx hemopexin 8 NM_022519 Serpina1 clade A (alpha-1 antiproteinase, antitrypsin), member 1 8 NM_001108802 Speg SPEG complex locus 7Figure 3
Interaction of differentially expressed genes between vil-high group and diabetic group from String software.Angptl3,Bhmt, andPon1 are circled in red.


### 3.8. Confirmation with qPCR

From the gene expression array results, we found thatPon1 andBhmt were in “metabolic pathway” (KEGG ID: rno01100), andAngptl3 andPon1 were in several GO terms such as “positive regulation of lipid catabolic process” (GO: 0050996), “triglyceride homeostasis” (GO: 0070328), “cholesterol metabolic process” (GO: 0008203), “cholesterol homeostasis” (GO: 0042632), “response to hypoxia” (GO: 0001666), and “acylglycerol homeostasis” (GO: 0055090). So, we focused on these three genes in further study. As shown in Figure 4, the relative mRNA levels of angiopoietin-like 3(Angptl3) and betaine-homocysteine S-methyltransferase(Bhmt) in diabetic rats were significantly higher than those of normal rats (P<0.01), whereas the expression of these two genes was significantly reduced in vildagliptin-treated groups compared with those in diabetic rats (P<0.01). Conversely, paraoxonase 1(Pon1) decreased in the diabetic group. However, Pon1 increased in vildagliptin-treated rats (P<0.01). These results were consistent with the microarray results.Figure 4
Confirmation of three representative differentially expressed genes by qPCR. Values are mean ± SD(n=6).Angptl3: angiopoietin-like 3;Bhmt: betaine-homocysteine S-methyltransferase;Pon1: paraoxonase 1. P∗∗<0.01 versus control group; P##<0.01 versus diabetic group. vil-low: low dose of vildagliptin; vil-high: high dose of vildagliptin.


## 3.1. Vildagliptin Moderated Blood Glucose and Insulin Resistance

After 12 weeks of treatment, vildagliptin significantly reduced fasting blood glucose AUC in OGTT (P<0.05, Figures 1(b) and 1(f)). Additionally, fasting serum insulin and HOMA-IR index in the vildagliptin-treated group were lower than in the diabetic group (P<0.05, Figures 1(c) and 1(d)).Figure 1
Effect of vildagliptin on metabolic parameters in rats. (a) Body weight, (b) fasting blood glucose, (c) insulin, (d) homeostasis model assessment (HOMA-IR), (e) blood glucose in oral glucose tolerance test (OGTT), (f) area under the curve (AUC) in OGTT, (g) total cholesterol, (h) triglyceride, (i) high-density lipoprotein cholesterol (HDL-C), (j) low-density lipoprotein cholesterol (LDL-C), and (k) relaxation responses to Ach in aortic rings. Values are mean ± SD (n=6), P∗<0.05, P∗∗<0.01, versus control group; P#<0.05, P##<0.01 versus diabetic group. vil-low: low dose of vildagliptin; vil-high: high dose of vildagliptin.
(a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k)


## 3.2. Vildagliptin Decreased Blood Lipid Levels

Vildagliptin decreased TC and LDL-C levels in diabetic rats (P<0.01, Figures 1(g) and 1(j)).


## 3.3. Vildagliptin Depressed Vasorelaxation Responses of Aortic Rings to Ach

As shown in Figure1(k), endothelium-dependent vasodilation was impaired in diabetic rats compared with normal rats. Treatment with vildagliptin ameliorated this impairment (P<0.01).


## 3.4. Identification of Differentially Expressed Genes

After filtering genes that had a cutoff of a 1.5-fold change or greater, we found that 150 genes were upregulated and 120 genes were downregulated in the vil-high group compared with the diabetic group (P<0.05).


