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. Rabbi ts immunized with GLP-1(7-36)amide conjugated to bovine serum albumin with glutaraldehyde produced antisera containing high-affinity antibod ies directed against an epitope that included the free amidated C-term inus 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 immunorea ctivity in extracts of rat hypothalamus, pancreas and intestine were d etermined 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 t otal 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 t otal 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 specim en of terminal ileum contained HPLC-identified GLP-1(1-36)amide (13% o f 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)am ide 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 pmo l/l and rose to a maximum of 15.4 +/- 3.0 pmol/l 10 min after intragas tric glucose 1 g/kg, illustrating the modest level of plasma responses of amidated forms of GLP-1. (C) 1997 Elsevier Science B.V.