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Background: Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin., Objectives: To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs., Methods: Eight healthy young men (aged 18-28 years, body mass index range, 20.6-26.2 kg/m(2)) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin., Results: With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14-C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs., Conclusions: These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity.

Background: Ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin is found in the circulation in two forms: acyl- and desacyl-ghrelin. Acyl- and desacyl-ghrelin concentrations increase at night, when cortisol concentrations are low. Acute ghrelin administration increases ACTH and cortisol concentrations and a feedback loop between the ghrelin and ACTH-cortisol axis has been postulated. A previous study showed that exogenously induced hypercortisolism for 5 days decreased plasma ghrelin concentrations., Objective: The objective of the study was to determine whether a 4-hour infusion of hydrocortisone given at a time of low endogenous cortisol concentrations (11:00 pm to 3:00 am) acutely suppresses acyl- and desacyl-ghrelin., Methods: Eight healthy young men aged (mean ± SD) 21.5 ± 2.7 years with a body mass index of 22.4 ± 2.5 kg/m(2) were studied in a single-blind, placebo-controlled study during two separate overnight admissions on the Clinical Research Unit. The volunteers received either a 4-hour (11:00 pm to 3:00 am) infusion of hydrocortisone or a saline infusion. The hydrocortisone infusion rate was 0.3 mg/kg·h for the initial 3 minutes, 0.24 mg/kg·h for 9 minutes, and then 0.135 mg/kg·h until the end of the infusion. Plasma acyl- and desacyl-ghrelin concentrations (in-house two site sandwich assay) and ACTH, cortisol, insulin, GH, and glucose levels were measured every 10 minutes for 16 hours (5:00 pm to 9:00 am)., Results: The mean differences (lower 95% limit; upper 95% limit) between the saline infusion and hydrocortisone infusion for acyl- and desacyl-ghrelin concentrations were not significantly different from zero. The infusion period (11:00 pm to 3:00 am) was as follows: acyl-ghrelin, 0.22 (-7.39; 7.83) (P = 1.00); desacyl-ghrelin, -3.36 (-17.66; 10.95) (P = 1.00). The postinfusion period (3:00-7:00 am) was as follows: acyl-ghrelin, 8.68 (1.07; 16.28); (P = .056); desacyl-ghrelin, 8.75 (-5.56; 23.05) (P = .403)., Conclusions: A short-term increase in circulating cortisol concentrations by exogenous hydrocortisone infusion does not suppress circulating nocturnal acyl- or desacyl-ghrelin concentrations. Thus, it is likely that the diurnal pattern of ghrelin secretion is under circadian control and not directly regulated by cortisol.

Background: Acyl-ghrelin is thought to have both orexigenic effects and to stimulate GH release. A possible cause of the anorexia of aging is an age-dependent decrease in circulating acyl-ghrelin levels., Objectives: The purpose of the study was to compare acyl-ghrelin and GH concentrations between healthy old and young adults and to examine the relationship of acyl-ghrelin and GH secretion in both age groups., Methods: Six healthy older adults (age 62-74 y, body mass index range 20.9-29 kg/m(2)) and eight healthy young men (aged 18-28 y, body mass index range 20.6-26.2 kg/m(2)) had frequent blood samples drawn for hormone measurements every 10 minutes for 24 hours. Ghrelin was measured in an in-house, two-site sandwich ELISA specific for full-length acyl-ghrelin. GH was measured in a sensitive assay (Immulite 2000), and GH peaks were determined by deconvolution analysis. The acyl-ghrelin/GH association was estimated from correlations between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-minute interval preceding each GH burst., Results: Twenty-four-hour mean (±SEM) GH (0.48 ± 0.14 vs 2.2 ± 0.3 μg/L, P < .005) and acyl-ghrelin (14.7 ± 2.3 vs 27.8 ± 3.9 pg/mL, P < .05) levels were significantly lower in older adults compared with young adults. Twenty-four-hour cortisol concentrations were higher in the old than the young adults (15.1 ± 1.0 vs 10.6 ± 0.9 μg/dL, respectively, P < .01). The ghrelin/GH association was more than 3-fold lower in the older group compared with the young adults (0.16 ± 0.12 vs 0.69 ± 0.04, P < .001)., Conclusions: These results provide further evidence of an age-dependent decline in circulating acyl-ghrelin levels, which might play a role both in the decline of GH and in the anorexia of aging. Our data also suggest that with normal aging, endogenous acyl-ghrelin levels are less tightly linked to GH regulation.

Context: Ghrelin is the endogenous agonist for the growth hormone secretagogue receptor (GHS-R). Intravenous administration of ghrelin induces insulin resistance and hyperglycemia and increases the levels of free fatty acids (FFA)., Objective: To investigate whether these effects are mediated directly by ghrelin in skeletal muscle tissue., Design: This study was single blinded, randomized, and placebo controlled. Eight healthy men (25.5 ± 3.1 years) received 240 min of intraarterial ghrelin infusion (4.2 ng × kg(-1) × min(-1)) into one femoral artery and intraarterial placebo infusion into the contralateral artery. Simultaneous blood samples were drawn from both femoral veins and muscle biopsies were obtained from both legs during both a basal period and during a hyperinsulinemic and euglycemic clamp period., Results: Ghrelin significantly elevated venous FFA levels and venous dilution of palmitate, suggestive of increased lipolysis. Glucose metabolism was unchanged, and there were no direct effects on pertinent enzymes in the insulin signaling cascade. The metabolic clearance rate of acyl ghrelin was 12.5 ± 3.3 ml × kg(-1) × min(-1). Acyl and desacyl ghrelin levels both increased., Conclusions: The results of this study suggest that ghrelin may stimulate lipolysis directly in skeletal muscle.

