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Departments of Metabolic Medicine (M.A.C., C.W.L.R., R.L.B., A.P., M.P., M.A.G., S.R.B.) and Dietetics (S.M.E., G.S.F.), Imperial College London, Hammersmith Hospital Campus, London, United Kingdom W12 0NN
Address all correspondence and requests for reprints to: Prof. Stephen R. Bloom, Department of Metabolic Medicine, Imperial College London at Hammersmith Campus, Du Cane Road, London, United Kingdom W12 0NN. E-mail: s.bloom{at}imperial.ac.uk.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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Human OXM was synthesized by Bachem (St. Helens, UK). The Limulus amebocyte lysate test for pyrogen (Associates of Cape Cod, Liverpool, UK) was negative, and the peptide was sterile on culture. Saline (0.9%) was supplied by Bayer (Haywards Heath, UK), and Hemaccel by Beacon (Tunbridge Wells, UK).
Subjects
Thirteen healthy volunteers (seven men and six women), aged 19–27 yr (mean ± SEM, 20.2 ± 0.7) with a body mass index between 20.4–27.1 kg/m2 (mean ± SEM, 22.5 ± 0.9), were recruited by advertisement at Hammersmith Hospital Campus, Imperial College London. Ethical approval was obtained from the Hammersmith Hospitals’ Trust research ethics committee. The subjects gave informed written consent, and the study was performed in accordance with the Declaration of Helsinki. Criteria for exclusion included smoking, substance abuse, pregnancy, medication (except for the oral contraceptive pill), medical or psychiatric illness, and abnormalities detected on physical examination and screening investigations (electrocardiogram, full blood count, urea and electrolytes, liver function tests, and fasting glucose). Potential subjects were screened by a dietician to exclude those with a high level of restrained eating, as assessed by the Dutch Eating Behavior Questionnaire (14), and disordered eating, as assessed by the Eating Attitudes Test (15). Subjects also completed a 3-d diet diary to determine their usual eating habits before acceptance into the study. Food preferences were assessed using a nine-point hedonic scale to ensure that food offered at the buffet lunch was acceptable.
Protocol
The study design was a double-blind, placebo-controlled, cross-over (Fig. 1
). Each subject received an iv infusion of OXM (3.0 pmol/kg·min) and a control infusion of saline (0.9%), at least 7 d apart, in random order. Female subjects were studied in the follicular phase of the menstrual cycle to control for the potential effect of cycle phase on food intake and appetite sensation (16). Subjects completed a diary of food consumption for 2 d preinfusion and 24 h postinfusion. They refrained from alcohol and strenuous exercise for 24 h before and after the study day. Subjects maintained a similar diet for 48 h before each infusion. They consumed an identical meal and then fasted from 2100 h on the night before each infusion. They were permitted to drink water. Subjects were studied in pairs and were separated by a screen. Throughout the study they were encouraged to relax by reading or watching video films. Clocks were removed from the study room to limit the potential effect of awareness of time on expectation of food and appetite sensation.
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At 15 min before termination of the infusion (t75), subjects were offered a buffet meal, which was provided in excess such that all appetites would be satisfied. Water was freely available. Energy intake was determined by weighing food and water pre- and postprandially. The duration of the meal was timed, and subjects rated the palatability of the food by VAS. Subjects remained in the study room until t240. They continued to complete VAS until 0900 h the following morning and recorded their food intake in diaries for 24 h after the buffet meal (until 1300 h the following day). Food diaries were analyzed by a dietician blinded to the study, and energy intake was calculated with the aid of the Dietplan program (Forestfield Software Ltd., West Sussex, UK).
Analysis of OXM degradation in human plasma
To determine whether infused OXM is converted into other molecular forms, plasma taken at the plateau of OXM infusion (t60) was fractionated with Sephadex G-50 Superfine on a 60 x 0.9-cm column (Amersham Pharmacia Biotech, Uppsala, Sweden). The column was eluted at a flow rate of 3.2 ml/h at 4 C in 0.06 M phosphate buffer containing 0.2 M NaCl and 0.3% (vol/vol) BSA, and 0.7-ml fractions were collected. To determine the relative elution coefficient (Kav) of OLI dextran blue (molecular weight, 2,000,000; 30 mg/ml; Kav = 0), horse heart cytochrome c (molecular weight, 12,384; 30 mg/ml; Kav = 0.26) and [125I]Na (Kav = 1.0) were added to each sample (n = 3), and samples were loaded in a volume of 0.8 ml. The elution profile of plasma OXM was determined by RIA for OLI.
