1 Department of Metabolic Medicine, Imperial College Faculty of Medicine, Hammersmith Hospital, London, U.K
2 Department of Dietetics, Imperial College Faculty of Medicine, Hammersmith Hospital, London, U.K
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ABSTRACT |
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The prevalence of obesity is rapidly increasing worldwide; currently >65% of adults in the U.S. are overweight (1). Although even a modest weight loss can improve the health of obese individuals, efforts to treat the obesity pandemic have been unsuccessful. Novel therapeutic targets are urgently required.
Several gut hormones have been found to modulate appetite (24). Oxyntomodulin is a peptide product of the proglucagon gene released from the L-cells of the small intestine in response to food ingestion (5). Oxyntomodulin has been reported to reduce food intake by 19.3% during an intravenous infusion administered to normal-weight humans, an effect that continues for >12 h after infusion (6). Furthermore, in rodents, repeated intraperitoneal administration over 7 days has been associated with reduced white adipose tissue and a highly significant reduction in weight compared with controls (7). Thus, oxyntomodulin may offer a novel treatment for human obesity.
We hypothesized that self-administered subcutaneous oxyntomodulin would induce weight loss, reduce appetite, and alter the levels of adipose hormones in overweight and obese volunteers investigated in a 4-week community-based study.
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RESEARCH DESIGN AND METHODS |
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Ethical approval was obtained from the Riverside Medical Ethics Committee (reference no. 3397). All subjects gave informed written consent, and the study was performed in accordance with the Declaration of Helsinki.
The study followed a double-blind, placebo-controlled, parallel-group protocol designed to investigate the effect of oxyntomodulin on body weight, food intake, and adipose hormones. Oxyntomodulin was synthesized by Bachem U.K., sterile for culture and negative for pyrogen, as previously described (10). A subcutaneous dose of 400 nmol was chosen, which allowed us to achieve the same blood concentration as a previous human intravenous infusion study that demonstrated a reduction in calorie intake (6).
From recruitment, subjects were instructed to continue their current level of exercise and usual diet in a regimen of three meals per day. For 1 week before the study, all subjects self-administered saline injections three times daily, 30 min preprandially, into their abdominal subcutaneous tissue. All subjects prepared their injections by adding 0.25 ml sterile water to vials containing freeze-dried sodium chloride and self-administered injections using a 27-gauge needle. Subjects who failed to comply with the protocol during this week were removed from the study.
Subjects were randomly allocated to the control or treatment group by an independent investigator. Randomization was performed by stratification using the parameters BMI and age; subsequent randomization into the control or treatment group was performed by coin toss. The control and treatment groups were therefore matched for BMI and age and had similar mean height and weight measurements (Table 1). The estimated energy requirements for the control and treatment groups were not significantly different.
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Energy intake was measured during three lunchtime study meals on days 1, 2, and 29. The day before each study meal, subjects refrained from alcohol and strenuous exercise and fasted overnight. Subjects first ingested a fixed 200-kcal breakfast and 210 min later self-administered their injection (designated as t = 0). A meal of known energy content was provided in excess quantity 30 min later, and subjects were requested to eat until they felt comfortably full. On day 1, all subjects administered a saline injection. On days 2 and 29, the control group administered saline and the treatment group administered oxyntomodulin. Visual analog scales, 100 mm in length, were completed throughout the study meal day to evaluate subjective feelings of hunger, nausea, and meal palatability.
Blood samples were taken at t = 30, 0, 15, 30, 60, 90, 150, 210, and 240 min to screen for any effect of oxyntomodulin on other analytes (Table 3). All blood samples were collected from an antecubital fossa cannula into lithium/heparin tubes (LIP, Cambridge, U.K.) containing 2,000 kallikrein inhibitor units of aprotinin (Trasylol; Bayer) and stored on ice. After centrifugation, plasma was immediately separated and stored at 20°C until being analyzed. Blood pressure and pulse were measured before each blood sample on each of the study meal days.
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Statistical analyses.
Combined data are expressed as means ± SE. Data were compared using paired t tests for within-group analyses and unpaired t tests for between-group analyses. The changes in body weight, food data, and adipose hormones were normally distributed and therefore analyzed using parametric analyses. Serial measurements of pulse and blood pressure over time were compared using two-way ANOVA. The integrated area under the curve for incremental plasma insulin was calculated using the trapezoid rule. Prism Version 3.0 was used for statistical calculations, and P < 0.05 was considered significant.
