The Physiological Activities Study Of Insulinotropic Peptides From Three Amphibian Skins

Insulinotropic peptides are a series of bioactive peptides which can regulate the insulin secretion of isletβ-cells.The investigation of their structural characterization, physiological activities and clinical application provides new treating remedy and novel therapy for typeâ…¡diabetic patient.Synchronously,insulinotropic toxins can be used as molecular probes to study the signal regulation of insulin secretion,as well as toxicological characteristic of some toxins.Today,the pharmacological action and clincal medication studies of glucagon like peptide-1(GLP-1) and its analogs are greatly focused and proceed rapidly,and many insulinotropic peptides have been identified from the toxins of insects,amphibians and reptiles by foreign scientists.However,in our country,few research work has been done to isolate and characterize insulinotropic peptides from natural animals,yet.Abound biological resources in our country award us great advantage to explore this research field, therefore,the job to exploit the methods and techniques of purifying insulinotropic peptides is of great importance and long-term sense.Three species of frogs(Odorrana grahami,Amolops loloensis and Rana nigrovittata), collected in Yunnan province,were used as experimental animals for this thesis.Skin secretions were purified by Sephadex G-50 gel filtertion and reverse phase high performance liquid chromatography(RP-HPLC),and more than ten peaks with insulin-releasing activity were obtained Fast atom bombardment(FAB) mass spectrometry was carried out to detect their molecular weights.Complete peptide sequencing was undertaken by Edman degradation on an Applied Biosystem spulsed liquid-phase sequencer,and eight amino acid sequences were obtained and respectively named as odorranain-G(13 amino-peptide),odorranain-L1(21 amino-peptide), odorranain-X1(16 amino-peptide),odorranain-X2(16 amino-peptide),odorranain-Y(11 amino-peptide),odorranain-Z(20 amino-peptide),amolopin(16 amino-peptide) and nigroain-A(16 amino-peptide).Directional cDNA library was constructed with plasmid cloning kit,and gene translations from cDNA clones matched well with the results of Edman degradation sequencing.To test insulin-releasing property of the purified peptides, primary cultured rat islets and INS-1 insulinoma cell lines were both used,with insulin concentrations determined by radioimmunoassay.While invesgating the stimulatory mechanism of excytosis,[Ca²⁺]_i ofβ-cell was measured with Fluo-3 and laser confocal microscope.However,no evident Ca²⁺influx increase revealed that the stimulation of insulin secretion probably not via activation of Ca²⁺ channel.Hither,we identified the antimicrobial activity of odorranain-G and predicted its exellent potential to treat diabetes associated complications.Odorranain-Z was also characterized as a double-functional peptide,and its anti-angiogenesis action may be used to cure proliferative diabetic retinopathy(PDR).In conclusion,our research firstly build up the technology of isolation,structural identification and physiological testing of insulinotropic peptides from natural amphibian animals.The primary structures of eight novel insulinotropic peptides with multiple bioactivities were successfully elucidated,which were significantly different from other known.Thereinto,six isolated peaks are purified from Odorrana grahami,indicating the skin secretion of this species is a precious biological resource.Odorranain-Y is merely a 11 amino-acid peptide,which is the smallest insulinotropic peptide.The pharmacological actions and physiological properties investigation of insulinotropic peptides leads to new antidiabetic therapy.