The dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin functions as antioxidant on human endothelial cells exposed to chronic hyperglycemia and metabolic high-glucose memory

Abstract

Dipeptidyl peptidase-4 inhibitors are widely used in type 2 diabetes. Endothelium plays a crucial role maintaining vascular integrity and function. Chronic exposure to high glucose drives to endothelial dysfunction generating oxidative stress. Teneligliptin is a novel dipeptidyl peptidase-4 inhibitor with antioxidant properties. This study is aimed to verify a potential protective action of teneligliptin in endothelial cells exposed to high glucose. Human umbilical vein endothelial cells were cultured under normal (5 mmol/L) or high glucose (25 mmol/L) during 21 days, or at high glucose during 14 days followed by 7 days at normal glucose, to reproduce the high-metabolic memory state. During this period, different concentrations of teneligliptin (0.1, 1.0 and 3.0 µmol/L) or sitagliptin (0.5 µmol/L) were added to cells. Ribonucleic acid and protein expression were assessed for antioxidant response, proliferation, apoptosis and endoplasmic reticulum stress markers. Teneligliptin promotes the antioxidant response in human umbilical vein endothelial cells, reducing ROS levels and inducing Nrf2-target genes messenger ribonucleic acid expression. Teneligliptin, but not sitagliptin, reduces the expression of the nicotine amide adenine dinucleotide phosphate oxidase regulatory subunit P22 ^-phox , however, both blunt the high glucose-induced increase of TXNIP. Teneligliptin improves proliferation rates in human umbilical vein endothelial cells exposed to high glucose, regulating the expression of cell-cycle inhibitors markers (P53, P21 and P27), and reducing proapoptotic genes (BAX and CASP3), while promotes BCL2 expression. Teneligliptin ameliorates high glucose-induced endoplasmic reticulum stress reducing the expression of several markers (BIP, PERK, ATF4, CHOP, IRE1a and ATF6). Teneligliptin has antioxidant properties, ameliorates oxidative stress and apoptotic phenotype and it can overcome the metabolic memory effect, induced by chronic exposure to high glucose in human endothelial cells.

Keywords: Antioxidant; DPP-4 inhibitors; Diabetes; Endothelial dysfunction; High glucose; Metabolic memory; Teneligliptin and sitagliptin.

Fig. 1

Teneligliptin effects on HUVECs viability after exposure to high-glucose conditions. HUVEC cells were incubated during 21 days under three different glucose conditions: NG (normoglycemia-5 mmol/L), HG (hyperglycemia-25 mmol/L) and NM (metabolic high-glucose memory-alternatively 5 and 25 mmol/L). Different doses of teneligliptin (0.1, 1.0 and 3.0 µmol/L) and sitagliptin (0.5 µmol/L) were used. Cell viability (%) was examined by an MTT assay. Bars represent mean ± SEM for three independent experiments

Fig. 2

Teneligliptin effects on HG stress markers: ROS, NAD(P)H oxidase subunits and TXNIP in HUVECs cultured under HG/HM conditions. HUVECs were maintained under NG, HG or HM conditions during 21 days. During the exposure, teneligliptin was added chronically to the medium at 0.1, 1.0 or 3.0 μmol/L. We used as control the DPP-4 inhibitor sitagliptin at the concentration of 0.5 μmol/L. a ROS production was stained by 20 μM H₂DCFDA for 30 min, and its oxidation product (DCF) fluorescence indicated ROS formation. b,c Total cellular RNA was isolated from HUVECs and mRNAs encoding for NOX4, P22 ^−phox and TXNIP genes were assessed by qRT-PCR and expressed relative to ACTB. *p < 0.05 and **p < 0.01 vs. NG. #p < 0.05 vs. HG. $p < 0.05 vs. HM. Bars represent mean ± SEM for six independent experiments

Fig. 3

Teneligliptin effects on the antioxidant gene expression in HUVECs cultured under HG/HM conditions. Total cellular RNA was isolated from HUVECs after 21 days in culture in NG, HG or NM. Cells were treated with different doses of teneligliptin (0.1, 1.0 and 3.0 µmol/L) or sitagliptin (0.5 µmol/L). mRNA encoding for the indicated genes was assessed by qRT-PCR and expressed relative to GAPDH or ACTB. *p < 0.05 vs. NG. #p < 0.05 vs. HG. $p < 0.05 vs. HM. Bars represent mean ± SEM of six independent experiments

Fig. 4

Teneligliptin effects on proliferation in HUVECs cultured under HG/HM conditions. HUVECs were maintained under NG, HG or HM conditions during 21 days. During the exposure, teneligliptin was added chronically to the medium at 0.1, 1.0 or 3.0 μmol/L. We used as control the DPP-4 inhibitor sitagliptin at the concentration of 0.5 μmol/L. a,b Total cellular RNA was isolated from HUVECs and mRNAs encoding for NOX4 and P22 ^−phox genes were assessed by qRT-PCR and expressed relative to GADPH or ACTB. c Protein expression of CASPASE 3 and P21 was assessed by western blot. The panels show a representative image of different independent experiments. Densitometric values were normalized to ACTB and represented relative to the control cells (NG), normalized to 1. d HUVECs proliferation was examined by measuring BrdU incorporation. *p < 0.05 and **p < 0.001 vs. NG. #p < 0.05 and ##p < 0.001 vs. HG. $p < 0.05 and $$p < 0.001 vs. HM. Bars represent mean ± SEM for six independent experiments

Fig. 5

Teneligliptin effects on ER function in HUVECs cultured under HG/HM conditions. HUVECs were maintained under NG, HG or HM conditions during 21 days. During the exposure, teneligliptin was added chronically to the medium at 0.1, 1.0 or 3.0 μmol/L. We used as control the DPP-4 inhibitor sitagliptin at the concentration of 0.5 μmol/L. Total cellular RNA was isolated from HUVECs and mRNAs encoding for the different ER stress markers were assessed by qRT-PCR and expressed relative to ACTB. *p < 0.05 and vs. NG. #p < 0.5 vs. HG. $p < 0.5 vs. HM. Bars represent mean ± SEM for six independent experiments