VPAC1受体对能量平衡作用的初探及其重组激动剂的高效制备
摘要
目的：VPAC1受体是神经多肽PACAP与VIP的共同受体，介导多种重要的生物学功能。本文利用VPAC1受体特异激动剂，采用高脂高糖制备的肥胖小鼠模型，开展VPAC1受体参与小鼠能量代谢平衡调节的功能研究；并在细胞水平对VPAC1受体调节能量代谢的机制进行初步探索，最后利用基因工程原理和技术实现重组VPAC1特异激动剂的高效制备，为VPAC1受体及其激动剂的深入开发与应用奠定基础。
方法：首先利用化学合成的VPAC1特异激动剂，研究VPAC1受体参与小鼠能量代谢的调节作用：高脂高糖喂养小鼠，制备肥胖模型，通过测定体重、脂肪、血糖耐受、血脂等指标检测VPAC1特异激动剂抑制肥胖及其对肥胖小鼠的减肥功能。为研究VPAC1调节能量代谢的作用机制，Westernblot检测VPAC1受体在小鼠3T3-L1前脂肪细胞及成熟脂肪细胞的表达情况，油红O染色检测VPAC1特异激动剂对小鼠3T3-L1前脂肪细胞分化的作用，并利用分离的小鼠脂肪细胞检测VPAC1受体特异激动剂对脂肪分解的作用。依照已有的VPAC1受体特异激动剂序列，PCR两步法合成VPAC1特异激动剂基因，利用IMPACT(Intein Mediated Purification with an Affinity Chitin-bindingTag)蛋白表达纯化系统，构建工程菌pTWIN-VPAC1-ER2566，并实现目的多肽单柱高效纯化。
结果：利用VPAC1受体特异激动剂，对VPAC1受体对肥胖小鼠能量代谢平衡调节作用的研究发现：与对照组相比，VPAC1特异激动剂有效降低小鼠体重，减少小鼠体内脂肪的积存，提高小鼠对糖的应激力，增强小鼠的葡萄糖耐受，降低小鼠的血糖血脂，显示VPAC1特异激动剂具有显著的抑制肥胖及促使肥胖小鼠减肥的功能。在细胞水平的作用机制研究发现：3T3-L1前脂肪细胞分化为脂肪细胞后，VPAC1受体表达量显著提高；VPAC1特异激动剂不仅促进3T3-L1前脂肪细胞向脂肪细胞分化，而且有效促进小鼠原代脂肪细胞的脂肪分解。最后本文还成功构建了工程菌pTWIN-VPAC1-ER2566，利用IMPACT系统实现1g发酵菌体收获0.2mg纯度超过95％的重组VPAC1受体特异激动剂，重组多肽经SDS-PAGE及激光飞行质谱鉴定与化学合成标准品分子量相同。
结论：本研究首次利用高脂高糖肥胖小鼠模型研究VPAC1受体参与调节能量代谢的作用，研究结果显示VPAC1受体特异激动剂具有显著的抑制肥胖及促进肥胖小鼠减肥的作用。对其作用机制的细胞水平的深入研究表明VPAC1受体在分化的3T3-L1脂肪细胞内特异高效表达，并且VPAC1受体特异激动剂有效促进脂肪细胞的分化及脂肪细胞中脂肪的分解。本研究还通过基因工程技术实现了重组的VPAC1特异激动剂的高效制备，为VPAC1受体生物学功能的深入研究及VPAC1特异激动剂的开发应用奠定了基础。通过同源建模建立了结合状态的特异激动剂多肽和VPAC1受体N端144个氨基酸的三级结构，并初步分析了受体与激动剂的结合区域。
Objetive: VPAC1 receptor is co-receptor of neural polypeptide PACAP and VIP,mediates lots of important biological functions. The regulation function of VPAC1 onthe energy metabolism was researched in this paper, utilizing the classic VPAC1receptor agonist and the alimentary obesity mouse model made by high calory feeding.The mechanism of these effects mediated by VPAC1 was explored tentatively atcellular level. Finally, high-efficient preparation of recombination VPAC1 agonist wasaccomplished,using genetic engineering principle and technology, which laid thefoundation for the further study and development of the biology function of VPAC1receptor and its agonist.
