亚血红素六肽抗2型糖尿病作用及机理研究
摘要
本实验室设计并合成含His的抗坏血酸过氧化物酶肽类模拟物DhHP-6,其清除过氧化物的活性可达3.9x103U·μmol-1(天然微过氧化物酶MP-11的93%),并经体外细胞实验证明其能够抑制细胞凋亡。Exendin-4是胰高血糖素样肽-1的受体激动剂,能够抵抗DPP-Ⅳ酶水解而具有较长的半衰期,是已上市的治疗2型糖尿病的新药。Exenatide为合成的exendin-4。本实验首先从促进细胞增殖、抗凋亡两方面证明DhHP-6体外对胰岛β细胞具有显著的保护作用。然后将DhHP-6和exenatide进行配比之后作用于胰岛β细胞,经初步实验得到最佳配比并进一步发现两种多肽对促进β细胞增殖具有协同作用。本论文为开发更为有效的治疗2型糖尿病的肽类药物提供了有价值的数据。
Diabetes mellitus (DM) is a serious disease challenging human health. Because of its increasing incidence these years, World Health Organization (WHO) recognized DM as one of the main chronic human diseases to be prevented. DM is a clinical syndrome due to the beta cells of the pancreas being unable to produce sufficient insulin. The main forms of DM are type 1 and type 2. Type 1 is insulin-dependent DM , but type 2 is insulin-independent DM. The main causes and mechanisms of DM disease are beta cell dysfunction, defects in insulin secretion and apoptosis of beta cells resulting from glucose toxicity, fat toxicity and oxidation damage. Patients with DM have the obvious resistance to insulin. Moreover, both type 1 and type 2 diabetes have obvious genetic heterogeneity and are affected by various environmental factors.
Many drugs of T2DM treatment promote insulin secretion from the beta cells of the pancreas. However, this kind of drugs has a great side-effect that long-term treatment will result in insulin over-secretion and thereby loss of beta cell functions.
Beta cell functions of T2DM reduce continuingly with increasing diagnosis years.Whatever any therapy is applied, we can not prevent the continuing reduction of beta cell function later. Thus, a new appropriate therapeutic way is required for beta cell protection.
Exendin-4 has 53 percentage of homology with human GLP-1 and high affinity to GLP-1 receptor. The effect of its reducing blood sugar is as 5530 times as GLP-1. Since it is protected from the hydrolysis of DPP-IV, its half life in blood plasma is 26 minutes. Because exendin-4 can increase the cytoplasm of beta cell and restore its function, it has the unique advantage of treating T2DM.
MP-8, MP-9 and MP-11 from the hydrolysis of cytochrome C (Cyt C) bind to hemachrome covalently and contain a histidine (his) residue in the peptide so that they have effective prevention and cure for various disease due to radical damage. Our research group designed and synthesized an analogue of anti-bad blood acid peroxide enzyme containing his residue, DhHP-6, which has the similar structure of MP from Cyt C. The activity of cleaning peroxides arrives to 3.9x103U·μmol-1 (93% of natural peroxide enzyme, MP-11). Moreover, in vitro experiment showed that it could protect cell from oxidized damage and thereby inhibit cell apoptosis. Thus, DhHP-6 has a potential role and perspective in preventing and curing the disease due to cell function damage.
I used insulinoma cell line, RINm-5F to enhance the activity of cell propagation and anti-apoptosis in this work. It showed that DhHP-6 played a critical role in protecting in vitro beta cells.
First of all, MTT assays were utilized to measure the ability of enhancing beta cells propagation by DhHP-6. It was shown that compared with control group, the number of beta cells after DhHP-6 treatment was significantly increased. Moreover, the increasing percentage of cell propagation was increased with the increase of DhHP-6 concentration. Therefore, DhHP-6 could enhance beta cell propagation and have more effective role than exenatide(synthetic exendin-4).
