Isolation of a new chicken pancreatic hormone (glucagon like peptide) and its physiological functions

• Project/Area Number: 05660328
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Applied animal science
• Research Institution: Kobe university
• Principal Investigator: HASEGAWA Shin Kobe university, Graduate school of science and technology, Professor, 大学院・自然科学研究科, 教授 (60107985)
• Project Period (FY): 1993 – 1994
• Project Status: Completed (Fiscal Year 1994)
• Budget Amount: ¥2,300,000 (Direct Cost: ¥2,300,000); Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
• Keywords: Chicken pancreatic hormone / Glucagon like peptide / Glucagon / cDNA cloning / Monoclonal antibody / Lipid metabolism / Protein metabolism / Carbohydrate metabolism / アフィニティクロマトグラフィー / 初代培養肝細胞 / 遊離脂肪細胞 / 脂質代謝系 / 炭水化物代謝系 / 酵素的DNA増幅法 / 組み換えシャトルベクター / 酵母発現

Research Abstract

cDNA clones coding for glucagon were isolated from a chicken pancreas cDNA library, and the nucleotide and amino acid sequences were determined. The amino acid sequence of chicken glucagon was HSQGTFTSDYSKYLDSR-RAQDFVQWLMST,which was contained in the 151-amino acid long precursor, being preceded by a signal sequence and an amino-terminal peptide(NH2-peptide)and followed by an intervening peptide and a GLP-I.Chicken preproglucagon, however, lacked GLP-II and intervening peptide II which have been shown to be contained in mammalian glucagon precursors. The DNA encoding chicken glucagon like peptide was synthesized by PCR method to construct a synthetic glucagon like peptide gene preceded by ATG coding for methionine and followed by TAA coding for the stop codon. The nucleotide sequence of glucagon like peptide was designed on the basis of preferential codon usage in Saccharomyces cerevisiae. Recombimant chicken glucagon lilke peptide was successfully produced with a high level of express ion in yeast as a fusion protein with MBP.The synthetic gene was designed to release glucagon like peptide, which does not contain methionine residues, from fusion protein with CNBr that specifically cleaves the peptide bond on the carboxyl side of the methionine residue. The resulting glucagon like peptide was purified to homogeneity by a combination of reverse-phase HPLC and ion-exchange HPLC.The yield of intact glucagon like peptide obtained from 11 of culture was approximately 1.5 mg. The structure of recombinant chicken glucagon like peptide was confirmed by HPLC and amino acid composition analysis. We produced monoclonal chicken glucagon like peptide antibodies, and tried to isolate chicken glucagon like peptide from chicken pancreas. Finally, we evaluated the effect of recombinant chicken glucagon like peptide on in vitro lipolysis in isolated adipocytes and on in vivo glycogenolysis in liver in chicks, by comparison to chicken glucagon and porcine glucagon, and indicated a definite role for chicken glucagon lilke peptide in regulating carbohydrate and lipid metabolism in chicks.