Teneligliptin enhances the beneficial effects of GLP-1 in endothelial cells exposed to hyperglycemic conditions

High-glucose-induced oxidative stress contributes to cardiovascular endothelial damage in diabetes. Glucagon-like peptide 1 (GLP-1) is beneficial to endothelial cells, but its effects are diminished when cells are continuously exposed to high glucose. Teneligliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that prevents oxidative stress, apoptosis and the metabolic memory effect. We explored the potential additive effects of Teneligliptin and GLP-1 in hyperglycemia-damaged endothelial cells. Human umbilical vein endothelial cells (HUVECs) were exposed to normal-glucose (5 mmol/L) or high-glucose (HG, 25 mmol/L) for 21 days, or to HG for 14 days followed by normal-glucose for 7 days (HM). These cells were continually treated with Teneligliptin 3.0 μmol/L, alone or in combination with an acute dose of GLP-1 50 nmol/L. DPP-4 was upregulated under hyperglycemic conditions, but Teneligliptin reduced DPP-4 expression and activity. Simultaneous Teneligliptin and GLP-1 synergistically increased the antioxidant response and reduced ROS levels in HG- and HM-exposed HUVECs. Concurrent treatment also enhanced cell proliferation, reduced apoptotic gene expression and ameliorated endoplasmic reticulum stress in HG- and HM-exposed HUVECs. Thus, long-term Teneligliptin treatment reduced DPP-4 levels and activity in HUVECs exposed to chronic hyperglycemia. Moreover, Teneligliptin enhanced the beneficial effects of GLP-1 on oxidative stress, proliferation, apoptosis and endoplasmic reticulum homeostasis.


INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a major chronic illness causing a series of pathological complications that reduce the quality of life. Cardiovascular disease (CVD) is the most important cause of death in diabetic patients [1]. Hyperglycemia, the major factor implicated in CVD in diabetic patients [2], damages the endothelium mainly by overstimulating the production of reactive oxygen species (ROS) [3], thus increasing oxidative stress and fostering endothelial dysfunction (ED). Lowering glucose levels is not sufficient to switch off the self-perpetuating intracellular pro-oxidant process [4], which is the basis of diabetic cardiovascular complications [5]. This pathogenic mechanism can be partly explained by the "metabolic memory," defined as the perpetuation of vascular damage despite the achievement of improved glycemic control [4].
The inhibition of dipeptidyl peptidase-4 (DPP-4) activity and/or the use of degradation-resistant GLP-1 analogues has been reported to improve endothelial function in clinical settings [14]. DPP-4, also known as CD26, is a 110-kDa type-II transmembrane glycoprotein that can cleave a variety of substrates, including the incretin hormones GLP-1 and gastric inhibitory polypeptide (GIP) [6], from the amino-terminus of a polypeptide with proline or alanine in the second position [15] ( Figure 1A). DPP-4 is present on the surface of different cells types [6], but can also be found in the circulation in a soluble form (sDPP-4) [16] upon its cleavage from the cell membrane, a process called "shedding" [17].
DPP-4 inhibitor (DPP-4i) drugs preclude the catalytic activity of DPP-4, which otherwise rapidly inactivates the intestinal hormone GLP-1 [6]. Among DPP-4i drugs, Teneligliptin has recently been commercialized. The efficacy and safety of long-term Teneligliptin monotherapy and combination therapy have been evaluated in T2DM patients [18,19]. In a previous paper, we demonstrated that Teneligliptin has an intrinsic antioxidant capacity in human umbilical vein endothelial cells (HUVECs) exposed to high-glucose (HG) conditions, as it reduces ROS levels and initiates the transcriptional cascade of antioxidant genes [20]. This DPP-4i also enhances proliferation, reduces apoptosis and improves ER homeostasis under the same stress conditions [20].
In the present study, we explored the possible additive effects of GLP-1 and Teneligliptin on the endothelial response, by examining the antioxidant response, proliferation rate and ER stress in HUVECs exposed to HG conditions.