## 3.5. GO Term Enrichment Analysis of Differentially Expressed Genes

In general, 91 GO terms were significantly enriched (P<0.05, Table 2). The top 20 most significant biological process (BP) terms are shown in Figure 2 (P<0.01). They were negative regulation of endopeptidase activity, epoxygenase P450 pathway, acute-phase response, blood coagulation, oxidation-reduction processes, cytoskeleton organization, negative regulation of fibrinolysis, microtubule-based processes, phosphatidylcholine metabolic processes, fibrinolysis, organ regeneration, complement activation classical pathway, inflammatory response, positive regulation of lipid catabolic process, triglyceride homeostasis, aging, cellular response to interferon gamma, cholesterol metabolic process, cholesterol homeostasis, and tissue regeneration.Table 2
The enriched GO terms with differentially expressed genes(P<0.05).
Term ID Term name Count P-value Fold Enrichment catalogue Genes GO:0010951 negative regulation of endopeptidase activity 16 7.22 × 10−10 8.425 Biology Process KNG1, MUG2, MUG1, SERPINA10, PZP, C5, AHSG, AMBP, SERPINA3N, SERPINF2, SERPINA4, SERPINC1, ITIH4, HRG, ITIH2, SERPIND1 GO:0019373 epoxygenase P450 pathway 7 5.88 × 10−7 21.821 Biology Process CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2C13, CYP2A1, CYP2A2 GO:0006953 acute-phase response 7 7.97 × 10−6 14.356 Biology Process KNG1, MUG1, CRP, ITIH4, SAA4, SERPINA1, AHSG GO:0007596 blood coagulation 8 2.08 × 10−5 9.446 Biology Process F13B, F12, SERPINA10, C9, F9, SERPIND1, CPB2, PLG GO:0055114 oxidation-reduction process 23 3.29 × 10−5 2.753 Biology Process ALDH8A1, CYP2B3, CYP2J4, HSD3B5, DECR2, RGD1304810, CYP2D3, EGLN1, KMO, P4HTM, VAT1, IYD, CYP2C6V1, TDO2, CYP3A23/3A1, HIF1AN, CYP4F4, CYP2C23, CYP2C13, SH3BGRL3, RDH16, CYP2A1, CYP2A2 GO:0007010 cytoskeleton organization 10 3.95 × 10−5 6.089 Biology Process CCL24, TNIK, CAP2, TUBB2A, SVIL, CAMSAP1, STRIP2, TUBB6, TUBA1A, TUBB4B GO:0051918 negative regulation of fibrinolysis 4 6.93 × 10−5 44.533 Biology Process SERPINF2, HRG, CPB2, PLG GO:0007017 microtubule-based process 6 1.68 × 10−4 11.405 Biology Process TUBB2A, TUBB5, TUBB6, TUBA1A, DCTN1, TUBB4B GO:0046470 phosphatidylcholine metabolic process 4 6.75 × 10−4 22.267 Biology Process APOA5, PON1, GPLD1, LIPC GO:0042730 fibrinolysis 4 8.35 × 10−4 20.782 Biology Process F12, HRG, CPB2, PLG GO:0031100 organ regeneration 7 0.00110 5.995 Biology Process APOA2, BAAT, ADH1, ALDOC, APOA5, PRPS2, AHSG GO:0006958 complement activation, classical pathway 5 0.00123 10.532 Biology Process C9, C5, CRP, C4BPB, C4BPA GO:0006954 inflammatory response 12 0.00158 3.149 Biology Process CCL24, KNG1, TLR10, SERPINA3N, TNFRSF10B, MUG1, CCL21, C5, HRH4, CCL9, SERPINA1, CXCR3 GO:0050996 positive regulation of lipid catabolic process 3 0.00236 38.967 Biology Process APOA2, APOA5, ANGPTL3 GO:0070328 triglyceride homeostasis 4 0.00431 11.990 Biology Process APOC4, APOA5, LIPC, ANGPTL3 GO:0007568 aging 11 0.00745 2.721 Biology Process ADRB3, CYP3A23/3A1, CRYAB, ENDOG, ALDOC, CRP, SPINK1, IGFBP1, ATP5G3, SREBF2, HTR2A GO:0071346 cellular response to interferon-gamma 5 0.00766 6.388 Biology Process CCL24, SERPINA3N, CCL21, CCL9, CFH GO:0008203 cholesterol metabolic process 5 0.00811 6.285 Biology Process