Applied Biochemistry and Biotechnology

, Volume 179, Issue 1, pp 59–74 | Cite as

Construction of a Fusion Peptide 5rolGLP-HV and Analysis of its Therapeutic Effect on Type 2 Diabetes Mellitus and Thrombosis in Mice

  • Zaizhong Ni
  • Yaofang Zhang
  • Haisong Wang
  • Yiming Wei
  • Baicheng Ma
  • Junfeng Hao
  • Peipei Tu
  • Huikun Duan
  • Xiaodan Li
  • Pingzhe Jiang
  • Xiaofeng Ma
  • Bin Wang
  • Ri Wu
  • Jianhong Zhu
  • Minggang LiEmail author


Glucagon-like peptide-1 (GLP-1), is currently used to treat type 2 diabetes mellitus and hirudin (HV), plays an important role in controlling thrombosis and cardiovascular diseases. This investigation aimed to develop a fusion peptide 5rolGLP-HV which combined functions of rolGLP-1 and rHV to treat diabetes and thrombosis. In this study, we constructed a fusion gene including five copies of rolGLP-1 and one copy of rHV (5rolGLP-HV). The optimum expression conditions of 5rolGLP-HV in a soluble form were 0.8 mM IPTG induction when OD600 reached 0.6–0.8 and further growing at 25 °C for 9 h. Isolated rolGLP-1 and rHV were acquired by trypsin digestion in vitro, and the concentration of them was determined by HPLC in vivo. Oral administration of 5rolGLP-HV significantly decreased the levels of blood glucose, GHbA1C, TC, and TG in diabetic mice at the time of 3 weeks compared to the saline-treated group (p < 0.05), while the insulin level was reversed significantly (p < 0.05). 5rolGLP-HV treatment significantly shortened the length of thrombus in thrombosis mice compared to the saline-treated group (p < 0.01). These results indicated that 5rolGLP-HV had dual-function in treating diabetes and preventing thrombosis.


5rolGLP-HV Diabetes Thrombosis Expression Purification Dual-function treatment 



Type 2 diabetes mellitus


Dipeptidyl peptidase IV


Recombinant oral long-acting GLP-1






Area under the curve


Optical density


Oral glucose tolerance test


Glycosylated hemoglobin A1c


Total cholesterol




Fasting blood-glucose


Antithrombin units



This study is supported by the Key Technologies R&D Program of Tianjin (14ZCZDSY00013).