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RESULTS |
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Effect of oxyntomodulin on body weight.
Repeated preprandial subcutaneous injection of oxyntomodulin resulted in a 2.4 ± 0.4% reduction in body weight, compared with a 0.5 ± 0.6% reduction in the control group (P = 0.0129) over the 4-week study period. This represented a significant weight loss of 2.3 ± 0.4 kg in the treatment group and 0.5 ± 0.5 kg weight loss in the control group (P = 0.0106) (Fig. 1). After 2 weeks, interim data demonstrated a significant weight loss of 1.1 ± 0.3 kg in the treatment group (P = 0.0343), which increased to 1.4 ± 0.4 kg after 3 weeks (P = 0.0331). Thus, on average, there was an additional 0.45-kg weight loss per week in the oxyntomodulin group.
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Only two subjects in each group failed to complete the study meal protocol. These subjects did not consume the same food type at each study meal. In the interest of accuracy, these subjects were excluded from food intake and appetite analysis before unblinding. Thus, study meal data were analyzed with n = 10 for the control group and n = 12 for the treatment group.
Plasma levels of oxyntomodulin.
After an injection of oxyntomodulin, plasma OLI increased to a peak of 972 ± 165 pmol/l at 30 min (Fig. 3). In comparison, on the days that subjects administered saline, the mean baseline level of OLI was 97.4 ± 5.5 pmol/l and the peak postprandial level of OLI was 116.5 ± 10.4 pmol/l at 150 min.
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Hormone, lipid, and glucose levels.
There was a statistically significant increase in preprandial insulin levels 30 min after subcutaneous administration of oxyntomodulin, but the increase was small compared with the effect of meal ingestion and not sufficient to affect plasma glucose levels (Table 3). This effect on insulin was present on both day 2 and day 29 (Fig. 4). The area under the curve for the incremental insulin level was nonsignificantly lower on day 2 (18.0 ± 0.9 nmol · min1 · l1; P = 0.1378) and significantly lower on day 29 (14.9 ± 0.7 nmol · min1 · l1; P = 0.0398) over the 4-h postinjection period compared with the day saline was administered (day 1: 21.6 ± 1.1 nmol · min1 · l1).
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Adverse events.
Short-lived minor discomfort at the injection site was reported by 8 of 14 of the subjects in the treatment group and 5 of 12 of the control group. In total, 6.9% of all oxyntomodulin injections and 6.6% of all saline injections were reported to cause minor discomfort; thus discomfort did not appear related to the substance injected.
Transient mild nausea was reported with 3% of oxyntomodulin injections compared with 0.2% of all saline injections (P = 0.0389). These data included subject Y, who experienced nausea associated with 13 of the first 47 injections. The dose subject Y administered was halved, and the incidence of nausea decreased to 2 out of the remaining 37 injections. On day 29, subject Y had a peak OLI level of 868 pmol/l, compared with the mean peak for the group of 972 ± 619 pmol/l, despite taking only half the dose of oxyntomodulin. A second participant, subject X, was removed from the treatment group after day 2 because of adverse effects and was not included in the analysis; after each of the initial three oxyntomodulin injections, subject X had significant nausea that interfered with daily activities. This subjects plasma OLI levels peaked at 2,140 pmol/l 15 min postinjection and were significantly higher than the rest of the treatment group over the entire 4-h study period (P < 0.0001 by two-way ANOVA). Another subject regularly experienced nausea for 2 days during her midmenstrual cycle and was included in the treatment group data in the interest of stringency.
The incidence of all other reported adverse events was <2% and not significantly different between the treatment and control groups. There were no effects of oxyntomodulin self-administration on pulse rate or systolic or diastolic blood pressure as measured during the study meal days.
Follow-up data.
The treatment group regained 1.1 ± 0.4 kg 2 weeks after terminating the oxyntomodulin injections. The control group gained 0.5 ± 0.2 kg and were 0.0 ± 0.6 kg different from their initial weight. The difference in weight loss between the control and treatment group was no longer significant 2 weeks after the end of the study period (P = 0.134). One subject in the control group and one in the treatment group were lost to follow-up after the end of the study; therefore, the follow-up data were analyzed with n = 11 in the control group and n = 13 in the treatment group.