APOA2, PON1, LIPC, ANGPTL3, SREBF2 GO:0042632 cholesterol homeostasis 5 0.00906 6.089 Biology Process CAV3, APOA2, APOA5, LIPC, ANGPTL3 GO:0042246 tissue regeneration 4 0.00996 8.907 Biology Process SERPINA10, APOA5, IGFBP1, PLG GO:0046330 positive regulation of JNK cascade 5 0.0112 5.730 Biology Process DIXDC1, CCL21, SERPINF2, MAP3K10, TPD52L1 GO:0050921 positive regulation of chemotaxis 3 0.0134 16.700 Biology Process CCL21, C5, CXCR3 GO:0006956 complement activation 3 0.0153 15.587 Biology Process C8A, C5, CFH GO:2000649 regulation of sodium ion transmembrane transporter activity 3 0.0153 15.587 Biology Process CAV3, SCN1B, SCN2B GO:0042573 retinoic acid metabolic process 3 0.0173 14.613 Biology Process ALDH8A1, ADH1, RDH16 GO:0009395 phospholipid catabolic process 3 0.0217 12.989 Biology Process APOA2, ENPP2, ANGPTL3 GO:0001666 response to hypoxia 9 0.0230 2.598 Biology Process CRYAB, ANG, CAMK2G, ALDOC, CRP, SERPINA1, EGLN1, PAK1, LIPC GO:0055117 regulation of cardiac muscle contraction 3 0.0241 12.305 Biology Process CAV3, P2RX4, CALM3 GO:0055090 acylglycerol homeostasis 2 0.0254 77.933 Biology Process APOA5, ANGPTL3 GO:0034370 triglyceride-rich lipoprotein particle remodeling 2 0.0254 77.933 Biology Process APOA2, APOA5 GO:0031116 positive regulation of microtubule polymerization 3 0.0265 11.690 Biology Process CAV3, MAP1B, DCTN1 GO:0032956 regulation of actin cytoskeleton organization 4 0.0287 5.995 Biology Process DIXDC1, HRG, PAK1, SH3BGRL3 GO:0048247 lymphocyte chemotaxis 3 0.0317 10.627 Biology Process CCL24, CCL21, CCL9 GO:0070098 chemokine-mediated signaling pathway 4 0.0363 5.469 Biology Process CCL24, CCL21, CCL9, CXCR3 GO:0045959 negative regulation of complement activation, classical pathway 2 0.0378 51.956 Biology Process C4BPB, C4BPA GO:0002542 Factor XII activation 2 0.0378 51.956 Biology Process KNG1, F12 GO:0009725 response to hormone 5 0.0381 3.936 Biology Process ANG, APOA5, LIPC, ANGPTL3, SREBF2 GO:0042311 vasodilation 3 0.0402 9.352 Biology Process KNG1, P2RX4, ALB GO:0006631 fatty acid metabolic process 4 0.0413 5.196 Biology Process BAAT, DECR2, LIPC, ANGPTL3 GO:0007399 nervous system development 7 0.0428 2.741 Biology Process PLXNA4, SCN2B, CAMK2G, KREMEN1, MAP1B, DPYSL3, NUMBL GO:0048675 axon extension 3 0.0431 8.992 Biology Process SEMA7A, MAP1B, POU4F3 GO:0072659 protein localization to plasma membrane 4 0.0485 4.871 Biology Process CAV3, TNIK, FGF13, CDH1 GO:0006935 chemotaxis 4 0.0485 4.871 Biology Process CCL24, ENPP2, C5, CXCR3 GO:0006810 transport 6 0.0489 3.017 Biology Process P2RX4, DYNC1LI1, AFM, LOC360919, ALB, LOC500473 GO:0072562 blood microparticle 22 1.84 × 10−18 14.631 Cellular Components KNG1, GC, C9, MUG1, APCS, C4BPA, PLG, AHSG, C8A, AMBP, APOA2, AFM, SERPINF2, ALB, HPX, APOA5, PON1, CFH, SERPINC1, ITIH4, HRG, ITIH2 GO:0005615 extracellular space 49 2.19 × 10−11 2.922 Cellular Components MUG2, MUG1, PZP, CRP, GPLD1, SPINK1, ANPEP, AZGP1, APOA2, ANG, SEMA7A, SERPINA4, APOA5, CFH, SERPINA1, SEMA3B, KNG1, F12, APCS, LOC360919, F9, C8A, AMBP, SERPINA3N, SERPINF2, SCGB2A2, GC, SERPINA10, ENPP2, C5, CCL9, AHSG, CCL24, ALB, CCL21, SERPINC1, ANGPTL3, C4BPB, DPYSL3, C4BPA, PLG, AFM, HPX, PON1, METRNL, SERPIND1, IGFBP1, LIPC, CPB2 GO:0034364 high-density lipoprotein particle 7 4.31 × 10−8 