TRPV1-mediated UCP2 upregulation ameliorates hyperglycemia-induced endothelial dysfunction

Abstract

Background: Diabetic cardiovascular complications are characterised by oxidative stress-induced endothelial dysfunction. Uncoupling protein 2 (UCP2) is a regulator of mitochondrial reactive oxygen species (ROS) generation and can antagonise oxidative stress, but approaches that enhance the activity of UCP2 to inhibit ROS are scarce. Our previous studies show that activation of transient receptor potential vanilloid 1 (TRPV1) by capsaicin can prevent cardiometabolic disorders. In this study, we conducted experiments in vitro and in vivo to investigate the effect of capsaicin treatment on endothelial UCP2 and oxidative stress. We hypothesised that TRPV1 activation by capsaicin attenuates hyperglycemia-induced endothelial dysfunction through a UCP2-mediated antioxidant effect.

Methods: TRPV1(-/-), UCP2(-/-) and db/db mice, as well as matched wild type (WT) control mice, were included in this study. Some mice were subjected to dietary capsaicin for 14 weeks. Arteries isolated from mice and endothelial cells were cultured. Endothelial function was examined, and immunohistological and molecular analyses were performed.

Results: Under high-glucose conditions, TRPV1 expression and protein kinase A (PKA) phosphorylation were found to be decreased in the cultured endothelial cells, and the effects of high-glucose on these molecules were reversed by the administration of capsaicin. Furthermore, high-glucose exposure increased ROS production and reduced nitric oxide (NO) levels both in endothelial cells and in arteries that were evaluated respectively by dihydroethidium (DHE) and DAF-2 DA fluorescence. Capsaicin administration decreased the production of ROS, restored high-glucose-induced endothelial dysfunction through the activation of TRPV1 and acted in a UCP2-dependent manner in vivo. Administration of dietary capsaicin for 14 weeks increased the levels of PKA phosphorylation and UCP2 expression, ameliorated the vascular oxidative stress and increased NO levels observed in diabetic mice. Prolonged dietary administration of capsaicin promoted endothelium-dependent relaxation in diabetic mice. However, the beneficial effect of capsaicin on vasorelaxation was absent in the aortas of UCP2(-/-) mice exposed to high-glucose levels.

Conclusion: TRPV1 activation by capsaicin might protect against hyperglycemia-induced endothelial dysfunction through a mechanism involving the PKA/UCP2 pathway.

Figure 1

Activation of TRPV1 up-regulates UCP2 through PKA phosphorylation. A, B and C: Representative western blot images showing protein expressions of TRPV1 (A), p-PKA/PKA (B) and UCP2 (C) in endothelial cells cultured with normal-glucose (NG, glucose 5.5 mmol/L), high-glucose (HG, glucose 30 mmol/L), HG+capsaicin (HG+Cap, Cap 1 μmol/L), HG+Cap+5’-iodo-resiniferatoxin (HG+Cap+iRTX, iRTX 1 μmol/L), HG+Cap+KT5720 (2 μmol/L) or HG+Cap+Genipin (10 μmol/L); #P<0.05, ##P<0.01 versus NG group; *P<0.05, **P<0.01 versus HG group; ΔP<0.05, ΔΔP<0.01 versus HG+Cap group. Data are mean ± SEM. Each n = 3.

Figure 2

The effect of TRPV1 activation on the production of ROS and NO through the PKA/UCP2 pathway. A and B: Representative western blot images (A) and summary data (B) showing P22^phox protein level in endothelial cells cultured with normal-glucose (NG, glucose 5.5 mmol/L), high-glucose (HG, glucose 30 mmol/L), HG+capsaicin (HG+Cap, Cap 1 μmol/L), HG+Cap+5’-iodo-resiniferatoxin (HG+Cap+iRTX, iRTX 1 μmol/L), HG+Cap+KT5720 (2 μmol/L), HG+Cap+Genipin (10 μmol/L). ##P <0.01 versus NG group; **P <0.01 versus HG group; ΔΔP <0.01 versus HG+Cap group; Data are mean ± SEM. Each n = 3. C-F: Representative endothelial cells stained by DHE (C and E) and DAF-2 DA (D and F) cultured with NG, HG, HG+Cap, HG+Cap+iRTX, HG+Cap+KT5720, HG+Cap+Genipin. ##P <0.01 versus NG group; **P <0.01 versus HG group; ΔΔP <0.01 versus HG+Cap group. Data are mean ± SEM from 4 independent experiments. The scale bar indicates 50 μm.

