Korean J Physiol Pharmacol.  2021 Sep;25(5):425-437. 10.4196/kjpp.2021.25.5.425.

Sitagliptin attenuates endothelial dysfunction independent of its blood glucose controlling effect

• Chang XM
• Xiao F
• Pan Q
• Wang XX
• Guo LX

• KMID: 2519405
• DOI: http://doi.org/10.4196/kjpp.2021.25.5.425

Abstract

Although the contributions of sitagliptin to endothelial dysfunction in diabetes mellitus were previously reported, the mechanisms still undefined. Autophagy plays an important role in the development of diabetes mellitus, but its role in diabetic macrovascular complications is unclear. This study aims to observe the effect of sitagliptin on macrovascular endothelium in diabetes and explore the role of autophagy in this process. Diabetic rats were induced through administration of high-fat diet and intraperitoneal injection of streptozotocin. Then diabetic rats were treated with or without sitagliptin for 12 weeks. Endothelial damage and autophagy were measured. Human umbilical vein endothelial cells were cultured either in normal glucose or in high glucose medium and intervened with different concentrations of sitagliptin. Rapamycin was used to induce autophagy. Cell viability, apoptosis and autophagy were detected. The expressions of proteins in c-Jun N-terminal kinase (JNK)-Bcl-2-Beclin-1 pathway were measured. Sitagliptin attenuated injuries of endothelium in vivo and in vitro. The expression of microtubuleassociated protein 1 light chain 3 II (LC3II) and beclin-1 were increased in aortas of diabetic rats and cells cultured with high-glucose, while sitagliptin inhibited the over-expression of LC3II and beclin-1. In vitro pre-treatment with sitagliptin decreased rapamycin-induced autophagy. However, after pretreatment with rapamycin, the protective effect of sitagliptin on endothelial cells was abolished. Further studies revealed sitagliptin increased the expression of Bcl-2, while inhibited the expression of JNK in vivo . Sitagliptin attenuates injuries of vascular endothelial cells caused by high glucose through inhibiting over-activated autophagy. JNK-Bcl-2-Beclin-1 pathway may be involved in this process.

Keyword

Apoptosis; Autophagy; Human umbilical vein endothelial cell; Sitagliptin phosphate; Streptozotocin diabetes

Figure

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  Fig. 1 Sitagliptin improved blood glucose and damage of aortas in diabetic rats. (A) Variation of FBG in the duration of treatments. (B) Variation of random blood glucose in the period of treatments. (C) The arrows point to endothelial cells. H&E staining of aortas (×200). (D) The thickness of artery wall of rats in each group. (E) Serum VCAM-1 level of rats in each group. (F–J) Representative Western blot bands for the protein levels of E-selection, ICAM-1, VWF, and TM in sitagliptin-treated rats. Data are expressed as means ± SD, n = 5–8. FBG, fasting blood glucose; H&E, hematoxylin and eosin; VCAM-1, vascular adhesion molecule-1; DM, diabetes mellitus; ICAM-1, intercellular adhesion molecule 1; VWF, Von Willebrand factor; TM, thrombomodulin. *p < 0.05 vs. control group, ***p < 0.001 vs. control group, #p < 0.05 vs. DM group, ###p < 0.001 vs. DM group.

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  Fig. 2 Sitagliptin inhibited autophagy in aortas in diabetic rats. (A) Representative pictures of immunohistochemistry of LC3 (×200). (B, C) Representative Western blot bands for the protein expression of LC3. (D, E) Representative Western blot bands for the protein expression of beclin-1. (F, G) Representative Western blot bands for the protein expression of LAMP1/2 of aortas in Sitagliptin-treated diabetic rats. (H) The Cathepsin B/D activity results of aortas in Sitagliptin-treated diabetic rats were presented. n = 4–5. LC3, microtubule-associated protein 1 light chain 3; LAMP1/2, lysosomal-associated membrane protein 1/2; DM, diabetes mellitus. *p < 0.05 vs. control group, **p < 0.01 vs. control group, #p < 0.05 vs. DM group, ##p < 0.01 vs. DM group, ###p < 0.001 vs. DM group.