Hormone measurements
All samples were assayed in duplicate and within one assay to eliminate interassay variation. Plasma OLI, glucagon, peptide YY (PYY), insulin, glucagon-like peptide-1 (GLP-1), and ghrelin were measured using established in-house RIAs. The OLI assay (5) could detect changes of 10 pmol/liter (95% confidence limit) with an intraassay variation of 5.7%. The PYY assay (18) could detect changes of 2 pmol/liter (95% confidence limit) with an intraassay variation of 5.8%. The PYY antibody was specific for the C-terminal of PYY and reacted fully with human PYY-(3–36). The insulin assay (19) could detect changes of 6 pmol/liter (95% confidence limit) with an intraassay variation of 5.4%. The GLP-1 assay (19) could detect changes of 8 pmol/liter (95% confidence limit) with an intraassay variation of 6.1%. The GLP-1 antibody was specific for N-terminal amidated GLP-1 and did not cross-react with GLP-1-(1–37), GLP-1-(1–36), or GLP-1-(7–37). The ghrelin assay (20) could detect changes of 10 pmol/liter (95% confidence limit) with an intraassay variation of 9.5%. Plasma leptin was measured using a human leptin RIA kit (Linco Research, Inc., St. Charles, MO).
Statistical analyses
Combined data are represented as the mean ± SEM. The integrated area under the curve was calculated using the trapezoid rule. Comparisons of areas under the curve and food consumption between OXM and saline groups were by paired t test. VAS were compared using the Wilcoxon signed-rank test. P < 0.05 was considered significant.
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Results |
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OXM infusion significantly reduced energy intake at the buffet meal by 19.3 ± 5.6% (reduction vs. saline, 921 ± 251 kJ; P < 0.01). Twelve of the 13 subjects studied showed a decrease in energy intake with OXM infusion (Fig. 2). There was no obvious cause for the failure of response in one subject. OXM infusion significantly reduced cumulative 12-h energy intake by 11.3 ± 6.2% (reduction vs. saline, 1528 ± 666 kJ; P < 0.05; Fig. 3). Cumulative 24-h energy intake was not significantly altered (saline, 12,740 ± 1,532 kJ; OXM, 11,589 ± 1,243 kJ). OXM did not change water consumption or the proportion of energy obtained from different macronutrients, either at the buffet meal or in the subsequent 24 h.
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During infusion of saline, VAS for hunger did not change significantly throughout the fasting period (Fig. 4), whereas OXM infusion caused a significant fall in hunger (incremental area under the curve t0 to t75: saline, +273 ± 128 mm/min; OXM, -374 ± 185 mm/min; P < 0.05). The decrease in hunger after the buffet meal was similar on saline and OXM infusion days, and hunger scores remained similar thereafter. The duration of the meal was significantly reduced by OXM (saline, 19.2 ± 1.3 min; OXM, 15.1 ± 1.8 min; P < 0.05). There was no significant effect of OXM on VAS for satiety, prospective food consumption, nausea, or meal palatability (data not shown).
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Infusion of OXM elevated plasma OLI from 62 ± 5 pmol/liter to a peak of 907 ± 32 pmol/liter at t60 (Fig. 5). In comparison, on the saline infusion day, consumption of the buffet meal led to a peak postprandial OLI level of 151 ± 18 pmol/liter at 195 min. Gel permeation analysis of plasma samples during OXM infusion (Fig. 6) demonstrated a single immunoreactive peak eluting in the same position as synthetic OXM (Kav = 0.6). Thus, intact full-length OXM was the principle circulating form.
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During the saline infusion, plasma ghrelin levels increased throughout the fasting period (t0, 461 ± 32 pmol/liter; t75, 484 ± 35 pmol/liter) and decreased postprandially (t225, 357 ± 28 pmol/liter). However, during infusion of OXM, fasting levels of ghrelin decreased before the meal (t0, 482 ± 33 pmol/liter; t75, 435 ± 35 pmol/liter), and there was a further postprandial reduction in ghrelin (t225, 356 ± 31 pmol/liter). Hence, plasma ghrelin before the buffet meal was significantly reduced by OXM infusion compared with saline (mean change in ghrelin from t0 to t75: saline, +24 ± 10 pmol/liter; OXM, -47 ± 11 pmol/liter; P < 0.0001; Fig. 7). The suppression of plasma ghrelin due to OXM infusion represents 44 ± 10% of the postprandial decrease in ghrelin on the corresponding saline infusion day (mean postprandial decrease, 155 ± 19 pmol/liter).
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There was no significant effect of OXM infusion on fasting plasma levels of PYY, insulin, glucagon, GLP-1, or leptin (Table 1). Plasma concentrations of leptin in female subjects were higher than in males, as previously reported. As expected, there was a significant postprandial rise in PYY for both saline (t75, 14 ± 1 pmol/liter; t225, 28 ± 2 pmol/liter; P < 0.0001) and OXM (t75, 14 ± 1 pmol/liter; t225, 29 ± 2 pmol/liter; P < 0.0001) infusions. There was also a significant postprandial rise in plasma insulin (saline: t75, 36 ± 5 pmol/liter; t225, 242 ± 30 pmol/liter; P < 0.0001; OXM: t75, 37 ± 5 pmol/liter; t225, 219 ± 38 pmol/liter; P < 0.0001) and GLP-1 (saline: t75, 47 ± 6 pmol/liter; t225, 86 ± 7 pmol/liter; P < 0.0001; OXM: t75, 41 ± 6 pmol/liter; t225, 86 ± 11 pmol/liter; P < 0.0001). There was no significant difference between saline and OXM infusions in the magnitude of the postprandial rise in PYY, insulin, or GLP-1. In addition, there was no significant postprandial change in plasma glucagon or leptin.
