Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A GLP-1 analog lowers ER stress and enhances protein folding to ameliorate homocysteine-induced endothelial dysfunction

Abstract

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases and increases mortality in type 2 diabetic patients. HHcy induces endoplasmic reticulum (ER) stress and oxidative stress to impair endothelial function. The glucagon-like peptide 1 (GLP-1) analog exendin-4 attenuates endothelial ER stress, but the detailed vasoprotective mechanism remains elusive. The present study investigated the beneficial effects of exendin-4 against HHcy-induced endothelial dysfunction. Exendin-4 pretreatment reversed homocysteine-induced impairment of endothelium-dependent relaxations in C57BL/6 mouse aortae ex vivo. Four weeks subcutaneous injection of exendin-4 restored the impaired endothelial function in both aortae and mesenteric arteries isolated from mice with diet-induced HHcy. Exendin-4 treatment lowered superoxide anion accumulation in the mouse aortae both ex vivo and in vivo. Exendin-4 decreased the expression of ER stress markers (e.g., ATF4, spliced XBP1, and phosphorylated eIF2α) in human umbilical vein endothelial cells (HUVECs), and this change was reversed by cotreatment with compound C (CC) (AMPK inhibitor). Exendin-4 induced phosphorylation of AMPK and endothelial nitric oxide synthase in HUVECs and arteries. Exendin-4 increased the expression of endoplasmic reticulum oxidoreductase (ERO1α), an important ER chaperone in endothelial cells, and this effect was mediated by AMPK activation. Experiments using siRNA-mediated knockdown or adenoviral overexpression revealed that ERO1α mediated the inhibitory effects of exendin-4 on ER stress and superoxide anion production, thus ameliorating HHcy-induced endothelial dysfunction. The present results demonstrate that exendin-4 reduces HHcy-induced ER stress and improves endothelial function through AMPK-dependent ERO1α upregulation in endothelial cells and arteries. AMPK activation promotes the protein folding machinery in endothelial cells to suppress ER stress.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Vascular function of mouse aortae upon acute Hcy exposure.
Fig. 2: Vascular function of the conduit and small arteries of the mice with diet-induced HHcy.
Fig. 3: ROS level of mouse aortae ex vivo and in vivo.
Fig. 4: Ex4-activated AMPK and eNOS in HUVECs and aortae from the mice with diet-induced HHcy.
Fig. 5: ER stress-induced oxidative stress in endothelial cells.
Fig. 6: ER stress and ER chaperone of endothelial cells.
Fig. 7: siRNA-mediated ERO1α knockdown in HUVECs.
Fig. 8: ERO1α overexpression in HUVECs and mouse aortae.
Fig. 9: The endothelial benefits mediated by exendin-4.

References

  1. 1.

    Ganguly P, Alam SF. Role of homocysteine in the development of cardiovascular disease. Nutr J. 2015;14:6.

    Article  Google Scholar 

  2. 2.

    Shenoy V, Mehendale V, Prabhu K, Shetty R, Rao P. Correlation of serum homocysteine levels with the severity of coronary artery disease. Indian J Clin Biochem. 2014;29:339–44.

    CAS  Article  Google Scholar 

  3. 3.

    Hoogeveen EK, Kostense PJ, Jakobs C, Dekker JM, Nijpels G, Heine RJ, et al. Hyperhomocysteinemia increases risk of death, especially in type 2 diabetes. Circulation. 2000;101:1506–11.

    CAS  Article  Google Scholar 

  4. 4.

    Cheng Z, Yang X, Wang H. Hyperhomocysteinemia and endothelial dysfunction. Curr Hypertens Rev. 2009;5:158–65.

    CAS  Article  Google Scholar 

  5. 5.

    Xu J, Zou MH. Molecular insights and therapeutic targets for diabetic endothelial dysfunction. Circulation. 2009;120:1266–86.

    Article  Google Scholar 

  6. 6.

    Looker HC, Fagot-Campagna A, Gunter EW, Pfeiffer CM, Sievers ML, Bennett PH, et al. Homocysteine and vitamin B12 concentrations and mortality rates in type 2 diabetes. Diabetes Metab Res Rev. 2007;23:193–201.

    CAS  Article  Google Scholar 

  7. 7.

    Bhatia P, Gupta S, Sharma S. Homocysteine excess and vascular endothelium dysfunction: delineating the pathobiological mechanisms. Int J Pharmacol. 2014;10:200–12.

    CAS  Article  Google Scholar 

  8. 8.

    Liu L, Liu J, Huang Y. Protective effects of glucagon-like peptide 1 on endothelial function in hypertension. J Cardiovasc Pharmacol. 2015;65:399–405.