Secondly, I constructed the model cells to induce the apoptosis of RINm-5F cells by glucose and sodium palmite co-application. MTT assays, flow cytometry and real time-PCR were utilized to test the anti-apoptosis activities of DhHP-6 and exenatide. The results showed that the livability of the model cells was reduced; flow cytometry indicated that the apoptosis peak rate of model cells was increased compared with the control cells; compared to the control cells, the livability of the drug treatment cells was significantly increased and the effect of beta cell protection was increased with the increase of DhHP-6 concentration treatment; compared to the model cells, the apoptosis peak rate of drug treatment cells was reduced to the level of control cells. Thus, DhHP-6 can inhibit the apoptosis induced by glucose and dissociated fatty acids. Moreover, its valid concentration for treatment was lower than exenatide. The resultes of real time-PCR showed that,DhHP-6 and exenatide could preventβ-cell apoptosis induced by FFA and glucose partly through sirt1 and bcl-2 involved Pathway.
DhHP-6 and exenatide could promote the RINm-5F cell propagation and inhibit its apoptosis separately, but underlying different mechanism. This work further studied whether these two peptides had the cooperating effect on treating beta cells.
DhHP-6 and exenatide were mixed to treat the beta cells. The results showed that when exenatide and DhHP-6 was mixed by 4:1(μM), the mixture had the best effect on promoting beta cell propagation. However, when the mixture concentration was 1:2.5(μM), anti-apoptosis activity was best. It was further shown that these two peptides had the cooperating effect on enhancing the beta cell propagation but not on inhibiting the beta cell apoptosis.
The results above indicated that DhHP-6 and exenatide could play a role in protecting beta cells separately. Furthermore, their mixture by certain proportion could also protect beta cells and maybe have cooperating effect on enhangcing beta cells propagation。which could give the contribution to developing the new effective drugs for T2DM.
Diabetes mellitus (DM) is a serious disease challenging human health. Because of its increasing incidence these years, World Health Organization (WHO) recognized DM as one of the main chronic human diseases to be prevented. DM is a clinical syndrome due to the beta cells of the pancreas being unable to produce sufficient insulin. The main forms of DM are type 1 and type 2. Type 1 is insulin-dependent DM , but type 2 is insulin-independent DM. The main causes and mechanisms of DM disease are beta cell dysfunction, defects in insulin secretion and apoptosis of beta cells resulting from glucose toxicity, fat toxicity and oxidation damage. Patients with DM have the obvious resistance to insulin. Moreover, both type 1 and type 2 diabetes have obvious genetic heterogeneity and are affected by various environmental factors.
Many drugs of T2DM treatment promote insulin secretion from the beta cells of the pancreas. However, this kind of drugs has a great side-effect that long-term treatment will result in insulin over-secretion and thereby loss of beta cell functions.
Beta cell functions of T2DM reduce continuingly with increasing diagnosis years.Whatever any therapy is applied, we can not prevent the continuing reduction of beta cell function later. Thus, a new appropriate therapeutic way is required for beta cell protection.
Exendin-4 has 53 percentage of homology with human GLP-1 and high affinity to GLP-1 receptor. The effect of its reducing blood sugar is as 5530 times as GLP-1. Since it is protected from the hydrolysis of DPP-IV, its half life in blood plasma is 26 minutes. Because exendin-4 can increase the cytoplasm of beta cell and restore its function, it has the unique advantage of treating T2DM.
MP-8, MP-9 and MP-11 from the hydrolysis of cytochrome C (Cyt C) bind to hemachrome covalently and contain a histidine (his) residue in the peptide so that they have effective prevention and cure for various disease due to radical damage. Our research group designed and synthesized an analogue of anti-bad blood acid peroxide enzyme containing his residue, DhHP-6, which has the similar structure of MP from Cyt C. The activity of cleaning peroxides arrives to 3.9x103U·μmol-1 (93% of natural peroxide enzyme, MP-11). Moreover, in vitro experiment showed that it could protect cell from oxidized damage and thereby inhibit cell apoptosis. Thus, DhHP-6 has a potential role and perspective in preventing and curing the disease due to cell function damage.
I used insulinoma cell line, RINm-5F to enhance the activity of cell propagation and anti-apoptosis in this work. It showed that DhHP-6 played a critical role in protecting in vitro beta cells.