  1. 1.
    Ferroni, P., Basili, S., Falco, A., & Davi, G. (2004). Platelet activation in type 2 diabetes mellitus. Journal of Thrombosis and Haemostasis, 2(8), 1282–1291.CrossRefGoogle Scholar
  2. 2.
    Ajjan, R., & Grant, P. J. (2006). Coagulation and atherothrombotic disease. Atherosclerosis, 186(2), 240–259.CrossRefGoogle Scholar
  3. 3.
    Ajjan, R. A., & Ariens, R. A. (2009). Cardiovascular disease and heritability of the prothrombotic state. Blood Reviews, 23(2), 67–78.CrossRefGoogle Scholar
  4. 4.
    Lennox, R., Porter, D.W., Flatt, P.R., Holscher, C., Irwin, N., Gault, V.A. (2014). Comparison of the independent and combined effects of sub-chronic therapy with metformin and a stable GLP-1 receptor agonist on cognitive function, hippocampal synaptic plasticity and metabolic control in high-fat fed mice. Neuropharmacology, 8622–8630.Google Scholar
  5. 5.
    Dailey, M. J., & Moran, T. H. (2013). Glucagon-like peptide 1 and appetite. Trends in Endocrinology and Metabolism, 24(2), 85–91.CrossRefGoogle Scholar
  6. 6.
    Samson, S. L., Sathyanarayana, P., Jogi, M., Gonzalez, E. V., Gutierrez, A., Krishnamurthy, R., Muthupillai, R., Chan, L., & Bajaj, M. (2011). Exenatide decreases hepatic fibroblast growth factor 21 resistance in non-alcoholic fatty liver disease in a mouse model of obesity and in a randomised controlled trial. Diabetologia, 54(12), 3093–3100.CrossRefGoogle Scholar
  7. 7.
    Mack, C. M., Moore, C. X., Jodka, C. M., Bhavsar, S., Wilson, J. K., Hoyt, J. A., Roan, J. L., Vu, C., Laugero, K. D., Parkes, D. G., et al. (2006). Antiobesity action of peripheral exenatide (exendin-4) in rodents: effects on food intake, body weight, metabolic status and side-effect measures. International Journal of Obesity, 30(9), 1332–1340.CrossRefGoogle Scholar
  8. 8.
    Parlevliet, E. T., de Leeuw van Weenen, J. E., Romijn, J. A., & Pijl, H. (2010). GLP-1 treatment reduces endogenous insulin resistance via activation of central GLP-1 receptors in mice fed a high-fat diet. American Journal of Physiology, Endocrinology and Metabolism, 299(2), E318–E324.Google Scholar
  9. 9.
    Parlevliet, E. T., Wang, Y., Geerling, J. J., Schroder-Van der Elst, J. P., Picha, K., O’Neil, K., Stojanovic-Susulic, V., Ort, T., Havekes, L. M., Romijn, J. A., et al. (2012). GLP-1 receptor activation inhibits VLDL production and reverses hepatic steatosis by decreasing hepatic lipogenesis in high-fat-fed APOE*3-Leiden mice. PLoS ONE, 7(11), e49152.CrossRefGoogle Scholar
  10. 10.
    Gao, M., Tian, H., Ma, C., Gao, X., Guo, W., & Yao, W. (2010). Expression, purification, and C-terminal site-specific PEGylation of cysteine-mutated glucagon-like peptide-1. Applied Biochemistry and Biotechnology, 162(1), 155–165.CrossRefGoogle Scholar
  11. 11.
    Tahrani, A. A., Piya, M. K., Kennedy, A., & Barnett, A. H. (2010). Glycaemic control in type 2 diabetes: targets and new therapies. Pharmacology and Therapeutics, 125(2), 328–361.CrossRefGoogle Scholar
  12. 12.
    Green, B. D., Liu, H. K., McCluskey, J. T., Duffy, N. A., O’Harte, F. P., McClenaghan, N. H., & Flatt, P. R. (2005). Function of a long-term, GLP-1-treated, insulin-secreting cell line is improved by preventing DPP IV-mediated degradation of GLP-1. Diabetes, Obesity & Metabolism, 7(5), 563–569.CrossRefGoogle Scholar
  13. 13.
    Reimann, F., & Gribble, F. M. (2002). Glucose-sensing in glucagon-like peptide-1-secreting cells. Diabetes, 51(9), 2757–2763.CrossRefGoogle Scholar
  14. 14.
    Drucker, D. J., & Nauck, M. A. (2006). The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet, 368(9548), 1696–1705.CrossRefGoogle Scholar
