Combination of Selective Immunoassays and Mass Spectrometry to Characterize Preproghrelin-Derived Peptides in Mouse Tissues

Abstract

Preproghrelin is a prohormone producing several preproghrelin-derived peptides with structural and functional heterogeneity: acyl ghrelin (AG), desacyl ghrelin (DAG), and obestatin. The absence of selective and reliable assays to measure these peptides simultaneously in biological samples has been a limitation to assess their real proportions in tissues and plasma in physiological and pathological conditions. We aimed at reliably measure the ratio between the different preproghrelin-derived peptides in murine tissues using selective immunoassays combined with a highly sensitive mass spectrometry method. AG-, DAG-, and obestatin-immunopositive fractions from the gastrointestinal tract of adult wild-type and ghrelin-deficient mice were processed for analysis by mass spectrometry (MS) with a Triple Quadrupole mass spectrometer. We found that DAG was predominant in mouse plasma, however it only represented 50% of total ghrelin (AG+DAG) production in the stomach and duodenum. Obestatin plasma levels accounted for about 30% of all circulating preproghrelin-derived peptides, however, it represented <1% of total preproghrelin-derived peptides production (AG+DAG+Obestatin) in the stomach. Assays were validated in ghrelin-deficient mice since neither ghrelin nor obestatin immunoreactivities were detected in their stomach, duodenum nor plasma. MS analyses confirmed that obestatin-immunoreactivity in stomach corresponded to the C-terminal amidated form of the peptide but not to des(1–10)-obestatin, nor to obestatin-Gly. In conclusion, specificity of ghrelin and obestatin immunoreactivities in gastrointestinal tissues using selective immunoassays was validated by MS. Obestatin was less abundant than AG or DAG in these tissues. Whether this is due to inefficient processing rate of preproghrelin into mature obestatin in gastrointestinal mouse tissues remains elusive.

Unexpected Association of Desacyl-Ghrelin with Physical Activity and Chronic Food Restriction: A Translational Study on Anorexia Nervosa

“…To better decipher the potential interaction between daily physical activity and metabolic alterations, we used a preclinical mouse model of chronic food restriction associated with voluntary physical activity, in which metabolic parameters have been characterised previously following either a short- (2 weeks) or long-term (8 weeks) protocol [ 45 , 46 ]. In the present study, AG and DAG plasma concentrations were assessed at different key stages of the protocol, using selective and sensitive immunoassays [ 29 ]. We first assessed such variations in mice submitted to a two-week food restriction that had the ability to run in a wheel.…”

“…To our knowledge, no study described the impact of physical activity on DAG. Here, we benefited from the development of selective and sensitive assays, validated both in humans (personal data) and rodents [ 29 ]. Our results converge in both rodents and humans and highlight the importance of the balance between AG and DAG.…”

“…Original studies using competition assays showed that DAG accounts for 80–90% of total circulating ghrelin [ 28 ]. More recently, using very selective sandwich immunoassays for AG and DAG, DAG accounted for 76% of the total circulating ghrelin (i.e., AG+DAG) [ 29 ]. Despite a number of studies reporting a physiological role for DAG, usually opposite to AG [ 27 , 30 , 31 , 32 ], its role remains elusive.…”

The ups and downs of growth hormone secretagogue receptor signaling

“…Plasma ghrelin can be either degraded by proteases or des‐acylated by serum esterases [37,38]. Ghrelin des‐acylation generates desacyl‐ghrelin, whose plasma levels are ~ 5‐ to 20‐fold higher than ghrelin [39,40]. Some evidence indicates that desacyl‐ghrelin is also produced in the stomach and that its secretion is regulated by norepinephrine and glucose in vitro , in a similar extent to what was shown for ghrelin [41].…”

“…The proposed proLEAP2 processing enzyme is furin, a calcium-dependent serine endoprotease that cleaves peptides at the C-terminal end of paired basic residues and usually processes precursors in the constitutive secretory pathway [50]. A recent study found that LEAP2 is also processed into LEAP2 [38][39][40][41][42][43][44][45][46][47] in the intestinal epithelium of humans and mice [51]. LEAP2's amino acid sequence is highly conserved in mammals, particularly the N-terminal end, where the initial six residues are identical across species [52,53].…”