First of all, MTT assays were utilized to measure the ability of enhancing beta cells propagation by DhHP-6. It was shown that compared with control group, the number of beta cells after DhHP-6 treatment was significantly increased. Moreover, the increasing percentage of cell propagation was increased with the increase of DhHP-6 concentration. Therefore, DhHP-6 could enhance beta cell propagation and have more effective role than exenatide(synthetic exendin-4).
Secondly, I constructed the model cells to induce the apoptosis of RINm-5F cells by glucose and sodium palmite co-application. MTT assays, flow cytometry and real time-PCR were utilized to test the anti-apoptosis activities of DhHP-6 and exenatide. The results showed that the livability of the model cells was reduced; flow cytometry indicated that the apoptosis peak rate of model cells was increased compared with the control cells; compared to the control cells, the livability of the drug treatment cells was significantly increased and the effect of beta cell protection was increased with the increase of DhHP-6 concentration treatment; compared to the model cells, the apoptosis peak rate of drug treatment cells was reduced to the level of control cells. Thus, DhHP-6 can inhibit the apoptosis induced by glucose and dissociated fatty acids. Moreover, its valid concentration for treatment was lower than exenatide. The resultes of real time-PCR showed that,DhHP-6 and exenatide could preventβ-cell apoptosis induced by FFA and glucose partly through sirt1 and bcl-2 involved Pathway.
DhHP-6 and exenatide could promote the RINm-5F cell propagation and inhibit its apoptosis separately, but underlying different mechanism. This work further studied whether these two peptides had the cooperating effect on treating beta cells.
DhHP-6 and exenatide were mixed to treat the beta cells. The results showed that when exenatide and DhHP-6 was mixed by 4:1(μM), the mixture had the best effect on promoting beta cell propagation. However, when the mixture concentration was 1:2.5(μM), anti-apoptosis activity was best. It was further shown that these two peptides had the cooperating effect on enhancing the beta cell propagation but not on inhibiting the beta cell apoptosis.
The results above indicated that DhHP-6 and exenatide could play a role in protecting beta cells separately. Furthermore, their mixture by certain proportion could also protect beta cells and maybe have cooperating effect on enhangcing beta cells propagation。which could give the contribution to developing the new effective drugs for T2DM.
引文
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[2] R.P. Robertson, J.S. Harmon .Free Radical Biology & Medicine .2006,41:177 -184
[3] Meneghini L. Why and how to use insulin therapy earlier in the management of type 2 diabetes. South Med J., 2007, 100(2):164-174
[4] FedericiM , H ribalM , Perego L , et al. High glucose causes apoptosis in cultured human pancreatic islets of langerhans. Diabetes, 2001, 50: 1290-1301
[5] J.Chen,F.M.Couto,A.H.Minn,A.Shalev,Exenatide inhibits β-cell apoptosis by decreasing thioredoxin-interacting protein, Biochem. Biophys. Res.Commun. 2006,346:1067-1074
[6] P. Christian Schulze, Jun Yoshioka, et al . J Biol Chem 2004,279(29): 30369-30374
[7] Tajiri Y, Grill V. Aminoguanidine exerts a beta-cell function preserving effect in high glucose cultured beta-cells ( INS-1) . Nit J Exp Diabetes Res ,2000 , 1 : 111-119
[8] 惠晓丽,王惠芳,姚孝礼. β细胞凋亡与2 型糖尿病[J]. 国外医学·内分泌学分册, 2005, 25 ( 1) : 22 -25.118-121
[9] Santangelo C, Matarrese P, Masella R, et al. Hepatocyte growth factor protects rat RINm5F cell line against free fatty acid-induced apoptosis by counteracting oxidative stress. J Mol Endocrinol, 2007, 38(1-2):147-158