    CAS  Article  Google Scholar 

  9. 9.

    Jakubowski H. Homocysteine modification in protein structure/function and human disease. Physiol Rev. 2019;99:555–604.

    CAS  Article  Google Scholar 

  10. 10.

    Ji C, Kaplowitz N. Hyperhomocysteinemia, endoplasmic reticulum stress, and alcoholic liver injury. World J Gastroenterol. 2004;10:1699–708.

    CAS  Article  Google Scholar 

  11. 11.

    Lenna S, Han R, Trojanowska M. Endoplasmic reticulum stress and endothelial dysfunction. IUBMB Life. 2014;66:530–7.

    CAS  Article  Google Scholar 

  12. 12.

    Nadkarni P, Chepurny OG, Holz GG. Regulation of glucose homeostasis by GLP-1. Prog Mol Biol Transl Sci. 2014;121:23–65.

    CAS  Article  Google Scholar 

  13. 13.

    Li Y, Li X, Zheng X, Tang L, Xu W, Gong M. Disulfide bond prolongs the half-life of therapeutic peptide-GLP-1. Peptides. 2011;32:1400–7.

    Article  Google Scholar 

  14. 14.

    Garber AJ. Long-acting glucagon-like peptide 1 receptor agonists: a review of their efficacy and tolerability. Diabetes Care. 2011;34:S279–84.

    CAS  Article  Google Scholar 

  15. 15.

    Liu L, Liu J, Wong WT, Tian XY, Lau CW, Wang YX, et al. Dipeptidyl peptidase 4 inhibitor sitagliptin protects endothelial function in hypertension through a glucagon–like peptide 1–dependent mechanism. Hypertension. 2012;60:833–41.

    CAS  Article  Google Scholar 

  16. 16.

    Oeseburg H, deBoer RA, Buikema H, van derHarst P, vanGilst WH, Silljé HHW. Glucagon-like peptide 1 prevents reactive oxygen species–induced endothelial cell senescence through the activation of protein kinase A. Arterioscler Thromb Vasc Biol. 2010;30:1407–14.

    CAS  Article  Google Scholar 

  17. 17.

    Bretón‐Romero R, Weisbrod RM, Feng B, Holbrook M, Ko D, Stathos MM, et al. Liraglutide treatment reduces endothelial endoplasmic reticulum stress and insulin resistance in patients with diabetes mellitus. J Am Heart Assoc. 2018;7:e009379.

    Article  Google Scholar 

  18. 18.

    Cheang WS, Tian XY, Wong WT, Lau CW, Lee SST, Chen ZY, et al. Metformin protects endothelial function in diet-induced obese mice by inhibition of endoplasmic reticulum stress through 5′ adenosine monophosphate–activated protein kinase–peroxisome proliferator–activated receptor δ pathway. Arterioscler Thromb Vasc Biol. 2014;34:830–6.

    CAS  Article  Google Scholar 

  19. 19.

    Cheang WS, Wong WT, Zhao L, Xu J, Wang L, Lau CW, et al. PPARδ is required for exercise to attenuate endoplasmic reticulum stress and endothelial dysfunction in diabetic mice. Diabetes. 2017;66:519–28.

    CAS  Article  Google Scholar 

  20. 20.

    Cunha DA, Ladrière L, Ortis F, Igoillo-Esteve M, Gurzov EN, Lupi R, et al. Glucagon-like peptide-1 agonists protect pancreatic beta-cells from lipotoxic endoplasmic reticulum stress through upregulation of BiP and JunB. Diabetes. 2009;58:2851–62.

    CAS  Article  Google Scholar 

  21. 21.

    Kilkenny C, Browne W, CuthillI C, Emerson M, Altman DG. NC3Rs Reporting Guidelines Working Group. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol. 2010;160:1577–9.

    CAS  Article  Google Scholar 

  22. 22.

    Wong WT, Tian XY, Xu A, Yu J, Lau CW, Hoo RLC, et al. Adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetic mice. Cell Metab. 2011;14:104–15.

    CAS  Article  Google Scholar 

  23. 23.

    Wong SL, Lau CW, Wong WT, Xu A, Au CL, Ng CF, et al. Pivotal role of protein kinase Cδ in angiotensin II-induced endothelial cyclooxygenase-2 expression: a link to vascular inflammation. Arterioscler Thromb Vasc Biol. 2011;31:1169–76.

    CAS  Article  Google Scholar 

  24. 24.