  15. 15.
    Holst, J. J. (2007). The physiology of glucagon-like peptide 1. Physiological Reviews, 87(4), 1409–1439.CrossRefGoogle Scholar
  16. 16.
    Baggio, L. L., & Drucker, D. J. (2007). Biology of incretins: GLP-1 and GIP. Gastroenterology, 132(6), 2131–2157.CrossRefGoogle Scholar
  17. 17.
    Ben-Shlomo, S., Zvibel, I., Shnell, M., Shlomai, A., Chepurko, E., Halpern, Z., Barzilai, N., Oren, R., & Fishman, S. (2011). Glucagon-like peptide-1 reduces hepatic lipogenesis via activation of AMP-activated protein kinase. Journal of Hepatology, 54(6), 1214–1223.CrossRefGoogle Scholar
  18. 18.
    Madonna, R., & De Caterina, R. (2011). Cellular and molecular mechanisms of vascular injury in diabetes—part I: pathways of vascular disease in diabetes. Vascular Pharmacology, 54(3-6), 68–74.CrossRefGoogle Scholar
  19. 19.
    Honardoost, M., Sarookhani, M. R., Arefian, E., & Soleimani, M. (2014). Insulin resistance associated genes and miRNAs. Applied Biochemistry and Biotechnology, 174(1), 63–80.CrossRefGoogle Scholar
  20. 20.
    Standl, E., Muller, M., & Schnell, O. (2009). The impact of glucose-lowering therapy on cardiovascular outcomes. Best Practice & Research Clinical Endocrinology & Metabolism, 23(3), 401–411.CrossRefGoogle Scholar
  21. 21.
    Markwardt, F. (1985). Pharmacology of hirudin: one hundred years after the first report of the anticoagulant agent in medicinal leeches. Biomedica Biochimica Acta, 44(7-8), 1007–1013.Google Scholar
  22. 22.
    Donella-Deana, A., Varro, A., Dockray, G. J., & Pinna, L. A. (1991). Substitution of phosphotyrosine for sulphotyrosine in biologically active peptides. Enzymatic phosphorylation of a progastrin peptide confers immunoreactivity reminiscent of the sulphated derivative. Biochimica et Biophysica Acta, 1095(1), 75–77.CrossRefGoogle Scholar
  23. 23.
    Lombardi, A., De Simone, G., Galdiero, S., Staiano, N., Nastri, F., & Pavone, V. (1999). From natural to synthetic multisite thrombin inhibitors. Biopolymers, 51(1), 19–39.CrossRefGoogle Scholar
  24. 24.
    Nowak, G., & Markwardt, F. (1991). Hirudin in disseminated intravascular coagulation. Haemostasis, 21(Suppl), 1142–1148.Google Scholar
  25. 25.
    Jiang, W., Li, W., Hong, Y., Wang, S., Fang, B. (2015). Cloning, expression, mutagenesis library construction of glycerol dehydratase, and binding mode simulation of its reactivase with ligands. Applied Biochemistry and Biotechnology.Google Scholar
  26. 26.
    Zafar, A., Aftab, M.N., Ud Din, Z., Aftab, S., Iqbal, I., Ul Haq, I. (2015). Cloning, purification and characterization of a highly thermostable amylase gene of thermotoga petrophila into Escherichia coli. Applied Biochemistry and Biotechnology.Google Scholar
  27. 27.
    Zhang, Y. F., Wei, Y. M., Ma, B. C., Qiao, K. Y., Ma, Z. H., Li, C., Ma, C., Ji, Y. L., Dong, Z., Hao, J. F., et al. (2013). Expression of rolGLP-HV in E-coli and its dual-function for the treatment of diabetes and thrombosis. International Journal of Peptide Research and Therapeutics, 19(3), 257–263.CrossRefGoogle Scholar
  28. 28.
    Zeng, X.Y., Dong, S., He, N.N., Jiang, C.J., Dai, Y., Xia, Y.F. (2015). Comparative pharmacokinetics of arctigenin in normal and type 2 diabetic rats after oral and intravenous administration. Fitoterapia, 105119–105126.Google Scholar
  29. 29.
    Rouse, R., Xu, L., Stewart, S., & Zhang, J. (2014). High fat diet and GLP-1 drugs induce pancreatic injury in mice. Toxicology and Applied Pharmacology, 276(2), 104–114.CrossRefGoogle Scholar
  30. 30.
