Uncoupling protein-2 mediates DPP-4 inhibitor-induced restoration of endothelial function in hypertension through reducing oxidative stress

Abstract

Aims: Although uncoupling protein 2 (UCP2) negatively regulates intracellular reactive oxygen species (ROS) production and protects vascular function, its participation in vascular benefits of drugs used to treat cardiometabolic diseases is largely unknown. This study investigated whether UCP2 and associated oxidative stress reduction contribute to the improvement of endothelial function by a dipeptidyl peptidase-4 inhibitor, sitagliptin, in hypertension.

Results: Pharmacological inhibition of cyclooxygenase-2 (COX-2) but not COX-1 prevented endothelial dysfunction, and ROS scavengers reduced COX-2 mRNA and protein expression in spontaneously hypertensive rats (SHR) renal arteries. Angiotensin II (Ang II) evoked endothelium-dependent contractions (EDCs) in C57BL/6 and UCP2 knockout (UCP2KO) mouse aortae. Chronic sitagliptin administration attenuated EDCs in SHR arteries and Ang II-infused C57BL/6 mouse aortae and eliminated ROS overproduction in SHR arteries, which were reversed by glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin 9-39, AMP-activated protein kinase (AMPK)α inhibitor compound C, and UCP2 inhibitor genipin. By contrast, sitagliptin unaffected EDCs in Ang II-infused UCP2KO mice. Sitagliptin increased AMPKα phosphorylation, upregulated UCP2, and downregulated COX-2 expression in arteries from SHR and Ang II-infused C57BL/6 mice. Importantly, exendin 9-39, compound C, and genipin reversed the inhibitory effect of GLP-1R agonist exendin-4 on Ang II-stimulated mitochondrial ROS rises in SHR endothelial cells. Moreover, exendin-4 improved the endothelial function of renal arteries from SHR and hypertensive patients.

Innovation: We elucidate for the first time that UCP2 serves as an important signal molecule in endothelial protection conferred by GLP-1-related agents. UCP2 could be a useful target in treating hypertension-related vascular events.

Conclusions: UCP2 inhibits oxidative stress and downregulates COX-2 expression through GLP-1/GLP-1R/AMPKα cascade.

FIG. 1.

Sitagliptin attenuates EDCs in SHR renal arteries. (A) In the presence of l-NAME, ACh elicited pronounced EDCs of renal arterial rings from SHR, which were inhibited by 2-week administration with sitagliptin. (B) EDCs in SHR arteries were attenuated or abolished by treatment with selective COX-2 inhibitor NS398 (1 μM) and DuP697 (3 μM) or selective TP receptor antagonist S18886 (0.1 μM) but unaffected by COX-1 inhibitor VAS (10 μM). COX-2 (C) and UCP2 (D) expressions in renal arteries from WKY and SHR. Results are means±SEM of six to eight experiments. *p<0.05 versus SHR vehicle, ^#p<0.05 versus WKY vehicle. l-NAME, NG-nitro-l-arginine methyl ester; EDCs, endothelium-dependent contractions; VAS, valeryl salicylate; ACh, acetylcholine; COX-2, cyclooxygenase-2; TP, thromboxane/prostanoid; UCP2, uncoupling protein 2; SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats.

FIG. 2.

Sitagliptin attenuates EDCs through the activation of GLP-1 receptor/AMPKα/UCP2 in SHR renal arteries. Effects of GLP-1 receptor antagonist exendin 9-39 (100 nM), AMPKα inhibitor compound C (10 μM), and UCP2 inhibitor genipin (1 μM) on EDCs in sitagliptin-treated SHR (A) and vehicle-SHR (B) groups. Level of AMPK phosphorylation (C) and expressions of UCP2 (D) and COX-2 (E) in SHR renal arteries. Results are means±SEM of four to eight experiments. *p<0.05 versus vehicle, ^#p<0.05 versus sitagliptin. Veh, vehicle; C, control; E, exendin 9-39; CC, compound C; G, genipin; AMPK, AMP-activated protein kinase; GLP-1, glucagon-like peptide 1.

FIG. 3.

