Neutral endopeptidase 24.11 is important for the degradation of both endogenous and exogenous glucagon in anesthetized pigs

Abstract

Glucagon has a short plasma t(1/2) in vivo, with renal extraction playing a major role in its elimination. Glucagon is degraded by neutral endopeptidase (NEP) 24.11 in vitro, but the physiological relevance of NEP 24.11 in glucagon metabolism is unknown. Therefore, the influence of candoxatril, a selective NEP inhibitor, on plasma levels of endogenous and exogenous glucagon was examined in anesthetized pigs. Candoxatril increased endogenous glucagon concentrations, from 6.3 +/- 2.5 to 20.7 +/- 6.3 pmol/l [COOH-terminal (C)-RIA, P < 0.05]. During glucagon infusion, candoxatril increased the t(1/2) determined by C-RIA (from 3.0 +/- 0.5 to 17.0 +/- 2.5 min, P < 0.005) and midregion (M)-RIA (2.8 +/- 0.5 to 17.0 +/- 3.0 min, P < 0.01) and reduced metabolic clearance rates (MCR; 19.1 +/- 3.2 to 9.4 +/- 2.0 ml.kg(-1).min(-1), P < 0.02, C-RIA; 19.2 +/- 4.8 to 9.0 +/- 2.3 ml.kg(-1).min(-1), P < 0.05, M-RIA). However, neither t(1/2) nor MCR determined by NH2-terminal (N)-RIA were significantly affected (t(1/2), 2.7 +/- 0.4 to 4.5 +/- 1.6 min; MCR, 30.3 +/- 6.4 to 28.5 +/- 9.0 ml.kg(-1).min(-1)), suggesting that candoxatril had no effect on NH2-terminal degradation but leads to the accumulation of NH2-terminally truncated forms of glucagon. Determination of arteriovenous glucagon concentration differences revealed that renal glucagon extraction was reduced (but not eliminated) by candoxatril (from 40.4 +/- 3.8 to 18.6 +/- 4.1%, P < 0.02, C-RIA; 29.2 +/- 3.1 to 14.7 +/- 2.2%, P < 0.02, M-RIA; 26.5 +/- 4.0 to 19.7 +/- 3.5%, P < 0.06, N-RIA). Femoral extraction was reduced by candoxatril when determined by C-RIA (from 22.7 +/- 2.4 to 8.0 +/- 5.1%, P < 0.05) but was not changed significantly when determined using M- or N-RIAs (10.0 +/- 2.8 to 4.7 +/- 3.7%, M-RIA; 10.5 +/- 2.5 to 7.8 +/- 4.2%, N-RIA). This study provides evidence that NEP 24.11 is an important mediator of the degradation of both endogenous and exogenous glucagon in vivo.

Exendin-4, but not glucagon-like peptide-1, is cleared exclusively by glomerular filtration in anaesthetised pigs

“…Studies were carried out in anaesthetised pigs using a protocol which has previously been used to reveal metabolic degradation of GLP-1 [6], glucose-dependent insulinotropic polypeptide (GIP) [16] and glucagon [17].…”

Identification of plasma protease derived metabolites of glucagon and their formation under typical laboratory sample handling conditions

“…Peaks 3, 4, and 5 were confirmed as glucagon [21][22][23][24][25][26][27][28][29], glucagon 20-29, and glucagon [19][20][21][22][23][24][25][26][27][28][29] to within 3.8 ppm. These peaks showed lower mass accuracies than the other metabolites, which is likely to be due to their lower abundances limiting mass spectrometer signal.…”

“…Glucagon [19][20][21][22][23][24][25][26][27][28][29] , or miniglucagon, has previously only been identified in liver plasma membranes [6] and pancreatic cells [7] , whereas glucagon [20][21][22][23][24][25][26][27][28][29] , and glucagon [21][22][23][24][25][26][27][28][29] have not been previously reported. Miniglucagon, has its own unique biological activity, which is opposite to that of gluagon , and thus acts as a modulator [8] .…”

“…Glucagon [1][2][3][4][5][6][7][8][9][10][11][12][13] and glucagon [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] , and the minor metabolites glucagon [1][2][3][4][5][6][7][8][9][10] , glucagon [14][15][16][17][18][19][20][21][22][23][24][25] and glucagon [23][24]…”

“…It could also help to explain the 7-fold difference in endogenous glucagon concentrations reported by immunoassays directed against the middle or C-terminal regions of glucagon [21] . LC/MS assays have the potential to circumvent such issues; however until recently [22][23] such assays were not sensitive enough to detect endogenous glucagon [24][25] [26] .…”