Markers of oxidative stress and hyperglycemiainduced damage are reduced by simultaneous Teneligliptin and GLP-1 treatment
HUVECs were exposed to different glucose conditions: continuous normal glucose (NG: 5 mmol/L) for 21 days, continuous HG (HG: 25 mmol/L) for 21 days, or metabolic memory (HM: continuous HG for 14 days, followed by NG for 7 days). Teneligliptin at 3.0 μmol/L was administered every 48 hours during this time, and GLP-1 was added at 50 nmol/L, alone or in combination with Teneligliptin, 1 hour before cell harvesting ( Figure 1B).
Under HG and HM conditions, the levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG, a marker of oxidative damage) were elevated. These increases were significantly ameliorated when cells were treated with Teneligliptin, but not with GLP-1. The decrease in 8-OH-dG levels was more evident after combined treatment with GLP-1 and DPP-4i, especially in the HM state, although it did not differ significantly from the reduction achieved with Teneligliptin or GLP-1 alone ( Figure 1C).
Among the mechanisms proposed to link hyperglycemia with CVD in diabetes, the activation of the protein kinase C (PKC) pathway is particularly important [5,21,22]. Exposure to HG and HM increased PKCβ mRNA levels ( Figure 1D) and PKCβ enzyme activity ( Figure 1E). Teneligliptin treatment, but not GLP-1 treatment, reversed these changes. The combination of the DPP-4i + GLP-1 did not further reduce PKCβ mRNA levels, but downregulated PKCβ activity further than Teneligliptin alone ( Figure 1D and 1E).
The mRNA levels of thioredoxin interacting protein (TXNIP), a biomarker of glucose-induced damage [23], were elevated in the HG and HM groups. GLP-1 and Teneligliptin alone did not counteract these increases; only their combination significantly dampened the increases in TXNIP ( Figure 1D).

The combination of Teneligliptin and GLP-1 improves the antioxidant response in HUVECs under HG and HM conditions
ROS levels were significantly greater in HG-and HM-exposed cells than in NG-exposed cells (Figure 2A). GLP-1 slightly reduced ROS production in the HG state ( * p=0.07) but had no apparent effect in the HM state, whereas Teneligliptin reduced ROS levels under both HG and HM conditions. A greater reduction in ROS production was evident after administration of the DPP-4i + GLP-1 (Figure 2A), suggesting that GLP-1 can exert antioxidant effects when it is not rapidly degraded.
To gain insight into the molecular mechanisms underlying this antioxidant effect, we examined the gene expression of the pro-oxidant subunits NOX4 and P22 -phox of NAD(P)H oxidase (NOX) [24], one of the molecules involved in ROS production [23]. The mRNA levels of NOX4 and P22 -phox increased similarly under HG and HM conditions ( Figure 2B). Although GLP-1 treatment was expected to reduce the levels of these transcripts under HG and HM conditions, as it did for NOX4 in the NG state ( * p=0.08), GLP-1 did not produce these positive effects. On the contrary, Teneligliptin reduced the mRNA levels of P22 -phox but not NOX4 under both HG and HM conditions. Of note, only the combination of the DPP-4i + GLP-1 reduced the expression of NOX4 under both hyperglycemic conditions, and further reduced the expression of P22 -phox ( Figure 2B).
As nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling is a major response to ROS overproduction, we analyzed the expression of three NRF2 target genes: HMOX, NQO1 and thioredoxin reductase (TXNRD) [25]. In the NG state, Teneligliptin and GLP-1 upregulated the gene expression of HMOX and NQO1,  and their combination further increased the expression of HMOX; however, TXNRD mRNA levels increased only after simultaneous treatment with the DPP-4i + GLP-1 ( Figure 2C). HG and HM treatment increased HMOX and NQO-1 mRNA levels, indicating a possible initiation of the antioxidant cascade. These increases were observed for TXNRD only in the HG state. Neither Teneligliptin nor GLP-1 alone exerted positive effects on the expression of these genes in the HG state, whereas Teneligliptin increased HMOX, NQO-1 and TXNRD levels in the HM state. Interestingly, DPP-4i + GLP-1 increased these three antioxidant transcripts under both HG and HM conditions ( Figure 2C), and the combination was more efficient than Teneligliptin alone for NQO-1 and TXNRD in the HG state.
Among the endogenous defense systems used by cells to reduce ROS levels, superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidases (GPXs) are important antioxidant enzymes that directly scavenge ROS, converting them into less reactive species [26]. In the NG state, Teneligliptin, but not GLP-1, increased SOD1 mRNA levels, and the combination of the DPP-4i + GLP-1 upregulated SOD2 and CAT ( Figure 2D). HG and HM treatment increased the mRNA levels of SOD1, SOD2 and GPX, but did not affect CAT levels ( Figure  2D). Teneligliptin, but not GLP-1, increased the gene expression of SOD2 under both HG and HM conditions, and of GPX in the HG state. Remarkably, only the combination of the DPP-4i + GLP-1 upregulated all the studied scavengers under both hyperglycemic conditions ( Figure 2D).

