Identification of amidated forms of GLP-1 in rat tissues using a highly sensitive radioimmunoassay.
The development of a sensitive radioimmunoassay (RIA) for C-terminally amidated forms of glucagon-like peptide-1 (GLP-1) is described. Rabbits immunized with GLP-1(7-36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibodies directed against an epitope that included the free amidated C-terminus of the peptide. These antisera could be used in a sensitive RIA (detection limit 0.1 fmol/tube) that measured GLP-1(7-36)amide and GLP-1(1-36)amide equally. Total concentrations of amidated GLP-1 immunoreactivity in extracts of rat hypothalamus, pancreas and intestine were determined by RIA, and resolved into GLP-1(7-36)amide, GLP-1(1-36)amide and unidentified cross-reacting substances by HPLC. Whereas only GLP-1(7-36)amide could be identified in the hypothalamus, in amounts that represented 55-94% of total glucagon-like immunoreactivity (GLI), the pancreas produced chiefly GLP-1(1-36)amide, representing 0.8-3.4% of total GLI, and only trace or undetectable amounts of GLP-1(7-36)amide (0-0.36% of total GLI). This argues against any role of intrapancreatic GLP-1(7-36)amide in the secretion of insulin. In the terminal ileum total amidated GLP-1 immunoreactivity represented 27-73% of total GLI, and in five of six specimens only GLP-1(7-36)amide could be identified on HPLC, in amounts representing 13-17% of total GLI. Only one specimen of terminal ileum contained HPLC-identified GLP-1(1-36)amide (13% of total GLI) in addition to GLP-1(7-36)amide (31% of total GLI). Acid-ethanol extraction of peptide-free rat plasma with added GLP-1(7-36)amide gave recoveries of 91+/-SEM 2% in the range 20-200 pmol/l. Basal plasma amidated GLP-1 in six unanaesthetized rats was 4.1+/-1.1 pmol/l and rose to a maximum of 15.4+/-3.0 pmol/l 10 min after intragastric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1.
van Delft
J
J
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Salamanca, Spain.
Uttenthal
L O
LO
Hermida
O G
OG
Fontela
T
T
Ghiglione
M
M
D000818:Animals; D005934:Glucagon / Q000737:chemistry* / Q000276:immunology; D052216:Glucagon-Like Peptide 1; D004763:Glucagon-Like Peptides; D005947:Glucose / Q000008:administration & dosage; D007031:Hypothalamus / Q000737:chemistry / Q000276:immunology; D007082:Ileum / Q000737:chemistry / Q000276:immunology; D007106:Immune Sera / Q000737:chemistry; D007441:Intubation, Gastrointestinal; D008297:Male; D009928:Organ Specificity; D010179:Pancreas / Q000737:chemistry / Q000276:immunology; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000097:blood / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D017208:Rats, Wistar
D007106:Immune Sera; D010446:Peptide Fragments; D010455:Peptides; C055453:glucagon-like peptide 1 (7-36)amide; D004763:Glucagon-Like Peptides; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D005947:Glucose