Sitagliptin reduces COX-2 expression via scavenging ROS in SHR renal arteries. Exendin 9-39 (100 nM), compound C (10 μM), or genipin (1 μM) reversed the inhibitory effect of sitagliptin (10 mg·kg⁻¹·day⁻¹) on ROS production by DHE staining (A), lucigenin-enhanced chemiluminescence assay (B), and EPR (C). Moreover, acute treatment with tempol (100 μM) or tiron (1 mM) plus DETCA (100 μM) (A) reduced ROS level, and genipin (1 μM) also reversed the ROS-lowering effect of exendin-4 (10 nM) (B, C). ROS scavenger tempol (100 μM) or tiron (1 mM) plus DETCA (100 μM) (D) and mitochondrial ROS scavengers MitoQ (100 nM), coenzyme Q10 (10 μM), or idebenone (10 μM) (E) reduced or abolished EDCs in SHR arteries. Tempol (100 μM), tiron (1 mM) plus DETCA (100 μM), coenzyme Q10 (10 μM), or idebenone (10 μM) reduced COX-2 mRNA (F) and protein expression (G) in SHR arteries. Results are means±SEM of four to eight experiments. *p<0.05 versus control, ^#p<0.05 versus sitagliptin or exendin-4. Ex9, exendin 9-39; CC, compound C; G, genipin; ex4, exendin-4; RLU, relative luminance unit; ROS, reactive oxygen species; EPR, electron paramagnetic resonance; DHE, dihydroethidium; DETCA, diethyldithiocarbamate.

FIG. 4.

Exendin-4 ameliorates endothelial dysfunction in SHR renal arteries. (A) Effects of GLP-1 analog exendin-4 (10 nM, 8 h) on EDCs in renal arteries from WKY and SHR. (B) Effects of exendin 9-39 (100 nM), compound C (10 μM), and genipin (1 μM) on exendin-4-induced reduction of EDCs. (C) Exendin-4 (ex4, 10 nM) reduced mitochondrial ROS production triggered by Ang II (100 nM) in primary cultured SHR endothelial cells, which was inhibited by cotreatment with exendin 9-39 (Ex9, 100 nM), compound C (CC, 10 μM), or genipin (1 μM). (D) Images showing mitochondrial ROS production under different treatments. Results are means±SEM of four to six experiments. ^†p<0.05 versus WKY control, *p<0.05 versus SHR control, ^#p<0.05 versus exendin-4. Ang II, angiotensin II. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

The critical role of UCP2 in the improvement of endothelial function by sitagliptin. Ang II unmasked EDCs, which were inhibited by cotreatment with sitagliptin (3 mg·kg⁻¹·day⁻¹) in C57BL/6 mice (A) but not in UCP2KO mice (B). Overexpression of UCP2 by adUCP2 diminished EDC in SHR renal arteries (C). Expressions of UCP2 and COX-2 in C57BL/6 mice (D, E) and UCP2KO mice (F). Results are means±SEM of five to eight experiments. *p<0.05 versus respective control; ^#p<0.05 versus Ang II or A, ^†p<0.05 versus Ang II+sitagliptin control. C or Ctrl, control; E, exendin 9-39; CC, compound C; G, genipin; AS, Ang II+sitagliptin; UCP2KO, UCP2 knockout; adUCP2, adenovirus UCP2.

FIG. 6.

Exendin-4 improves endothelial function in renal arteries from HT. UCP2 (A) and COX-2 (B) expressions in human renal arteries. (C) DHE fluorescence of ROS in human renal arteries. Results are means±SEM of four experiments. *p<0.05 versus NT; ^#p<0.05 versus HT. C, control; E, exendin-4; NT, normotensive patients; HT, hypertensive patients.

FIG. 7.

The proposed cellular mechanism for the protective effect of sitagliptin against endothelial dysfunction in hypertension. Ang II evoked EDCs by increasing ROS level/COX-2 expression. Sitagliptin elevates UCP2 expression via the activation of GLP-1 receptor/AMPKα through GLP-1-dependent pathway, leading to reduced ROS production and subsequent downregulation of COX-2 expression, thus attenuates EDCs in hypertension.