32.313 Cellular Components APOA2, APOC4, APOA5, PON1, GPLD1, SAA4, LIPC GO:0070062 extracellular exosome 66 6.06 × 10−8 1.957 Cellular Components ALDH8A1, CYP2J4, MUG1, TUBB2A, CRP, GPLD1, ANPEP, CKB, AZGP1, APOA2, DES, PACSIN3, ANG, SERPINA4, ITIH4, TUBB5, CFH, TUBB6, ITIH2, SEMA3B, SERPINA1, TUBA1A, KNG1, F12, TNIK, APCS, CRYAB, F9, METTL7A, VAT1, AMBP, C8A, RPS16, SERPINF2, BHMT, PRNP, SLC27A2, PRPS2, GC, C9, SERPINA10, ALDOC, C5, CDH1, KMO, AHSG, ALCAM, ALB, SERPINC1, DOPEY2, HRG, TUBB4B, COTL1, PLG, P2RX4, AFM, HPX, ARF3, PON1, CALM3, PAPPA2, METRNL, SERPIND1, SH3BGRL3, MYH14, CPB2 GO:0031090 organelle membrane 11 2.10 × 10−7 9.591 Cellular Components CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, CYP4F4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0030018 Z disc 9 1.24 × 10−4 6.037 Cellular Components CAV3, JPH2, DES, CRYAB, BAG3, PDLIM3, PAK1, HOMER1, FLNC GO:0014704 intercalated disc 6 4.60 × 10−4 9.232 Cellular Components CAV3, SCN1B, DES, ATP2A2, FGF13, PAK1 GO:0030426 growth cone 9 6.13 × 10−4 4.772 Cellular Components ANG, CRP, MAP1B, CALM3, DPYSL3, FGF13, MYH14, PAK1, HAP1 GO:0005874 microtubule 11 8.97 × 10−4 3.658 Cellular Components DYNC1LI1, TUBB2A, CAMSAP1, MAP1B, TUBB5, TUBB6, FGF13, TUBA1A, DCTN1, TUBB4B, GLYATL2 GO:0043034 costamere 4 0.00196 15.695 Cellular Components SVIL, SYNM, HOMER1, FLNC GO:0005576 extracellular region 19 0.00456 2.088 Cellular Components APCS, C9, CRP, NTN4, GPLD1, SAA4, C4BPB, FGF13, PTH2, C4BPA, PLG, LOC500473, CCL24, C8A, SERPINA3N, ALB, APOA5, HRG, LIPC GO:0005789 endoplasmic reticulum membrane 16 0.00504 2.258 Cellular Components CYP2B3, CDIPT, HSD3B5, CYP2D3, SREBF2, CYP2C6V1, UGT2B37, UGT2B17, CYP3A23/3A1, ATP2A2, CYP4F4, CYP2C23, CYP2C13, CYP2A1, CYP2A2, SLC27A2 GO:0030424 axon 12 0.00657 2.609 Cellular Components GC, ALCAM, P2RX4, CRYAB, ALDOC, MAP1B, FGF13, CDH1, MYH14, PAK1, HOMER1, HTR2A GO:0034361 very-low-density lipoprotein particle 3 0.0192 13.848 Cellular Components APOA2, APOC4, APOA5 GO:0043231 intracellular membrane-bounded organelle 16 0.0302 1.825 Cellular Components KNG1, CYP2B3, CAV3, CYP2J4, HSD3B5, GPLD1, PLG, SREBF2, AMBP, UGT2B17, CYP3A23/3A1, PON1, CYP2C23, UGT2A3, CYP2A1, SLC27A2 GO:0005856 cytoskeleton 8 0.0345 2.605 Cellular Components TNIK, DES, FILIP1, MAP1B, FLNC, COTL1, TPM2, HAP1 GO:0044216 other organism cell 2 0.0376 52.316 Cellular Components C4BPB, C4BPA GO:0033017 sarcoplasmic reticulum membrane 3 0.0457 8.719 Cellular Components JPH2, ATP2A2, CAMK2G GO:0004867 serine-type endopeptidase inhibitor activity 15 6.34 × 10−11 11.167 Molecular Function MUG2, PZP, MUG1, SERPINA10, SPINK1, AMBP, SERPINA3N, SERPINF2, SERPINA4, SERPINC1, ITIH4, HRG, ITIH2, SERPINA1, SERPIND1 GO:0070330 aromatase activity 9 2.65 × 10−8 18.039 Molecular Function CYP2C6V1, CYP2B3, CYP3A23/3A1, CYP4F4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0008392 arachidonic acid epoxygenase activity 8 1.97 × 10−7 18.393 Molecular Function CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0008395 steroid hydroxylase activity 8 5.35 × 10−7 16.035 Molecular Function CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2D3, CYP2C13, CYP2A1, CYP2A2 GO:0020037 heme binding 13 1.46 × 10−6 6.122 Molecular Function