The combination of Teneligliptin and GLP-1 improves the proliferative capacity and ER homeostasis of HUVEC cells under HG and HM conditions
GLP-1 has well-known proliferative properties in the NG state [27], but not under HG conditions [22]. HG and HM treatment of HUVECs increased the gene expression of P53, the classical regulator of proliferation pathways. Teneligliptin, but not GLP-1, reduced P53 mRNA levels, and the combination of the DPP-4i + GLP-1 accentuated this decrease ( Figure 3A). P21 and P27 mRNA levels increased under HG and HM conditions. Once again, GLP-1 did not counteract these increases, while Teneligliptin, alone or in combination with GLP-1, reduced P21 mRNA levels under HG and HM conditions. Moreover, only simultaneous administration of the DPP-4i + GLP-1 reduced P27 mRNA levels in the HM state ( Figure 3A).
To confirm these results, we performed a 5-bromodeoxyuridine (BrdU) assay. Teneligliptin treatment, alone or in combination with GLP-1, significantly increased the proliferation rates of cells maintained under HG and HM conditions, while GLP-1 alone did not ( Figure 3B).
We next assessed the apoptotic pathway. HG and HM exposure downregulated the anti-apoptotic gene BCL2, and upregulated the pro-apoptotic genes BAX and CASP3. GLP-1 administration increased BCL2 expression in the NG state, but not under HG and HM conditions. Teneligliptin, alone or in combination with the incretin hormone, increased BCL2 levels and reduced BAX and CASP3 levels under hyperglycemic conditions ( Figure  3A). These results were confirmed with a caspase 3 activity assay ( Figure 3C). We also observed that, in the HG state, simultaneous administration of the DPP-4i + GLP-1 had a more pronounced effect on caspase 3 activity than Teneligliptin or GLP-1 treatment alone ( Figure 3C).
For their survival, cells must respond to ER perturbations, which are also involved in the pathogenesis of diabetes [23]. We measured the gene expression of several markers of the unfolded protein response, and observed that: (1) binding immunoglobulin protein (BIP), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 and 6 (ATF4-ATF6), CCAAT/-enhancer-binding protein homologous protein (CHOP), and inositol-requiring enzyme 1 alpha (IRE1a) levels increased after HG and HM exposure; (2) Teneligliptin, but not GLP-1, reduced the levels of all the analyzed markers; and (3) the combination of the DPP-4i + GLP-1 further reduced the levels of BIP, PERK, CHOP and ATF6 in the HG state ( Figure 3D).