Method: First, the role of VPAC1 receptor in energy metabolism was researched withthe chemosynthesis VPAC1 agonist. The alimentary obesity mouse model wasestablished with high calory feeding, then the effects of VPAC1 agonist on theinhibition and curation of obesity were studyed by measuring the followingindexs:,weight, fat wet weight, total cholesterol (TC), triglyceride (TG), bloodglucose, low-density lipoprotein (LDL)of mice. To research the mechanism ofregulation function in energy metabolism, the expression VPAC1 receptor in 3T3-L1preadipocyte and adipocytes was determined. The role of VPAC1 agonist indifferentiation of 3T3-L1 from preadipocyte to adipocytes was determined by oil redO stain. Furthermore, the lipolytic effect of VPAC1 agonist in primary rat adipocyteswas detected. Finally, according to the VPAC1 agonist sequence,the gene coding theVPAC1 agonist was synthesized by two steps PCR. Engineering bacteriapTWIN-VPAC1-ER2566 was established,and high-efficient preparation of VPAC1agonist was accomplished with the IMPACT(Intein Mediated Purification with anAffinity Chitin-bindingTag) protein expressiong and purification system.
Results: Compared with the dissolvant control, VPAC1 agonist efficiently reducedthe mice weight and the fat accumulation in vivo, raised the tolerance for highglucose and enhance the glucose tolerance of mouse, and decreased blood glucose andblood fat of mouse. The results reveal that VPAC1 agonist played an important role inobesity inhibition and curation. Through the cellular experimens we found that, theexpression of VPAC1 receptor elevated obviously while the 3T3-L1 preadipocytecells were differentiated to adipocytes. VPAC1 agonist promoted the differentiation effect of 3T3-L1 cells from preadipocyte to adipocytes, and promoted the lipolyticeffect in primary rat adipocytes too.At last, Engineering bacteriapTWIN-VPAC1-ER2566 was established, and 0.2mg recombination VPAC1 agonistwith the purity of more than 95% was obtained from 1g fermentation product withIMPACT system. The molecular wieght of recombination VPAC1 agonist is proved thesame to standard substance by SDS-PAGE and mass spectrum.
Conclusion: It was the first time that the role of VPAC1 receptor in energy metabolismwas researched utilizing the alimentary obesity mouse model, and the results indicatedthat VPAC1 agonist play an important role in obesity inhibition and curation. Thefurther cellular level research found that the expression of VPAC1 receptor elevatedobviously in 3T3-L1 cells after the preadipocyte cells were differentiated toadipocytes. VPAC1 agonist promoted the differentiation of 3T3-L1 from preadipocyteto adipocytes, and promoted the lipolytic effects in primary rat adipocytes too.High-efficient preparation of recombination VPAC1 agonist was accomplished withgenetic engineering principle and technology, which laid the foundation for the furtherstudy and development of the biology function of VPAC1 receptor and its agonist.The modeling structure of VPAC1 receptor N-terminal domain (residues 1-144) andVPAC1 agonist was performed by Homology module, and binding region wasanalyzed based on the model.
方法：首先利用化学合成的VPAC1特异激动剂，研究VPAC1受体参与小鼠能量代谢的调节作用：高脂高糖喂养小鼠，制备肥胖模型，通过测定体重、脂肪、血糖耐受、血脂等指标检测VPAC1特异激动剂抑制肥胖及其对肥胖小鼠的减肥功能。为研究VPAC1调节能量代谢的作用机制，Westernblot检测VPAC1受体在小鼠3T3-L1前脂肪细胞及成熟脂肪细胞的表达情况，油红O染色检测VPAC1特异激动剂对小鼠3T3-L1前脂肪细胞分化的作用，并利用分离的小鼠脂肪细胞检测VPAC1受体特异激动剂对脂肪分解的作用。依照已有的VPAC1受体特异激动剂序列，PCR两步法合成VPAC1特异激动剂基因，利用IMPACT(Intein Mediated Purification with an Affinity Chitin-bindingTag)蛋白表达纯化系统，构建工程菌pTWIN-VPAC1-ER2566，并实现目的多肽单柱高效纯化。