    Shrivastava, A., Chaturvedi, U., Sonkar, R., Khanna, A. K., Saxena, J. K., & Bhatia, G. (2012). Antioxidant effect of Azadirachta indica on high fat diet induced diabetic Charles Foster rats. Applied Biochemistry and Biotechnology, 167(2), 229–236.CrossRefGoogle Scholar
  31. 31.
    Morsy, M.A., Heeba, G.H., Mahmoud, M.E. (2015). Ameliorative effect of eprosartan on high-fat diet/streptozotocin-induced early diabetic nephropathy in rats. European Journal of Pharmacology, 75090–75097.Google Scholar
  32. 32.
    Liu, C., Hu, M. Y., Zhang, M., Li, F., Li, J., Zhang, J., Li, Y., Guo, H. F., Xu, P., Liu, L., et al. (2014). Association of GLP-1 secretion with anti-hyperlipidemic effect of ginsenosides in high-fat diet fed rats. Metabolism - Clinical and Experimental, 63(10), 1342–1351.CrossRefGoogle Scholar
  33. 33.
    Shi, B. X., Li, J. C., Yu, A. P., Yuan, B., & Wu, C. S. (2006). Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris. Process Biochemistry, 41(12), 2446–2451.CrossRefGoogle Scholar
  34. 34.
    Ma, B., Tu, P., Zhao, X., Zhang, Y., Wang, Y., Ma, C., Ji, Y., Li, X., Abbas, S. A., & Li, M. (2013). Expression and purification of optimized rolGLP-1, a novel GLP-1 analog, in Escherichia coli BL21(DE3) and its good glucoregulatory effect on type 2 diabetic mice. Current Pharmaceutical Biotechnology, 14(11), 985–994.CrossRefGoogle Scholar
  35. 35.
    Hou, J., Yan, R., Yang, L., Wu, Z., Wang, C., Ding, D., Li, N., Ma, C., & Li, M. (2007). High-level expression of fusion protein containing 10 tandem repeated GLP-1 analogs in yeast Pichia pastoris and its biological activity in a diabetic rat model. Bioscience, Biotechnology, and Biochemistry, 71(6), 1462–1469.CrossRefGoogle Scholar
  36. 36.
    Hou, J., Yan, R., Ding, D., Yang, L., Wang, C., Wu, Z., Yu, X., Li, W., & Li, M. (2007). Oral administration of a fusion protein containing eight GLP-1 analogues produced in Escherichia coli BL21(DE3) in streptozotocin-induced diabetic rats. Biotechnology Letters, 29(10), 1439–1446.CrossRefGoogle Scholar
  37. 37.
    Cen, X., Ni, J., Tan, T., Liu, X., Li, C., Chen, J., Huang, Y., Zhu, S., & Bi, Q. (2006). Investigation on recombinant hirudin via oral route. Peptides, 27(4), 836–840.CrossRefGoogle Scholar
  38. 38.
    Fujita, M., Ito, Y., Hong, K., & Nishimuro, S. (1995). Characterization of nattokinase-degraded products from human fibrinogen or cross-linked fibrin. Fibrinolysis, 9(3), 157–164.CrossRefGoogle Scholar
  39. 39.
    Billings, P. C., St Clair, W. H., Maki, P. A., & Kennedy, A. R. (1992). Distribution of the Bowman Birk protease inhibitor in mice following oral administration. Cancer Letters, 62(3), 191–197.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Zaizhong Ni
    • 1
    • 2
  • Yaofang Zhang
    • 1
    • 2
  • Haisong Wang
    • 1
    • 2
  • Yiming Wei
    • 1
    • 2
  • Baicheng Ma
    • 1
    • 2
  • Junfeng Hao
    • 1
    • 2
  • Peipei Tu
    • 1
    • 2
  • Huikun Duan
    • 1
    • 2
  • Xiaodan Li
    • 1
    • 2
  • Pingzhe Jiang
    • 1
    • 2
  • Xiaofeng Ma
    • 1
    • 2
  • Bin Wang
    • 1
    • 2
  • Ri Wu
    • 1
    • 2
  • Jianhong Zhu
    • 3
  • Minggang Li
    • 1
    • 2
    • 4
    Email author
  1. 1.State Key Laboratory of Medicinal Chemical BiologyNankai UniversityTianjinChina
  2. 2.Key Laboratory for Bioactive Materials of the Ministry of Education, Institute of Molecular Biology, College of Life ScienceNankai UniversityTianjinChina
  3. 3.Department of Preventive MedicineWenzhou Medical UniversityWenzhouChina
  4. 4.Life Science CollegeNankai UniversityTianjinChina

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