Chronic Teneligliptin treatment reduces DPP-4 levels in the medium and DPP-4 gene/protein levels in HUVECs exposed to HG and HM conditions
Previous studies have demonstrated that plasma DPP-4 activity is elevated in patients with T2DM [28]. This could impair the incretin effect by accelerating the inactivation of GLP-1.
Analysis of DPP-4 activity in HUVECs revealed an increase under HG and HM conditions and a reduction after treatment with Teneligliptin, alone or in combination with GLP-1 ( Figure 4A). Moreover, Teneligliptin (alone or in combination with GLP-1) had no effect on DPP-4 mRNA levels in the NG state, but reduced them under HG and HM conditions ( Figure 4B). These results were confirmed by Western blot analysis: HG and HM exposure upregulated DPP-4 protein levels, and Teneligliptin alone strongly attenuated these increases ( Figure 4C).
Seeking insight into the effects of Teneligliptin on DPP-4, we quantified DPP-4 levels in the culture medium. Surprisingly, we observed that both HG and HM conditions increased the DPP-4 content of the medium. While GLP-1 did not reverse these increases, Teneligliptin, alone or in combination with the incretin hormone, significantly reduced the DPP-4 content of the culture medium ( Figure 4D).
To determine whether Teneligliptin prevents the release of soluble DPP-4, we analyzed the gene expression of matrix metalloproteinases (MMPs) 1, 2 and 14, as these enzymes are involved in the DPP-4 shedding mechanism in smooth muscle cells [29], and the latter two have been found to be elevated in obese mice [30]. In HUVECs, the mRNA levels of all three MMPs increased under HG and HM conditions. GLP-1 did not exert positive effects on these genes, and surprisingly increased their expression in the NG state. On the other hand, Teneligliptin, alone or in combination with GLP-1, attenuated the increases of all three MMPs in the HG state, and of MMP1 and MMP2 in the HM state ( Figure 4E).
The upstream regulators of MMPs include tumor necrosis factor α (TNFα), which upregulates MMP activity, and transforming growth factor β (TGFβ), which reduces MMP activity [31]. We analyzed the mRNA transcripts of these genes, and found that HG and HM conditions increased TNFα and TGFβ levels ( Figure  4F). Teneligliptin, alone or in combination with GLP-1, reduced TNFα but not TGFβ mRNA levels, while GLP-1 did not alter the expression of either gene ( Figure 4F).