结果：利用VPAC1受体特异激动剂，对VPAC1受体对肥胖小鼠能量代谢平衡调节作用的研究发现：与对照组相比，VPAC1特异激动剂有效降低小鼠体重，减少小鼠体内脂肪的积存，提高小鼠对糖的应激力，增强小鼠的葡萄糖耐受，降低小鼠的血糖血脂，显示VPAC1特异激动剂具有显著的抑制肥胖及促使肥胖小鼠减肥的功能。在细胞水平的作用机制研究发现：3T3-L1前脂肪细胞分化为脂肪细胞后，VPAC1受体表达量显著提高；VPAC1特异激动剂不仅促进3T3-L1前脂肪细胞向脂肪细胞分化，而且有效促进小鼠原代脂肪细胞的脂肪分解。最后本文还成功构建了工程菌pTWIN-VPAC1-ER2566，利用IMPACT系统实现1g发酵菌体收获0.2mg纯度超过95％的重组VPAC1受体特异激动剂，重组多肽经SDS-PAGE及激光飞行质谱鉴定与化学合成标准品分子量相同。
结论：本研究首次利用高脂高糖肥胖小鼠模型研究VPAC1受体参与调节能量代谢的作用，研究结果显示VPAC1受体特异激动剂具有显著的抑制肥胖及促进肥胖小鼠减肥的作用。对其作用机制的细胞水平的深入研究表明VPAC1受体在分化的3T3-L1脂肪细胞内特异高效表达，并且VPAC1受体特异激动剂有效促进脂肪细胞的分化及脂肪细胞中脂肪的分解。本研究还通过基因工程技术实现了重组的VPAC1特异激动剂的高效制备，为VPAC1受体生物学功能的深入研究及VPAC1特异激动剂的开发应用奠定了基础。通过同源建模建立了结合状态的特异激动剂多肽和VPAC1受体N端144个氨基酸的三级结构，并初步分析了受体与激动剂的结合区域。
Objetive: VPAC1 receptor is co-receptor of neural polypeptide PACAP and VIP,mediates lots of important biological functions. The regulation function of VPAC1 onthe energy metabolism was researched in this paper, utilizing the classic VPAC1receptor agonist and the alimentary obesity mouse model made by high calory feeding.The mechanism of these effects mediated by VPAC1 was explored tentatively atcellular level. Finally, high-efficient preparation of recombination VPAC1 agonist wasaccomplished,using genetic engineering principle and technology, which laid thefoundation for the further study and development of the biology function of VPAC1receptor and its agonist.
Method: First, the role of VPAC1 receptor in energy metabolism was researched withthe chemosynthesis VPAC1 agonist. The alimentary obesity mouse model wasestablished with high calory feeding, then the effects of VPAC1 agonist on theinhibition and curation of obesity were studyed by measuring the followingindexs:,weight, fat wet weight, total cholesterol (TC), triglyceride (TG), bloodglucose, low-density lipoprotein (LDL)of mice. To research the mechanism ofregulation function in energy metabolism, the expression VPAC1 receptor in 3T3-L1preadipocyte and adipocytes was determined. The role of VPAC1 agonist indifferentiation of 3T3-L1 from preadipocyte to adipocytes was determined by oil redO stain. Furthermore, the lipolytic effect of VPAC1 agonist in primary rat adipocyteswas detected. Finally, according to the VPAC1 agonist sequence,the gene coding theVPAC1 agonist was synthesized by two steps PCR. Engineering bacteriapTWIN-VPAC1-ER2566 was established,and high-efficient preparation of VPAC1agonist was accomplished with the IMPACT(Intein Mediated Purification with anAffinity Chitin-bindingTag) protein expressiong and purification system.
Results: Compared with the dissolvant control, VPAC1 agonist efficiently reducedthe mice weight and the fat accumulation in vivo, raised the tolerance for highglucose and enhance the glucose tolerance of mouse, and decreased blood glucose andblood fat of mouse. The results reveal that VPAC1 agonist played an important role inobesity inhibition and curation. Through the cellular experimens we found that, theexpression of VPAC1 receptor elevated obviously while the 3T3-L1 preadipocytecells were differentiated to adipocytes. VPAC1 agonist promoted the differentiation effect of 3T3-L1 cells from preadipocyte to adipocytes, and promoted the lipolyticeffect in primary rat adipocytes too.At last, Engineering bacteriapTWIN-VPAC1-ER2566 was established, and 0.2mg recombination VPAC1 agonistwith the purity of more than 95% was obtained from 1g fermentation product withIMPACT system. The molecular wieght of recombination VPAC1 agonist is proved thesame to standard substance by SDS-PAGE and mass spectrum.