CYP2B3, CYP2J4, CYP2D3, AMBP, CYP2C6V1, TDO2, CYP3A23/3A1, CYP4F4, CYP2C23, HRG, CYP2C13, CYP2A1, CYP2A2 GO:0008092 cytoskeletal protein binding 8 1.07 × 10−5 10.423 Molecular Function DES, PACSIN3, CRYAB, ALDOC, PDLIM3, CDH1, FLNC, ABCG2 GO:0005506 iron ion binding 13 1.11 × 10−5 5.031 Molecular Function CYP2B3, CYP2J4, CYP2D3, EGLN1, P4HTM, CYP2C6V1, CYP3A23/3A1, HIF1AN, CYP4F4, CYP2C23, CYP2C13, CYP2A1, CYP2A2 GO:0004866 endopeptidase inhibitor activity 5 5.40 × 10−4 13.028 Molecular Function MUG2, MUG1, C5, SERPINA1, AHSG GO:0017080 sodium channel regulator activity 5 6.95 × 10−4 12.214 Molecular Function CAV3, SCN1B, SCN2B, GPLD1, FGF13 GO:0016712 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen 5 8.78 × 10−4 11.495 Molecular Function CYP2B3, CYP2J4, CYP3A23/3A1, CYP2D3, CYP2A1 GO:0005200 structural constituent of cytoskeleton 6 0.00223 6.514 Molecular Function TUBB2A, TUBB5, TUBB6, SYNM, TUBA1A, TUBB4B GO:0016706 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors 4 0.00262 14.213 Molecular Function HIF1AN, RGD1304810, EGLN1, P4HTM GO:0008201 heparin binding 8 0.00268 4.283 Molecular Function SERPINA10, ANG, APOA5, SERPINC1, CFH, HRG, SERPIND1, LIPC GO:0016705 oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 5 0.00297 8.316 Molecular Function CYP2C6V1, CYP2B3, CYP2C23, EGLN1, CYP2C13 GO:0005102 receptor binding 12 0.00666 2.598 Molecular Function KNG1, HAO1, P2RX4, BAAT, ANG, LRRC4B, DECR2, HRG, HOMER1, SLC27A2, HAP1, PLG GO:0004497 monooxygenase activity 5 0.00944 6.013 Molecular Function CYP2C6V1, CYP2B3, CYP2C23, CYP2D3, CYP2C13 GO:0004622 lysophospholipase activity 3 0.01328 16.751 Molecular Function ENPP2, LIPC, GDPD1 GO:0042803 protein homodimerization activity 19 0.01474 1.845 Molecular Function RBPMS2, CRYAB, GIMAP7, CAMK2G, CRP, NR4A1, TPD52L1, ABCG2, AMBP, ADRB3, APOA2, HIF1AN, ANG, SERPINF2, ADH1, TENM3, PON1, RDH16, PRPS2 GO:0005543 phospholipid binding 5 0.0198 4.825 Molecular Function APOA2, APOA5, MAP1B, PON1, SYTL3 GO:0044325 ion channel binding 6 0.0233 3.693 Molecular Function CAV3, CALM3, FGF13, PRNP, HOMER1, HAP1 GO:0048020 CCR chemokine receptor binding 3 0.0239 12.342 Molecular Function CCL24, CCL21, CCL9 GO:0043395 heparan sulfate proteoglycan binding 3 0.0289 11.167 Molecular Function CFH, HRG, LIPC GO:0030215 semaphorin receptor binding 3 0.0289 11.167 Molecular Function SEMA7A, SEMA3B, SH3BGRL3 GO:0005509 calcium ion binding 16 0.0330 1.797 Molecular Function MATN2, F12, APCS, CALR4, TBC1D9, ENPP2, CRP, DECR2, F9, CDH1, P4HTM, ATP2A2, PON1, ITIH4, CALM3, SYTL3 GO:0008307 structural constituent of muscle 3 0.0371 9.771 Molecular Function PDLIM3, SYNM, TPM2 GO:0003779 actin binding 8 0.0406 2.511 Molecular Function GC, DIXDC1, CAP2, ANG, DIAPH3, MAP1B, COTL1, TPM2 GO:0015020 glucuronosyltransferase activity 3 0.0460 8.685 Molecular Function UGT2B37, UGT2B17, UGT2A3 GO:0005507 copper ion binding 4 0.0463 4.963 Molecular Function P2RX4, ANG, PRNP, ATP7B GO:0005515 protein binding 29 0.0484 1.427 Molecular Function CAV3, GC, SCN1B, ALDOC, CRP, CDH1, RHOV, CKB, A1CF, TUBB5, SERPINC1, SERPINA1, PAK1, TUBA1A, HAP1, TUBB4B, SCN2B, CRYAB, MAP1B, HRK, NR4A1, HOMER1, NUMBL, P2RX4, ATP2A2, CALM3, SLC27A2, PRPS2, HTR2AFigure 2
Enriched biological process for the differentially expressed genes between vil-high group and diabetic group.


