The Effect Of VPAC1 On Energy Homeostasis And The Effective Preparation Of The Recombinant Agonist Of VPAC1

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.