DISCUSSION
The main findings of the present study are: (i) Long-term treatment with Teneligliptin enhances the beneficial effects of GLP-1 in HUVECs exposed to hyperglycemic conditions, by reducing oxidative stress and improving the antioxidant response, proliferation and ER homeostasis.
(ii) HG and HM conditions cause the enduring upregulation of DPP-4 expression, and Teneligliptin dampens this harmful alteration.
An increase in the 8-OH-dG level is a reliable marker of DNA oxidative damage that persists after glucose normalization [32]. Teneligliptin alone reduced 8-OH-dG formation, and synergistically reduced it in the presence of GLP-1.
We confirmed the direct action of Teneligliptin in restoring ROS levels, increased by HG and HM states in HUVECs. Of note, the combination of the DPP-4i + GLP-1 was more potent than Teneligliptin alone in counteracting ROS production, demonstrating that simultaneous administration of the two drugs powerfully improves the redox state of the endothelium.
The activation of NRF2 is one of the most important cellular mechanisms regulating the expression of phase-II detoxifying enzymes. Oxidative stress stimuli, induced by HG and HM conditions, cause NRF2 accumulation in the nucleus, where it can upregulate the expression of its targets [33]. In our in vitro model, GLP-1 alone did not initiate the NRF2-induced antioxidant transcriptional cascade; only simultaneous administration of the DPP-4i + GLP-1 increased the expression of the NRF2 target genes HMOX, NQO-1 and TXNRD, and reduced the mRNA levels of TXNIP, which is recognized as a connection point in many molecular abnormalities induced by hyperglycemia [34].
ER stress is a major pathological feature induced by hyperglycemia [34], and was not reversed by GLP-1 treatment. Teneligliptin was able to dampen the expression of ER stress markers, and had more pronounced effects when co-administered with GLP-1, indicating that this treatment has both intrinsic effects and synergistic effects with the incretin hormone.
A common cellular outcome linked to hyperglycemia-induced oxidative and ER stress is apoptosis [34]. HG and HM conditions lowered the rate of cellular proliferation and increased apoptosis. The combination of GLP-1 and Teneligliptin restored the proproliferative and anti-apoptotic capacities of GLP-1, and even exceeded what the DPP-4i accomplished alone.
The mechanisms regulating the transcription and enzymatic activity of DPP-4 are of interest, but are not yet fully understood. The 5′-flanking region of the DPP-4 coding sequence has been found to contain DNA elements for gene expression [35]. The factors regulating the expression and tissue distribution of DPP-4 have been studied in several types of cancer, and it has been observed that hypoxia increases the surface levels of DPP-4 [36]. Metformin has also been identified as a previously unrecognized DPP-4i [37], although the mechanisms are not entirely understood and remain controversial.
Here, we have shown for the first time that prolonged treatment with Teneligliptin downregulates DPP-4 activity on two different levels ( Figure 5). On the one hand, Teneligliptin can reduce DPP-4 mRNA and protein levels under HG and HM conditions. In NG-exposed HUVECs, Teneligliptin reduced DPP-4 expression only at the protein level. This can be explained by the fact that Teneligliptin will only downregulate DPP-4 gene expression in response to an increased amount of DPP-4 protein, which is present only under HG and HM conditions. Interestingly, a recent study on human dermal microvascular endothelial cells (HMVECs) revealed a similar effect on DPP-4 of another DPP-4i, Linagliptin [38]. The study showed that both Sitagliptin and Linagliptin suppressed DPP-4 levels in a cell-free system; however, in TGFβ-treated endothelial cells, only Linagliptin (but not Sitagliptin) suppressed DPP-4 protein levels, demonstrating the differential and intrinsic drugspecific effects of DPP4i drugs [38]. Similarly, Takai et al. reported that Sitagliptin and Linagliptin had similar effects on blood glucose and plasma insulin levels in Zucker diabetic fatty rats; however, DPP-4 activity was significantly lower in the Linagliptin-treated rats than in the Sitagliptin-treated rats, in plasma as well as in vascular tissues [39]. This finding corroborates the hypothesis that, although all DPP-4i drugs have the same function, they may have unique drug-specific effects [38]. On the other hand, Teneligliptin indirectly inhibits the shedding of DPP-4 by downregulating MMP1, MMP2 and MMP14 gene expression via TNFα under hyperglycemic conditions: a decrease in these metalloproteinases reduces the release of the soluble form of DPP-4 by HUVECs. Consequently, treatment with this specific DPP-4i, by lowering the amount of soluble DDP-4, increases the halflife of GLP-1, which can then exert its positive actions on the vasculature ( Figure 5).
Although additional studies are needed to clarify the mechanisms of DPP-4 shedding in the context of diabetes, our results indicate that this DPP-4i could exert positive actions beyond the direct inhibition of the DPP-4 enzyme. This could be of interest in clinical practice, since different molecules of the same class have somewhat different effects on the development of cardiovascular complications [40]. For example, several clinical studies (SAVOR-TIMI53 for Saxaglipitin, EXAMINE for Alogliptin and TECOS for Sitagliptin) demonstrated no cardiovascular benefit of their respective DPP-4i drugs, although cardiovascular safety was not compromised [41][42][43]. In contrast, the newly published results of the SPEAD-A study, related to the effects of Alogliptin on T2DM patients without CVD history, revealed that Alogliptin reduced the progression of the carotid intimamedia thickness in patients [44]. It is important to note that, beyond their differences in clinical settings, certain DPP-4i drugs could have intrinsic properties not strictly linked to their class effects [45].
In conclusion, we have demonstrated that Teneligliptin enhances the beneficial effects of GLP-1 on the antioxidant response, ER function and cellular proliferation. Interestingly, Teneligliptin can reduce the DPP-4 content and activity of HUVECs, thus downregulating DPP-4 expression in a manner reported for only one other DPP-4i, Linagliptin [38].

Cell culture and experimental design
HUVECs were purchased from Lonza and cultured with an EGM™-2 Bulletkit™ (Lonza Ibérica S.A.U., Barcelona, Spain), along with the following supplemental growth factors: Epidermal Growth Factor, Hydrocortisone, human recombinant Fibroblast Growth Factor β, Heparin, 2% Fetal Bovine Serum, and Gentamicin/Amphotericin-B. Cells were cultured in supplemented medium at 37°C in a humidified atmosphere with 5% CO 2 . Cells were used between four and six passages. We seeded cells at an initial concentration of 8×10 4 cells/well in six-well plates, in order to obtain confluent plates at the end of the experiment and to prevent contact inhibition of cell growth. Twenty-four hours after being seeded, the cells were exposed to three different experimental glucose conditions: continuous normal glucose (NG -5 mmol/L) for 21 days; continuous high glucose (HG, 25 mmol/L) for 21 days; and metabolic memory (HM -continuous HG for 14 days, followed by NG for 7 days) [20,46,47]. Glucose monohydrate was purchased from Sigma-Aldrich (Química, S.L., Madrid, Spain) and was dissolved directly in NG culture medium to produce the HG medium.