## 3.6. Pathway Enrichment Analysis of Differentially Expressed Genes

In the pathway enrichment analysis, 12 pathway terms were significantly enriched (P<0.05, Table 3). The top 10 most significant pathway terms were complement and coagulation cascades, retinol metabolism, steroid hormone biosynthesis, chemical carcinogenesis, linoleic acid metabolism, inflammatory mediator regulation of TRP channels, Gap junction, prion diseases, metabolic pathways, and arachidonic acid metabolism.Table 3
The enriched Kegg pathway with differentially expressed genes(P<0.05).
Pathway ID Pathway name Count P-value Fold Enrichment Genes rno04610 Complement and coagulation cascades 16 7.92 × 10−14 14.904 KNG1, F12, C9, C5, F9, C4BPB, C4BPA, PLG, F13B, C8A, SERPINF2, CFH, SERPINC1, SERPINA1, SERPIND1, CPB2 rno00830 Retinol metabolism 12 3.00 × 10−8 9.697 CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, ADH1, CYP2C23, CYP2C13, UGT2A3, CYP2A1, RDH16, CYP2A2 rno00140 Steroid hormone biosynthesis 10 2.63 × 10−6 8.280 CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, HSD3B5, CYP2C23, CYP2D3, CYP2C13, UGT2A3 rno05204 Chemical carcinogenesis 9 5.47 × 10−5 6.633 CYP2C6V1, CYP2B3, UGT2B37, UGT2B17, CYP3A23/3A1, ADH1, CYP2C23, CYP2C13, UGT2A3 rno00591 Linoleic acid metabolism 5 3.01 × 10−3 8.179 CYP2C6V1, CYP2J4, CYP3A23/3A1, CYP2C23, CYP2C13 rno04750 Inflammatory mediator regulation of TRP channels 7 6.98 × 10−3 4.082 CYP2C6V1, CYP2J4, CAMK2G, CYP2C23, CALM3, CYP2C13, HTR2A rno04540 Gap junction 6 9.57 × 10−3 4.573 TUBB2A, TUBB5, TUBB6, TUBA1A, TUBB4B, HTR2A rno05020 Prion diseases 4 1.14 × 10−2 8.384 C8A, C9, C5, PRNP rno01100 Metabolic pathways 30 1.17 × 10−2 1.556 CYP2B3, CYP2J4, PGS1, CDIPT, TUSC3, ALDOC, HSD3B5, KMO, ANPEP, ATP5G3, CKB, TDO2, ADH1, CYP2C13, UGT2A3, CYP2C6V1, MAN2A2, HAO1, UGT2B37, UGT2B17, BAAT, CYP3A23/3A1, BHMT, PON1, CYP2C23, LIPC, RDH16, CYP2A1, CYP2A2, PRPS2 rno00590 Arachidonic acid metabolism 5 3.16 × 10−2 4.140 CYP2C6V1, CYP2B3, CYP2J4, CYP2C23, CYP2C13 rno04726 Serotonergic synapse 6 3.63 × 10−2 3.245 CYP2C6V1, CYP2J4, CYP2C23, CYP2D3, CYP2C13, HTR2A rno04360 Axon guidance 6 4.08 × 10−2 3.144 PLXNA4, SEMA7A, NTN4, SEMA3B, PAK1, UNC5C