[10] Poitout,Vincent, Robertson, et al. Secondary β-cell failure in type 2 diabetes-a convergence of glucotoxicity and lipotocicity[J]. Endocrinology, 2002, 143 (2):339
[11] Unger R H,Zhou Y T.Lipotoxicity of beta-cells in obesity and other causes of fatty acid spill over[J].Diabetes,2001,50(Suppl 1):S118-S121
[12] Lin K,Paterson A J,Chin E,et al.Glucose stimulates protein modification by O-linked GlcNAc in pancreatic β-cell:Link-age of O-linked GlcNAc toβ-cell death[J].Proc Natl Acad Sci USA,2000,97:2820-2825
[13] 孙国建, 李淑翠.游离脂肪酸与胰岛素抵抗、β细胞功能减退关系的研究进展.滨州医学院学报.2004,27(1):34-36
[14] R. Paul Robertson, Jamie S. Harmon. Pancreatic islet b-cell and oxidative stress: The importance ofglutathione peroxidase. FEBS,2007
[15] Desmond Jay, Hirofumi Hitomi, Kathy K. Griendling. Oxidative stress and diabetic cardiovascular complications. Free Radical Biology & Medicine 2006,40:183 -192
[16] Kaneto.H, Kajimoto. Y, et al. Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity. Diabetes 1999,48:2398-2406
[17] 李丙蓉,邓华聪 2 型糖尿病的病理生理机制 医学新知杂志2004年第14卷第2期
[18] Gerich , John E M D. Contributions insulin resistance and insulin secretory defects to the pathogenesis of type 2 diabetes mellitus. Mayo Clinic Proceedings , 2003 ,78(4) :447
[19] Louise A, Scrocchi Bess A, Marshall Sonya M, et al. Identification of glucagon-like peptide 1(GLP-1)actions essential for glucose homeostasis in mice with disruption of glp-1 receptor signaling. Diabetes, 1998, 47:632
[20] Orskov C, Rabenhoj L, Wettergren A, et al. Tissue and plasma concentrations of amidated and glycine-extented glucagon-like peptide 1 in humans. Diabetes, 1994, 43:535-539
[21] Mojsov S, Heinrich G, Wilson IB, Ravazzola M, Orci L, Habener JF: Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing. J Biol Chem,1986, 261:11880-11889
[22] Kreymann B, Williams G, Ghatei MA, Bloom SR: Glucagon like peptide 1 (7-36): a physiological incretin in man. Lancet,1987, ii:1300-1303
[23] C. Mark B. Edwards, Jeannie F. Todd,et al. Glucagon-Like Peptide 1 Has a Physiological Role in the Control of Postprandial Glucose in Humans. Diabetes, 1999,48:86-93
[24] Kieffer TJ, Habener JF. The glucagon-like peptides. Endocr Rev. 999, 20: 876-913
[25] Schirra J, Katschinski M, Weidmann C, et al. Gastric emptying and release of incretin homones after glucose ingestion in humans. J Clin Invest, 1996, 97: 91-103
[26] Gromada J, dissing S, Bokrist K, et al. Glucagon-lke peptide 1 increase cytoplasmic calcium in insulin-secreting β TC3-cells by enhancement of intracellur calcium mobilization. Diabetes, 1995, 44: 767-774
[27] Stoffel, M., Espinosa, R., III, Le Beau, M. M., & Bell, G. I. Human glucagon-like peptide-1 receptor gene. Localization to chromosome band 6p21 by fluorescence in situ hybridization and linkage of a highly polymorphic simple tandem repeat DNA polymorphism to other markerson chromosome 6. Diabetes 1993,42:1215-1218
[28] Haar EV, Lee SI, Band,hakavi S, Griffin TJ, Kim DH. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol, 2007, 9(3):316-323
[29] Buteau, J., Roduit, R., Susini, S., Prentki, M. Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increase stranscription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia 1999,42:856-864
[30] Yaney, G. C., Civelek, V. N., Richard, A. M., Dillon, J. S., Deeney, J. T.,Hamilton, J. A., et al. (2001). Glucagon-like peptide 1 stimulates lipolysis inclonal pancreatic beta-cells (HIT). Diabetes 2001,50:56-62
[31] Máire E. Doyle , Josephine M. Egan c,Mechanisms of action of glucagon-like peptide 1 in the pancreas .Pharmacology & Therapeutics (2007)
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