Conclusion: It was the first time that the role of VPAC1 receptor in energy metabolismwas researched utilizing the alimentary obesity mouse model, and the results indicatedthat VPAC1 agonist play an important role in obesity inhibition and curation. Thefurther cellular level research found that the expression of VPAC1 receptor elevatedobviously in 3T3-L1 cells after the preadipocyte cells were differentiated toadipocytes. VPAC1 agonist promoted the differentiation of 3T3-L1 from preadipocyteto adipocytes, and promoted the lipolytic effects in primary rat adipocytes too.High-efficient preparation of recombination VPAC1 agonist was accomplished withgenetic engineering principle and technology, which laid the foundation for the furtherstudy and development of the biology function of VPAC1 receptor and its agonist.The modeling structure of VPAC1 receptor N-terminal domain (residues 1-144) andVPAC1 agonist was performed by Homology module, and binding region wasanalyzed based on the model.
引文
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51. Goetzl EJ. et al. Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide(VIP). Proc Natl Acad Sci U S A. 2001 Nov 20; 98(24): 13854-13859.
52．汪家政，范明．《蛋白技术手册》，科学出版社，第三章，“蛋白浓度测定”，p43．
53. Ntambi JM, Kim YC. Adipocpte differentiation and geneexpression[J]. J Nutr,2000, 130(12): 31225-31265.
54. Manami Tsutsumi, Thomas H. Claus, Yin Liang, Yaxin Li, Ling Yang, Jian Zhu, et al.A Potent and Highly Selective VPAC2 Agonist Enhances Glucose-Induced Insulin Release and Glucose Disposal.Diabetes. 2002 May;51(5): 1453-60.
55. PHILIPPE GOURLET, ANDRE VANDERMEERS, PASCALE VERTONGEN, JEAN RATHE,PHILIPPE DE NEEF, JOHNNY CNUDDE, MAGALI WAELBROECK AND PATRICK ROBBERECHT.Development of High Affinity Selective V1P1 Receptor Agonists.Peptides, Vol. 18, No. 10, pp. 1539-1545, 1997
56. ALANINE SCANNING AND MOLECULAR MODELING OF THE PEPTIDE.Identification of Key Residues for Interaction of Vasoactive Intestinal Peptide with Human VPAC1 and VPAC2 Receptors and Development of a Highly Selective VPAC1 Receptor Agonist.THE JOURNALOF BIOLOGICAL CHEMISTRY.Vol. 275, No. 31, Issue of August 4, pp. 24003-24012, 2000.
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11. Kamaishi H, Endoh T, Suzuki TMultiple signal pathways coupling VIP and PACAP receptors to calcium channels in hamster submandibular ganglion neurons. Auton Neurosci. 2004 Mar 31;111(1):15-26.
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14. Mazzocchi G, Malendowicz LK, Neri G, Andreis PG, Ziolkowska A, Gottardo L, Nowak KW, Nussdorfer GG. Pituitary adenylate cyclase-activating polypeptide and PACAP receptor expression and function in the rat adrenal gland. Int J Mol Med. 2002 Mar;9(3): 233-43.
15. Sacchetti B, Lorenzini CA, Baldi E, Bucherelli C, Roberto M, Tassoni G, Brunelli M Pituitary adenylate cyclase-activating polypeptide hormone (PACAP) at very low dosages improves memory in the rat. Neurobiol Learn Mem. 2001 Jul;76(1):1-6.
16. Hashimoto H, Shintani N, Tanaka K, Mori W, Hirose M, Matsuda T, Sakaue M, Miyazaki J, Niwa H, Tashiro F, Yamamoto K, Koga K, Tomimoto S, Kunugi A, Suetake S, Baba A Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) Proc Natl Acad Sci U S A. 2001 Nov 6; 98(23): 13355-60. Epub 2001 Oct 30..
17. Yuhara A, Nishio C, Abiru Y, Hatanaka H, Takei N. PACAP has a neurotrophic effect on cultured basal forebrain cholinergic neurons from adult rats. Brain Res Dev Brain Res. 2001 Nov 26;131(1-2):41-5.
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19．董艳，何成，韩晞，垂体腺苷酸环化酶激活肽减轻大鼠缺血性脑水肿作用的实验研究，中国应用生理学杂志，2002，18(2)：121-31．
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22. Cazillis M, Gonzalez BJ, Billardon C, Lombet A, Fraichard A, Samarut J, Gressens P, Vaudry H, Rostene W. VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells. Eur J Neurosci. 2004 Feb;19(4):798-808.