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Discussion |
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We have now demonstrated that systemic administration of OXM significantly reduces food intake in healthy human subjects. Intravenous infusion of OXM reduced calorie intake by 19% at the buffet meal, and cumulative energy intake was decreased in the 12 h post infusion. Much smaller alterations in food consumption would lead to weight loss if sustained over the long term (32). However, there was no significant effect of OXM on cumulative 24-h energy intake. OXM did not affect enjoyment of the meal, which is important in view of its potential therapeutic use.
Ghrelin is a powerful stimulant of appetite in man (23), and preprandial rises in plasma ghrelin have been suggested to be a trigger for meal initiation (33). Hence, the novel finding that OXM infusion suppresses fasting plasma ghrelin is potentially important. Inhibition of the normal preprandial rise in ghrelin by OXM is likely to be one mechanism by which OXM infusion reduces appetite. This finding may also shed light on the poorly understood mechanism by which ghrelin levels are reduced postprandially. In rodents, fasting increases plasma ghrelin, whereas oral intake of glucose, but not water, decreases ghrelin secretion, suggesting that suppression of plasma ghrelin is related to ingestion of nutrients rather than stomach distension (34). Hence, OXM released in response to nutrient ingestion may contribute to the normal postprandial inhibition of plasma ghrelin. It is believed that only a proportion of total circulating ghrelin is the biologically active, octanoylated form (35). The effect of food consumption and OXM infusion may be to primarily reduce levels of this active ghrelin.
Intravenous infusion of OXM has been shown to inhibit gastric emptying in humans (36). Suppression of gastric emptying may lead to increased gastric distension, which may contribute to satiety by causing a sensation of fullness. In the current study, hunger scores were significantly reduced by OXM in the fasting state, when gastric distension is unlikely to be important. Hence, the reduction in appetite during the premeal period is unlikely to result from the effects of OXM on gastric emptying. The anorectic effect of OXM does not appear to be mediated by stimulation of the release of PYY or leptin, as concentrations of these hormones were unaffected by OXM infusion.
It is not clear which receptor mediates the appetite- suppressant effect of OXM. OXM has been shown to bind to the receptor for GLP-1 (GLP-1R) in vitro, but with much lower affinity than GLP-1 (37). In rats, we have previously shown that the anorectic effect of intracerebroventricular OXM is blocked by coadministration of the GLP-1R antagonist exendin-(9–39) (7). However, exendin-(9–39) might also block the binding of OXM at a putative OXM-specific receptor.
GLP-1 is a physiological incretin hormone (19) and as such potentiates the postprandial release of insulin. OXM might also be expected to increase insulin release by binding to the GLP-1R. In this study there was no effect of OXM infusion on fasting insulin levels. However, we cannot determine the postprandial insulin-stimulating effect of OXM infusion due to the different amounts of food consumed at the buffet and the potential effect of OXM on gastric emptying.
It is unclear where circulating OXM acts to reduce appetite. The reduction of food intake by intrahypothalamic injection of OXM (7) suggests that OXM may have direct effects on hypothalamic appetite-regulating centers. Indeed, circulating OXM may gain access to the hypothalamus at sites where the blood-brain barrier is deficient, such as the arcuate nucleus (38). This region, in particular, is thought to be important in sensing and integrating peripheral signals of energy homeostasis (39). Circulating OXM may also be detected by vagal nerve endings, which transmit afferent signals to the brain stem, as has recently been shown for ghrelin (40).
Infusion of OXM produced circulating levels of OLI comparable to the elevated concentrations seen in tropical sprue (9) and after jejuno-ileal bypass surgery for morbid obesity (10, 11). Therefore, OXM may contribute to the loss of appetite and weight loss observed in these conditions. We have demonstrated the anorectic effect of elevated circulating levels of OXM. However, it is possible that lower postprandial concentrations of OXM may contribute to the physiological reduction of appetite in normal individuals.
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Acknowledgments |
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Footnotes |
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Abbreviations: GLP-1, Glucagon-like peptide-1; GLP-1R, glucagon-like peptide-1 receptor; OLI, oxyntomodulin-like immunoreactivity; OXM, oxyntomodulin; PYY, peptide YY; VAS, visual analog scales.
Received March 11, 2003.
Accepted June 14, 2003.
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ghrelin) from baseline (t0) is shown. Infusion of OXM or saline from t0 to t90 is denoted by the bold horizontal line. Presentation of the buffet meal at t75 is represented by the vertical broken line (n = 13). *, P < 0.0001 (OXM vs. saline).