## 3.7. Gene Interaction Network

Based on 270 differentially expressed genes, a gene interaction network was analyzed by the String software. We found that 257 nodes and 338 edges were constructed (Figure3). Twenty-five genes had more than 5 edges. The top 10 genes by degree are listed in Table 4.Table 4
The top 10 genes from gene interaction analysis.
Gene Accession Gene Symbol gene name degree NM_019369 Itih4 inter-alpha-trypsin inhibitor heavy chain family, member 4 17 NM_053491 Plg plasminogen 16 NM_001014006 F12 coagulation factor XII (Hageman factor) 12 NM_001012027 Serpinc1 serpin peptidase inhibitor, clade C (antithrombin), member 1 11 NM_138514 Cyp2c13 cytochrome P450, family 2, subfamily c, polypeptide 13 9 NM_019287 Apob apolipoprotein B 8 ENSRNOT00000074103 Cyp2c6 cytochrome P450, family 2, subfamily C, polypeptide 6, variant 1 8 NM_053318 Hpx hemopexin 8 NM_022519 Serpina1 clade A (alpha-1 antiproteinase, antitrypsin), member 1 8 NM_001108802 Speg SPEG complex locus 7Figure 3
Interaction of differentially expressed genes between vil-high group and diabetic group from String software.Angptl3,Bhmt, andPon1 are circled in red.


## 3.8. Confirmation with qPCR

From the gene expression array results, we found thatPon1 andBhmt were in “metabolic pathway” (KEGG ID: rno01100), andAngptl3 andPon1 were in several GO terms such as “positive regulation of lipid catabolic process” (GO: 0050996), “triglyceride homeostasis” (GO: 0070328), “cholesterol metabolic process” (GO: 0008203), “cholesterol homeostasis” (GO: 0042632), “response to hypoxia” (GO: 0001666), and “acylglycerol homeostasis” (GO: 0055090). So, we focused on these three genes in further study. As shown in Figure 4, the relative mRNA levels of angiopoietin-like 3(Angptl3) and betaine-homocysteine S-methyltransferase(Bhmt) in diabetic rats were significantly higher than those of normal rats (P<0.01), whereas the expression of these two genes was significantly reduced in vildagliptin-treated groups compared with those in diabetic rats (P<0.01). Conversely, paraoxonase 1(Pon1) decreased in the diabetic group. However, Pon1 increased in vildagliptin-treated rats (P<0.01). These results were consistent with the microarray results.Figure 4
Confirmation of three representative differentially expressed genes by qPCR. Values are mean ± SD(n=6).Angptl3: angiopoietin-like 3;Bhmt: betaine-homocysteine S-methyltransferase;Pon1: paraoxonase 1. P∗∗<0.01 versus control group; P##<0.01 versus diabetic group. vil-low: low dose of vildagliptin; vil-high: high dose of vildagliptin.