23. Chang Q, Deng Y P, Liu S H, Morphologic study on PACAP protecting vascular smooth muscle cell from damage by LDL. Progress of Anatomical Sciences 1999; 5: 347-349.
24. Linden A, Hansson, L Andersson A, Palmqvist M, Arvidsson P, Lofdahl C-G, Larsson P, Lotvall J. Bronchodilation by an inhaled VPAC2 receptor agonist in patients with stable asthma. Thorax 2003;58:217-221. (http://thorax.bmjjournals.com/cgi/content/full/58/3/217)
25. West AP, McKinnell C, Sharpe RM, Saunders PT. Pituitary adenylate cyclase activating polypeptide can regulate testicular germ cell protein synthesis in vitro. J Endocrinol. 1995 Feb;144(2):215-23.
26. Guidone G, Muller D, Vogt K, Mukhopadhyay AK. Characterization of VIP and PACAP receptors in cultured rat penis corpus cavemosum smooth muscle cells and their interaction with guanylate cyclase-B receptors. Regul Pept. 2002 Oct 15;108(2-3):63-72.
27. Ko C, In Y H, Park-Sarge O K. Role of progesterone receptor activation in pituitary adenylate cyclase activating polypeptide gene expression in rato vary. Endocrinology, 1999,140(11):5185-5194.
28. Delgado M, Abad C, Martinez C, Juarranz MG, Leceta J, Ganea D, Gomariz RP. PACAP in immunity and inflammation. Ann N Y Acad Sci. 2003 May;992:141-57.
29. Abad C, Martinez C, Leceta J, Gomariz RP, Delgado M. Pituitary adenylate cyclase-activating polypeptide inhibits collagen-induced arthritis: an experimental immunomodulatory therapy. J Immunol. 2001 Sep 15;167(6):3182-9.
30. Martinez C, Abad C, Delgado M, Arranz A, Juarranz MG, Rodriguez-Henche N, Brabet P, Leceta J, Gomariz RP. Anti-inflammatory role in septic shock of pituitary adenylate cyclase-activating polypeptide receptor. Proc Nad Acad Sci U S A. 2002 Jan 22;99(2): 1053-8. Epub 2002 Jan 15.
31. Pozo D. VIP- and PACAP-mediated immunomodulation as prospective therapeutic tools. Trends Mol Med. 2003 May;9(5):211-7.
32. Hannibal J, Hindersson P, Knudsen SM, Georg B, Fahrenkrug J. The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptide-containing retinal ganglion cells of the retinohypothalamic tract. J Neurosci. 2002 Jan 1;22(1):RC191.
33. Akesson L, Ahren B, Manganiello VC, Hoist LS, Edgren G, Degerman E. Dual effects of pituitary adenylate cyclase-activating polypeptide and isoproterenol on lipid metabolism and signaling in primary rat adipocytes. Endocrinology. 2003 Dec;144(12):5293-9. Epub 2003 Aug 28.
34. Masanori Nakata, Seiji Shioda, Yoshitomo Oka, Ikuro Maruyama, Toshihiko Yada, Insulinotropin PACAP potentiates insulin-stimulated glucose uptake in 3T3 L1 cells. Peptides 1999 20 ::943-948.
35. Nakata M, Yada T. Physiological and therapeutic roles of PACAP in glucose metabolism and diabetes, Nippon Yakurigaku Zasshi. 2004 ;123(4):267-273.
36. Masanori Nakata, Daisuke Kohno, Norihito Shintani, Yuriko Nemoto,Hitoshi Hashimoto, Akemichi Baba, Toshihiko Yada,PACAP deficient mice display reduced carbohydrate intake and PACAP activates NPY-containing neurons in the rat hypothalamic arcuate nucleus. Neuroscience Letters 2004,370:252-256.
37. Sarah L. Gray, Kevin J. Cumming, Frank R. Jirik and Nancy M. Sherwood,Targeted Disruption of the Pituitary Adenylate Cyclase-Activating Polypeptide Gene Results in Early Postnatal Death Associated with Dysfunction of Lipid and Carbohydrate Metabolism, Molecular Endocrinology 15 (10): 1739-1747 2001.