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  Fig. 3 Sitagliptin protected HUVECs from injuries caused by high glucose in a dose-dependent manner. Cells were treated with normal glucose, high glucose (33 mM), different concentrations of sitagliptin (0, 0.1, 1, 10, and 100 μM). (A) Cell viability was detected by MTT assay. (B, C) Cell apoptosis was determined by flow cytometry. (D) The productions of IL-1α, IL-6, IL-8, and IL-18 in HG-induced HUVECs were determined by ELISA after treated by different concentrations of sitagliptin. HUVECs, Human umbilical vein endothelial cells; HG, high glucose; S, sitagliptin; IL-1α, interleukin 1 alpha; IL-6, interleukin 6; IL-8, interleukin 8; IL-18, interleukin 18; ELISA, enzyme-linked immunosorbent assay. *p < 0.05 vs. control group, ***p < 0.001 vs. control group. #p < 0.05 vs. HG group. ##p < 0.01 vs. HG group. ###p < 0.001 vs. HG group.

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  Fig. 4 Sitagliptin protected HUVECs through inhibiting over-activated autophagy. Cells were treated with normal glucose, high glucose, different concentrations of sitagliptin (0, 0.1, 1, 10, and 100 μM). The expression of LC3 was detected by Western blot (A, B). (C) Cell viability was detected after HG-induced HUVECs treated with rapamycin and sitagliptin. (D, E) Representative Western blot bands for the protein expression of LC3 and Beclin-1 in HG-induced HUVECs treated with rapamycin and sitagliptin. (F, G) Representative Western blot bands for the protein expression of LAMP1/2 in HG-induced HUVECs treated with rapamycin and sitagliptin. (H) The Cathepsin B/D activity results of in HG-induced HUVECs treated with rapamycin and sitagliptin were presented. (I) The productions of IL-1α, IL-6, IL-8, IL-18 in HG-induced HUVECs were determined by ELISA after treated with rapamycin and sitagliptin. HUVECs, Human umbilical vein endothelial cells; LC3, microtubule-associated protein 1 light chain 3; HG, high glucose; S, sitagliptin; RAPA, rapamycin; LAMP1/2, lysosomal-associated membrane protein 1/2; IL-1α, interleukin 1 alpha; IL-6, interleukin 6; IL-8, interleukin 8; IL-18, interleukin 18; ELISA, enzyme-linked immunosorbent assay. *p < 0.05 vs. control group, **p < 0.05 vs. HG + Sitagliptin group, ***p < 0.001 vs. control group or HG + Sitagliptin group, #p < 0.05 vs. HG group, ###p < 0.001 vs. HG group, ##p < 0.01 vs. HG group.

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  Fig. 5 Pretreatment with sitagliptin prevented autophagy in HUVECs exposed to rapamycin. (A–E) The protein levels of LC3, Beclin-1, LAMP1, and LAMP2 in Sitagliptin-pretreated HUVECs were determined using Western blot after exposed to rapamycin. (F, G) The Cathepsin B/D activity results in Sitagliptin-pretreated HUVECs were determined after exposed to rapamycin. HUVECs, Human umbilical vein endothelial cells; LC3, microtubule-associated protein 1 light chain 3; LAMP1/2, lysosomal-associated membrane protein 1/2. *p < 0.05 vs. RAPA-0.01 group. ***p < 0.001 vs. RAPA-0.01 group, ###p < 0.001 vs. RAPA-0.1 group, &&&p < 0.001 vs. RAPA-1 group, ^^^p < 0.001 vs. RAPA-10 group, $$$p < 0.001 vs. RAPA-100 group.

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  Fig. 6 Sitagliptin may regulate the expression of protein in JNK-Bcl-2-Beclin-1 pathway. The expressions of JNK (A, B), Bcl-2(C, D), Beclin-1(E, F) in thoracic aortas of rats in Control, DM, and DM + Sitagliptin groups. n = 4–5. JNK, c-Jun N-terminal Kinase; DM, diabetes mellitus. *p < 0.05 vs. control group, #p < 0.05 vs. DM group.

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