Figure 3

TRPV1 activation ameliorates high-glucose-induced endothelial dysfunction in a UCP2-dependent manner. A: Representative immunofluorescence images showing the co-expression of TRPV1, PKA and UCP2 in the aortas from wild type mice, particularly in the endothelium (Bar denotes 50 μm). B and C: Acetylcholine (1 nmol/L to 10 μmol/L)-induced endothelium-dependent relaxation of isolated aortic artery rings from wild type and TRPV1^-/- mice, pre-incubated with normal-glucose for 12 hours (NG, glucose 5.5 mmol/L), high-glucose (HG, glucose 30 mmol/L), HG+capsaicin (HG+Cap, Cap 1 μmol/L), HG+Cap+KT5720 (2 μmol/L); **P<0.01 versus NG group; ##P<0.01 versus HG group; ΔΔP<0.01 versus HG+Cap group. Data are mean ± SEM. Each n=6. D and E: Nitroglycerin (1 nmol/L to 10 μmol/L) -induced endothelium-independent relaxation of isolated aortic artery rings from wild type and TRPV1^-/- mice, after cultured for 12 hours with NG, HG, HG+Cap. Data are mean ± SEM. Each n =6. F and G: Representative data that Acetylcholine- and nitroglycerin-induced relaxation in the presence or absence of capsaicin (Cap, 1 μmol/L) in isolated aortic arteries rings from UCP2^-/- mice and wild type (WT) mice under high-glucose condition(HG). **P<0.01 HG + Cap versus HG group of WT, #P<0.05 HG group of WT versus HG group of UCP2^-/-. Data are mean ± SEM. Each n=6.

Figure 4

TRPV1 activation by dietary capsaicin promotes endothelial PKA phosphorylation and increases UCP2 levels in diabetic mice. Representative protein expression of TRPV1 (A and B), p-PKA/PKA (C and D) and UCP2 (E) levels in aorta or mesenteric arteries from db/db mice treated with normal diet (db/db Cont) or normal diet plus 0.01% capsaicin (db/db Cap) and the lean littermate control C57BL/KsJ mice treated with normal diet (WT Cont). Data are mean ± SEM. Each n = 3. ##P <0.01, #P <0.05 versus WT Cont group; *P <0.05 versus db/db Cont group.

Figure 5

TRPV1 activation by dietary capsaicin attenuates endothelial oxidative stress and increases the level of NO in diabetic mice. A and B: Representative protein expressions of p22^phox (A) and p-eNOS (B) in aortas from db/db mice treated with normal diet (db/db Cont) or normal diet plus 0.01% capsaicin (db/db Cap) and wild type mice treated with normal diet (WT Cont). ##P<0.01 versus the WT Cont group; *P<0.05 versus the db/db Cont group. Data are mean ± SEM. Each n = 3. C and D: Representative images and summary data detected by DHE (C) and DAF-2 DA (D) in mesenteric arteries from wild type and db/db mice treated with normal diet (WT Cont and db/db Cont) or normal diet plus 0.01% capsaicin (WT Cap and db/db Cap). #P<0.05 versus WT Cont group; ** P<0.01 versus WT Cap group; ΔΔP<0.01 versus db/db cont group. Data are mean ± SEM. Each n = 4. The scale bar indicates 50 μm.

Figure 6

Dietary capsaicin improves the endothelium-dependent relaxation in db/db mice. A and C: Quantification of the endothelium-dependent relaxation induced by acetylcholine treatment (1 nmol/L to 10 μmol/L) of aortas and mesenteric arteries from db/db mice given either a normal diet (Cont) or a normal diet plus 0.01% capsaicin (Cap) for 14 weeks. Data are expressed as the mean ± SEM from six rings, with six mice per group. *P<0.05, ** P<0.01 versus the db/db control group. B and D: The inhibitory effect of nitro-L-arginine methyl ester (L-NAME, 100 μM, 30min), on acetylcholine-induced maximal relaxation in aortas and mesenteric arteries from db/db mice with or without dietary capsaicin administration. Data are expressed as the mean ± SEM from six rings, with six mice per group. E and F: Representative data showing endothelium-independent relaxation in db/db mouse aortas and mesenteric arteries with or without dietary capsaicin administration. Data are expressed as the mean ± SEM from six rings, with six mice per group.

Figure 7

Upregulation of UCP2 by TRPV1 activation attenuates hyperglycemia-induced ROS production in ECs. The increased metabolism of glucose due to intracellular hyperglycemia leads to the overproduction of NADH, a critical component of the superoxide-generating mechanism in endothelial cells. Upregulation of mitochondrial UCP2 in response to elevated superoxide levels plays an active role in the feedback regulation of reactive oxygen species production that is associated with chronic oxidative stress. Activation of the endothelial TRPV1 channel in endothelial cells by dietary capsaicin mediates the phosphorylation of PKA and upregulates UCP2, thus inhibiting the activity of NADPH and decreasing ROS production in ECs.