38. Akesson L, Ahren B, Manganiello VC, Hoist LS, Edgren G, Degerman E. Dual Effects of Pituitary Adenylate Cyclase-Activating Polypeptide and Isoproterenol on Lipid Metabolism and Signaling in Primary Rat Adipocytes , Endocrinology. Endocrinology. 2003 Dec;144(12):5293-5299.
39. Laurence Lins, Alain Couvineau, Christiane Rouyer-Fessard, Pascal Nicole,et al.The Human VPAC1 Receptor.THE JOURNAL OF BIOLOGICAL CHEMISTRY.Vol. 276, No. 13, Issue of March 30, pp. 10153-10160, 2001.
40. Tamakawa H, Miyata A, Satoh K, et al.The augmentation of pituitary adenylate cyclase-activating polypeptide(PACAP) in streptozotocin-induced diabeticrats [J] Peptide ,1998 ,19:1497-1502.
41. Kojima M, Ito T, Oono T, et al.VIP attenuation of the severity of experimental pancreatitis is due to VPAC1 receptor-mediated inhibition of cytokine production.Pancreas. 2005 Jan;30(1):62-70.
42. M R Nicol, V J Cobb, B C Williams, S D Morley, S W Walker and J I Mason.Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical turnout cell line via VPAC1 receptors Journal of Molecular Endocrinology (2004) 32, 869-877.
43. Branch DR, Valenta LJ, Yousefi S, Sakac D, Singla R, Bali M, Sahai BM, Ma XZ. VPAC1 is a cellular neuroendocrine receptor expressed on T ceils that actively facilitates productive HIV-1 infection. AIDS. 2002 Feb 15;16(3): 309-19.
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46. Delgado M, Reduta A, Sharma V, et al. VIP/PACAP oppositely affects immature and mature dendritic cell expression of CD80/CD86 and the stimulatory activity for CD4(+) T cells[J]. J Leukoc Bio, 2004, 75(6): 1122-1130.
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48. Delgado M, Ganea D. VIP and PACAP enhance the in vivo generation of memory Th2 cells by inhibiting peripheral deletion of antigen2specific effectors[J]. Arch Physiol Biochem,2001,109(4): 372-376.
49. Voice J, Donnelly S ,Dorsam G, et al. c-Maf and J unB Mediation of Th2 Differentiation Induced by the Type 2 G Protein2Coupled Receptor (VPAC2) for vasoactive Intestinal peptide [J]. J Immunol, 2004, 172 (12): 7289-7296.
50. Voice J K, Grinninger C, Kong Y, et al. Roles of vasoactive intestinal peptide (VIP) in the expression of different immune phenotypes by wild Ⅱ type mice and T cell-targeted type Ⅱ VIP receptor transgenicmice[J]. J Immunol, 2003,17 (1): 308-314.
51. Goetzl EJ. et al. Enhanced delayed-type hypersensitivity and diminished immediate-type hypersensitivity in mice lacking the inducible VPAC2 receptor for vasoactive intestinal peptide(VIP). Proc Natl Acad Sci U S A. 2001 Nov 20; 98(24): 13854-13859.
52．汪家政，范明．《蛋白技术手册》，科学出版社，第三章，“蛋白浓度测定”，p43．
53. Ntambi JM, Kim YC. Adipocpte differentiation and geneexpression[J]. J Nutr,2000, 130(12): 31225-31265.
54. Manami Tsutsumi, Thomas H. Claus, Yin Liang, Yaxin Li, Ling Yang, Jian Zhu, et al.A Potent and Highly Selective VPAC2 Agonist Enhances Glucose-Induced Insulin Release and Glucose Disposal.Diabetes. 2002 May;51(5): 1453-60.
55. PHILIPPE GOURLET, ANDRE VANDERMEERS, PASCALE VERTONGEN, JEAN RATHE,PHILIPPE DE NEEF, JOHNNY CNUDDE, MAGALI WAELBROECK AND PATRICK ROBBERECHT.Development of High Affinity Selective V1P1 Receptor Agonists.Peptides, Vol. 18, No. 10, pp. 1539-1545, 1997
56. ALANINE SCANNING AND MOLECULAR MODELING OF THE PEPTIDE.Identification of Key Residues for Interaction of Vasoactive Intestinal Peptide with Human VPAC1 and VPAC2 Receptors and Development of a Highly Selective VPAC1 Receptor Agonist.THE JOURNALOF BIOLOGICAL CHEMISTRY.Vol. 275, No. 31, Issue of August 4, pp. 24003-24012, 2000.