Context: Ghrelin, an endogenous ligand for the GH secretagogue receptor, is an orexigenic peptide hormone produced primarily by the stomach. Recent studies suggest significant differences in the specificity of currently available ghrelin assays., Objective: The aim of the study was to compare four ghrelin assays (two commercially available and two developed by our group) of differing specificity, each used on the same set of more than 800 plasma samples from a human study., Design: Thirteen volunteers were sampled every 20 min for 6 h after consumption of one of three isocaloric drinks consisting of either 80% fat, 80% carbohydrate, or 80% protein. The samples were assayed by RIA for total and active ghrelin, as well as by sandwich assays for acyl and des-acyl ghrelin. The ghrelin profiles for each individual were smoothed using a statistical algorithm to lessen the effects of pulsatility and noise., Results: The sandwich assays for acyl and des-acyl ghrelin yielded ghrelin values that were lower than those from the corresponding RIAs. The ghrelin profiles after nutrient ingestion were similar, yet key differences among the four assays were apparent; in particular, percentage changes were significantly greater in the sandwich assays., Conclusions: The lower levels and greater relative changes in ghrelin values reported by the sandwich assays are consistent with greater assay specificity. When applied to the nutrient study, the sandwich assays were better able to distinguish the different responses to different nutrients than were the RIAs.

Context: Experimental studies in GH-deficient patients and in healthy subjects receiving somatostatin-infusion suggest that GH is an important regulator of substrate metabolism during fasting. These models may not adequately reflect the selective effects of GH, and GH receptor (GHR) blockade offers a new model to define the metabolic role of GH., Objective: The aim of this study was to investigate the impact of GHR blockade on substrate metabolism and insulin sensitivity during fasting., Design: We conducted a randomized, placebo-controlled, crossover study in 10 healthy young men., Intervention: After 36 h of fasting with saline or pegvisomant (GHR blockade), the subjects were studied during a 4-h basal period and 2.5-h hyperinsulinemic euglycemic clamp., Main Outcome: We measured whole-body and forearm glucose, lipid, and protein metabolism, peripheral insulin sensitivity, and acyl and desacyl ghrelin., Results: GHR blockade significantly suppressed circulating free fatty acids (1226 +/- 83 vs. 1074 +/- 65 micromol/liter; P = 0.03) and ketone bodies (3080 +/- 271 vs. 2015 +/- 235 micromol/liter; P

Context: Ghrelin is an orexigenic hormone that can increase body weight. Its circulating levels increase before meals and are suppressed after food ingestion. Understanding the effects of specific types of ingested macronutrients on ghrelin regulation could facilitate the design of weight-reducing diets., Objective: We sought to understand how ingestion of carbohydrates, proteins, or lipids affect acyl (bioactive) and total ghrelin levels among human subjects, hypothesizing that lipids might suppress ghrelin levels less effectively than do either carbohydrates or proteins., Design: This was a randomized, within-subjects cross-over study., Setting: The study was conducted at a University Clinical Research Center., Participants: There were 16 healthy human subjects included in the study., Interventions: Isocaloric, isovolemic beverages composed primarily of carbohydrates, proteins, or lipids were provided., Main Outcome Measures: The magnitude of postprandial suppression of total and acyl ghrelin levels (measured with a novel acyl-selective, two-site ELISA) was determined., Results: All beverages suppressed plasma acyl and total ghrelin levels. A significant effect of macronutrient class on decremental area under the curve for both acyl and total ghrelin was observed; the rank order for magnitude of suppression was protein more than carbohydrate more than lipid. Total ghrelin nadir levels were significantly lower after both carbohydrate and protein, compared with lipid beverages. In the first 3 postprandial hours, the rank order for acyl and total ghrelin suppression was carbohydrate more than protein more than lipid. In the subsequent 3 h, there was a marked rebound above preprandial values of acyl and total ghrelin after carbohydrate ingestion alone., Conclusions: These findings suggest possible mechanisms contributing to the effects of high-protein/low-carbohydrate diets to promote weight loss, and high-fat diets to promote weight gain.

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.

Context: The timing and frequency of GH secretory episodes is regulated by GHRH and somatostatin. This study provides evidence for amplification of these GH pulses by endogenous acyl-ghrelin., Design: Blood was sampled every 10 min for 26.5 h during a fed admission with standardized meals and also during the final 24 h of a 61.5-h fast. GH secretion profiles were derived from deconvolution of 10-min sampling data, and full-length acyl-ghrelin levels were measured using a newly developed two-site sandwich assay., Setting: The study was conducted at a university hospital general clinical research center., Participants: Participants included eight men with mean (+/- sd) age 24.5 +/- 3.7 yr (body mass index 24 +/- 2.1 kg/m(2))., Results: Correlations were computed between amplitudes of individual GH secretory events and the average acyl-ghrelin concentration in the 60-min interval preceding each GH burst. In the fed state, the peak correlations were positive for all subjects and significantly higher than in the fasting state when acyl-ghrelin levels declined [mean (+/- sem): 0.7 (0.04) vs. 0.29 (0.08), P = 0.017]. In addition, long-term fasting was associated with an increase in the GH secretory pulse mass and amplitude but not frequency [fed vs. fasting pulse mass: 0.22 (0.05) vs. 0.44 (0.06) microg/liter, P = 0.002; amplitude: 5.2 (1.3) vs. 11.8 (1.9) microg/liter/min, P = 0.034; pulses per 24 h: 19.4 (0.5) vs. 22.0 (1.4), P = 0.1]., Conclusion: Our data support the hypothesis that under normal conditions in subjects given regular meals endogenous acyl-ghrelin acts to increase the amplitude of GH pulses.