    Gou L, Zhao L, Song W, Wang L, Liu J, Zhang H, et al. Inhibition of miR-92a suppresses oxidative stress and improves endothelial function by upregulating heme oxygenase-1 in db/db Mice. Antioxid Redox Signal. 2018;28:358–70.

    CAS  Article  Google Scholar 

  25. 25.

    Ling WC, Liu J, Lau CW, Murugan DD, Mustafa MR, Huang Y. Treatment with salvianolic acid B restores endothelial function in angiotensin II-induced hypertensive mice. Biochem Pharmacol. 2017;136:76–85.

    CAS  Article  Google Scholar 

  26. 26.

    Tyagi N, Sedoris KC, Steed M, Ovechkin AV, Moshal KS, Tyagi SC. Mechanisms of homocysteine-induced oxidative stress. Am J Physiol Circ Physiol. 2005;289:H2649–56.

    CAS  Article  Google Scholar 

  27. 27.

    Wei R, Ma S, Wang C, Ke J, Yang J, Li W, et al. Exenatide exerts direct protective effects on endothelial cells through the AMPK/Akt/eNOS pathway in a GLP-1 receptor-dependent manner. Am J Physiol Metab. 2016;310:E947–57.

    Google Scholar 

  28. 28.

    Cheng CK, Bakar HA, Gollasch M, Huang Y. Perivascular adipose tissue: the sixth man of the cardiovascular system. Cardiovasc Drugs Ther. 2018;32:481–502.

    CAS  Article  Google Scholar 

  29. 29.

    Liu L, Liu J, Tian XY, Wong WT, Lau CW, Xu A, et al. Uncoupling protein-2 mediates DPP-4 inhibitor-induced restoration of endothelial function in hypertension through reducing oxidative stress. Antioxid Redox Signal. 2014;21:1571–81.

    CAS  Article  Google Scholar 

  30. 30.

    Zito E. ERO1: a protein disulfide oxidase and H2O2 producer. Free Radic Biol Med. 2015;83:299–304.

    CAS  Article  Google Scholar 

  31. 31.

    Feldman DE, Chauhan V, Koong AC. The unfolded protein response: a novel component of the hypoxic stress response in tumors. Mol Cancer Res. 2005;3:597–605.

    CAS  Article  Google Scholar 

  32. 32.

    Hogg PJ. Disulfide bonds as switches for protein function. Trends Biochem Sci. 2003;28:210–4.

    CAS  Article  Google Scholar 

  33. 33.

    Wright J, Birk J, Haataja L, Liu M, Ramming T, Weiss MA, et al. Endoplasmic reticulum oxidoreductin-1α improves folding and secretion of mutant proinsulin and limits mutant proinsulin-induced endoplasmic reticulum stress. J Biol Chem. 2013;288:31010–8.

    CAS  Article  Google Scholar 

  34. 34.

    Liu JQ, Zhang L, Yao J, Yao S, Yuan T. AMPK alleviates endoplasmic reticulum stress by inducing the ER-chaperone ORP150 via FOXO1 to protect human bronchial cells from apoptosis. Biochem Biophys Res Commun. 2018;497:564–70.

    CAS  Article  Google Scholar 

  35. 35.

    Pobre KFR, Poet GJ, Hendershot LM. The endoplasmic reticulum (ER) chaperone BiP is a master regulator of ER functions: getting by with a little help from ERdj friends. J Biol Chem. 2019;294:2098–108.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by Health and Medical Research Fund [Grant numbers 05162906 and 05161746], Early Career Scheme [Grant number 24122318], the National Natural Science Foundation of China [Grant numbers 91739103 and 91939302], and Hong Kong Research Grants Council [Grant numbers 14112919 and C4024-16W].

Author information

Affiliations

Authors

Contributions

CKC designed the study, performed experiments, analyzed the data, and wrote the manuscript. CWL and JYL performed some of the experiments. WCsC, CFN, and RCWM were involved in regular discussion and revised the manuscript. YH and XYT designed the study, analyzed the data, wrote the manuscript, and provided grant support.

Corresponding authors

Correspondence to Xiao Yu Tian or Yu Huang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Cheng, C.K., Luo, JY., Lau, C.W. et al. A GLP-1 analog lowers ER stress and enhances protein folding to ameliorate homocysteine-induced endothelial dysfunction. Acta Pharmacol Sin (2021). https://doi.org/10.1038/s41401-020-00589-x

Download citation

Keywords

  • homocysteine
  • GLP-1 analog
  • exendin-4
  • AMPK
  • ER stress
  • ER chaperone
  • oxidative stress
  • endothelial dysfunction

Search

Quick links