## 4. Discussion

In our study, we found that vildagliptin reduced blood glucose, TC, and LDL-C. In a clinical trial, sitagliptin-combined metformin add-on therapy led to greater improvement in HbA1c than the metformin monotherapy group after 18 weeks of treatment. Moreover, sitagliptin combined with metformin significantly reduced TG, TC, and LDL-C and increased HDL-C compared with the metformin group [13].We used aortic rings to test the Ach-induced endothelium-dependent vasodilation. In diabetic rats, Ach-induced endothelium-dependent vasodilation was impaired. Vildagliptin augmented endothelial function. Other DDP-4 inhibitors also had similar vasoprotective effects. Saxagliptin treatment for 8 weeks increased aortic nitric oxide (NO) release by 22% and reduced mean arterial pressure in spontaneously hypertensive rats [14]. Moreover, sitagliptin treatment for 2 weeks protected endothelial function and reduced systolic blood pressure in spontaneously hypertensive rats through GLP-1 signaling [15].In our research, the expression ofAngptl3 was downregulated in vildagliptin-treated rats.Angptl3 is a key regulator, which can inhibit lipoprotein lipase (LPL) activity [16, 17]. The enzyme LPL hydrolyzes TG to free fatty acids (FFA). ANGPTL3 overexpression mice had high plasma TG levels [18]. Patients with a loss-of-function mutation ofAngptl3 are characterized by low plasma TC, TG, HDL-C, and LDL-C [19, 20]. In 2008, Kathiresan et al. first identified an SNP site near ANGPTL3 that was associated with plasma TG levels in a genome-wide association study [21]. In obesity and T2D subjects, the ANGPTL3 level was higher than that in healthy subjects [22]. Moreover, the level of ANGPTL3 was increased in the livers of insulin-deficient and insulin-resistant diabetic mice [23]. In mice and monkeys, using ANGPTL3-specific antibodies led to reduced plasma TG [24–26]. The therapeutic targets of the inhibition activity of ANGPTL require further research to treat dyslipidemia [16, 17].We also found that the expression ofBhmt was reduced in the vildagliptin-treated group. Methionine (Met) was produced from methylate homocysteine (Hcy) by BHMT enzymes with betaine. Thus, BHMT can reduce Hcy levels and increase Met levels. Met can then be converted to S-adenosylmethionine (SAM).In vivo, SAM is a main methyl donor to regulate methionine metabolism in many reactions. An increase in BHMT activity and SAM levels was observed in the livers of both type 1 diabetic rats [27] and the type 2 diabetic model [28, 29]. Insulin treatment in the rat hepatoma cell line and STZ-induced diabetic rats can inhibit the excess expression of BHMT and SAM [28, 30].Moreover, our results showed that Pon1 was upregulated in the vildagliptin-treated group. Pon1 is an enzyme that has antioxidant functions. In serum, Pon1 is located in HDL. Pon1 protects LDL from oxidation and hydrolyzed oxidized LDL [31–34]. HDL levels are negatively associated with the risk of developing coronary artery disease (CAD), but high levels of oxidized LDL (oxLDL) in the aorta lead to cholesterol accumulation, foam cell formation, and atherosclerotic lesions [31, 35]. Low Pon1 expression accelerates aortic lesion development in mice [36]. In humans, serum PON level is reduced in patients with a history of myocardial infarction [32] and diabetic patients [37]. PON1 transgenic mice had decreased oxidative stress and atherosclerotic lesions [38, 39], whereas PON1 knockout mice had increased serum oxidative stress and were more susceptible to high-fat-diet-induced atherosclerosis [34, 40].


## 5. Conclusion

In conclusion, this study has confirmed that vildagliptin significantly attenuates endothelial dysfunction in diabetic rats. Importantly, our study indicates that vildagliptin may activatePon1 expression and inhibit the expression ofBhmt andAngptl3 in the aorta. This study may increase the understanding of the pathways that contribute to which DPP-4 inhibitor attenuates endothelial dysfunction. More studies are needed to investigate whether or not vildagliptin treatment directly altersAgptl3,Bhmt, andPon1 expression in the aorta of diabetic rats, independent of blood glucose (Figure 5).Figure 5
The possible mechanism of attenuation of vildagliptin on endothelial dysfunction. Vildagliptin attenuates endothelial dysfunction in diabetic rats through activatingPon1 expression in aorta to inhibit oxLDL production; inhibiting Bhmt expression to reduce Met and SAM production; inhibitingAngptl3 expression to activate LPL activity and thereby reduce plasma TG level. LDL: low-density lipoprotein; Pon1: paraoxonase 1; oxLDL: oxidized low-density lipoprotein; Hcy: homocysteine; Bhmt: betaine-homocysteine S-methyltransferase; Met: methionine, SAM: S-adenosylmethionine; TG: triglyceride.



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*Source: 3109251-2018-05-23.xml*