Novel ghrelin assays provide evidence for independent regulation of ghrelin acylation and secretion in healthy young men

Abstract

Context: Ghrelin, an acylated peptide hormone secreted from the gut, regulates appetite and metabolism. Elucidating its pattern of secretion in the fed and fasted states is important in the face of the obesity epidemic.

Objective: Our objective was to examine changes in circulating ghrelin and des-acyl ghrelin in response to meals and fasting using newly developed two-site sandwich assays and sample preservation protocols to allow specific detection of full-length forms.

Design: Ten-minute sampling was done for 26.5 h during a fed admission with standardized meals and on a separate admission during the final 24 h of a 61.5-h fast and continuing for 2.5 h after terminating the fast.

Setting: The study was conducted at the University Hospital General Clinical Research Center.

Participants: Eight male volunteers participated, mean +/- sd age 24.5 +/- 3.7 yr and body mass index 24 +/- 2.1 kg/m(2).

Main outcome measures: Ten-minute sampling profiles were assessed for ghrelin and des-acyl ghrelin, fed and fasting.

Results: In the fed state, ghrelin and des-acyl ghrelin showed similar dynamics; both were sharply inhibited by meals and increased at night. During fasting, ghrelin decreased to nadir levels seen postprandially, and des-acyl ghrelin remained near peak levels seen preprandially. Total full-length ghrelin (acyl plus des-acyl) levels remained unchanged.

Conclusions: Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.

Figure 1

Protocol for 10-min sampling on fed and fasting admissions. Strenuous daily exercise was restricted to less than 1 h/d. Screening included a medical history questionnaire, physical examination, and a fasting blood profile. Exclusion criteria included smoking, acute illness, or medications known to affect GH release. On the fed admission, the 26.5-h sampling period included four standardized meals. On the fasting admission, the matched sampling period included the final 24 h of a 61.5-h fast and then the response to breaking the fast. Samples were collected for measurement of ghrelin, des-acyl ghrelin, and BuChE. The subjects were allowed to sleep after 2100 h. Bkfst, Breakfast.

Figure 2

Dot-blot characterization of ghrelin antisera. Ghrelin and ghrelin fragments were spotted on nitrocellulose to test specificities of antisera used in sandwich assays as described in Subjects and Methods. Membrane strips dotted with antigens were probed with the three different antisera: C-Term, rabbit antiserum affinity purified to the ghrelin C terminus; Acyl, rabbit antiserum specific to an acylated ghrelin epitope; and Des, mouse monoclonal antiserum specific to a des-acylated ghrelin epitope. Spots were dotted with ghrelin fragments and controls as indicated in the figure. The acyl-specific antiserum did not recognize rat acyl(3–28), which lacks residues 1 and 2. Note that des(1–10) was included only in the test of the des-acyl-specific antiserum. A minor artifact of this experiment was that all peptides were dotted with an equal number of nanograms per spot; thus, the smaller fragments have more moles of the antigenic sites and generally appear darker.

Figure 3

Dose-response curves for ghrelin sandwich assays. A, Acyl-ghrelin assay; B, des-acyl ghrelin assay. Acyl-ghrelin (•) and des-acyl ghrelin (○) were tested in both assays at varying doses shown on a log scale. Controls for nonspecific cross-reactivity were tested in both assays at one dose (all shown as open squares). Ghrelin fragments tested at 20 ng/ml were acyl-ghrelin(1–5), acyl-ghrelin(1–10), acyl-ghrelin(1–14), acyl-ghrelin(1–18), ghrelin(17–28), and ghrelin(3–28) (rat). Other peptides tested at 100 nm (87–506 ng/ml depending on molecular weight) were GH-releasing peptide 6, motilin, galanin, somatostatin, cortistatin-14, cortistatin-17, GHRH, pituitary adenylate cyclase-activating peptide (1–38), secretin, gastric inhibitory polypeptide, PTH, and calcitonin. Although the acyl-ghrelin assay showed no response to des-acyl ghrelin or other tested peptides, the des-acyl ghrelin assay had 3% cross-reactivity to acyl-ghrelin and is an inherent property of the monoclonal antibody's specificity (note faint cross-reactivity seen in Fig. 2) and no significant response to other peptides. RFU, Relative fluorescence units.

Figure 4

A, Stability of ghrelin acylation in human blood samples. The percentage of ghrelin that was acylated in human blood at increasing incubation times was fit as a single-phase exponential decay. Average of six subjects with three incubation conditions each: AEBSF plus chilling on ice (—•—), AEBSF without ice (– –▪– –), and no AEBSF and no ice (···♦···). B, Inhibition of BuChE activity by HCl. Plasma was incubated 1 h with the indicated dose of HCl (microliters per milliliter plasma) and then diluted into a neutralizing buffer for assay. Blank indicates no enzyme negative control. The y-axis is percentage of activity without acid.

Figure 5

Profiles of plasma ghrelin and des-acyl ghrelin over 26.5 h on fed and fasting admissions, with 10-min sampling, mean ± sem of eight subjects, and each sample assayed in duplicate for both forms of ghrelin. A, Acyl-ghrelin of fed admission (black symbols and black line) and fasting admission (gray symbols and gray line). B, Des-acyl ghrelin of fed admission (black symbols and black line) and fasting admission (gray symbols and gray line). C, Fed acyl-ghrelin (black symbols and black line) and des-acyl ghrelin (gray symbols and gray line). Note that the scales are different, with des-acyl ghrelin present at higher levels. D, Fasting acyl-ghrelin (black symbols and black line) and des-acyl ghrelin (gray symbols and gray line). Bkfast, Breakfast.

Figure 6

A, Average levels of ghrelin (black bars) and des-acyl ghrelin (gray bars) on fed and fasting admissions. B, Average levels of serum BuChE activity on fed and fasting admissions. Activity is in units per milliliter relative to the activity of purified bovine BuChE. In both A and B, each bar represents the average of 144 time points per subject over a matched 24-h period, mean ± sem for eight subjects.