ROS measurement
The fluorescent probe 2',7'-Dichlorofluorescein diacetate (H 2 DCFDA) (Sigma-Aldrich Química, S.L., Madrid, Spain), was used to measure the intracellular production of ROS. Approximately 5×10 3 HUVECs were grown on clear, flat-bottomed, treated 96-well plates for 21 days under NG, HG or HM conditions. At the end of the experiment, the cells were treated with the indicated drugs, and the reactions were stopped by the staining of cells with 20 mM H 2 DCFDA for 30 minutes at 37°C. The intensity of H 2 DCFDA was kinetically measured on a fluorescent microplate reader (Synergy HT, BioTek Instruments, Inc., Winooski, Vermont, USA) in accordance with the manufacturer's recommendations.

Oxidative stress marker
The 8-OHdG content of HUVECs was determined with a Bioxytech 8-OHdG-EIA Kit (OXIS Health Products, Portland, OR, USA) in accordance with the manufacturer's recommendations. The assay was repeated three times, and each sample was run in triplicate.

PKC kinase activity
PKCβ kinase activity was measured with a PKC Kinase Activity Assay Kit (Abcam, Cambridge, UK) according to the manufacturer's instructions. HUVECs were cultured in NG, HG or HM conditions for 21 days, with or without Teneligliptin and/or GLP-1, and the medium was changed every 48 hours. At the end of incubation, the cells were lysed, the protein content was measured in Bradford assay buffer (Sigma-Aldrich Química, S.L., Madrid, Spain), and 30 μg of lysate was used to determine PKC-specific kinase activity. The assay was performed in triplicate, and the results are shown as arbitrary units (a.u.).

RNA isolation and qRT-PCR
Total RNA was isolated from HUVECs with a Total RNA Isolation Kit (Norgen Biotek Corp, Thorold, Ontario, Canada) in accordance with the manufacturer's instructions. First-strand cDNA was prepared from 1-2 μg of total RNA, the Superscript III RT Kit and random hexamer primers (Invitrogen, Carlsbad, CA, USA) in a total volume of 25 μL, according to the manufacturer's instructions. The reverse transcription reaction was carried out for 90 minutes at 50°C and for an additional 10 minutes at 55°C. Real-time PCR (qRT-PCR) was performed on an ABI Prism 7900 sequence detection system with SYBR Green reagents (Takara Bio Company, Clontech, Mountain View, CA, USA) and TaqMan® Gene Expression Master Mix (Life Technologies, Madrid, Spain).

BrdU incorporation
BrdU incorporation was determined with a Cell Proliferation ELISA colorimetric assay (Roche, Mannheim, Germany) according to the manufacturer's instructions. After sample treatment, cells were labeled overnight with BrdU, and then they were fixed and washed. An anti-BrdU-peroxidase working solution and substrate solution were added, and BrdU incorporation was quantified based on the absorbance at 370 nm in a microplate reader.

Caspase 3 activity
Caspase 3 activity was measured with a Caspase 3 Colorimetric Assay Kit (Abcam) according to the manufacturer's instructions. Briefly, after sample treatment, cells were labeled with the substrate DEVD (aspartic acid, glutamic acid, valine, aspartic acid)-pnitroaniline, and incubated at 37°C for 1 hour. Light emission from the chromophore p-nitroaniline was quantified with a microplate reader at 405 nm.

Protein extraction
Cells were harvested, and whole-cell lysates were prepared with radioimmunoprecipitation assay buffer (Sigma-Aldrich Química, S.L., Madrid, Spain) containing a protease and phosphatase inhibitor cocktail. The protein content of the lysates was determined with the Bradford reagent. www.impactjournals.com/oncotarget

Human CD26/DPP-4 quantification and activity
The concentration and activity of human CD26/ DPP-4 were analyzed in cell culture supernatants by means of a human CD26 ELISA Kit (Thermo Fisher Scientific, USA) and a fluorimetric DPP-4 Activity Assay Kit (Abcam, United Kingdom), respectively, according to the manufacturers' instructions. The assays were repeated three times, and each sample was processed in triplicate.