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DISCUSSION |
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The plasma OLI levels achieved by subcutaneous injection were similar to those achieved by an intravenous infusion of 3.0 pmol · kg1 · min1 in a study that reduced food intake by 19.3% in normal weight subjects (6). This anorectic effect was comparable with the effect observed in overweight subjects in the current study. This suggests that oxyntomodulin has an equipotent anorectic effect in normal weight and overweight subjects and that obesity does not confer resistance to the anorectic effect of oxyntomodulin.
A recent human intravenous infusion study demonstrated no effect of oxyntomodulin on insulin or glucose levels in normal-weight humans (6). In the current study, there was no evidence of a postprandial incretin effect. There was a small increase in preprandial plasma insulin after oxyntomodulin injection. However, this increase was not sufficient to alter plasma glucose levels. Other gut hormones, such as glucagon-like peptide, are known to have a powerful incretin effect (12). Indeed, glucagon-like peptide 1, while improving glucose tolerance when given to type 2 diabetic subjects (16), can cause hypoglycemia in nondiabetic subjects (17). There was no evidence of a change in baseline insulin, glucose, or lipid levels over the 4-week study period in this group of healthy obese subjects. Further studies are needed to assess to what degree long-term oxyntomodulin administration leads to an improvement in metabolic parameters in obese subjects with abnormal glucose and lipid metabolism.
The current study did not demonstrate an effect of oxyntomodulin on the circulating level of the orexigenic hormone ghrelin. It has been suggested that a proportion of the anorectic effect of intravenously administered oxyntomodulin is secondary to its suppressant effect on ghrelin (6). However, this does not seem a likely mechanism for the dose administered subcutaneously in the current study. The decrease in body weight was secondary to a significant reduction in energy intake. This reduction in energy intake was likely due to a reduction in appetite, as there was preservation of food satisfaction and therefore no evidence of taste aversion. Indeed, a previous study demonstrated that administration of intravenous oxyntomodulin reduces preprandial hunger scores in normal-weight humans (6). However, the current study did not demonstrate a change in subjective appetite scores before the meal after a subcutaneous injection of oxyntomodulin. This may have reflected a perception of early satiety rather than a reduced preprandial appetite or a failure of the visual analog scores as a tool of appetite measurement.
The self-administration of subcutaneous oxyntomodulin was well-tolerated by the subjects, and good compliance rates were achieved. This was a fixed initial dose study; a dose of 400 nmol oxyntomodulin may not be appropriate for all subjects. Indeed, subject Y experienced a much lower incidence of nausea after the dose was reduced. Subject X demonstrated extremely high plasma OLI levels after oxyntomodulin injection and experienced significant nausea. These subjects may have absorbed oxyntomodulin more rapidly or metabolized the peptide more slowly. Larger groups studies are needed to assess if this was an idiosyncratic response.
These preliminary data suggest that the administration of oxyntomodulin could be an effective treatment for obesity. The reduction in food intake seen at the end of the study was statistically no different to the reduction on day 2, indicating that the efficacy of oxyntomodulin was maintained. In addition, the rate of weight loss was consistent over the 4-week study period. Oxyntomodulin levels are increased in patients who have had jejunoileal bypass surgery (18,19). This sustained elevation of plasma oxyntomodulin may be one of the factors leading to successful weight reduction in these patients. There was no evidence of oxyntomodulin antibody formation from the assay data. Together, this suggests that administration of oxyntomodulin beyond the 4-week study period may be expected to result in continued weight loss. However, these data are limited by our small sample size and the 4-week duration of the study. Longer-term clinical trials involving larger numbers of participants are required to demonstrate the long-term efficacy of oxyntomodulin as an antiobesity therapy.
In summary, subcutaneous self-administration of oxyntomodulin three times daily in the community reduced body weight, decreased food intake, and altered the levels of adipose hormones in overweight and obese nondiabetic human subjects. The anorectic effect was well maintained over the 4-week study period, and the weight loss compared favorably with that achieved through other drug therapies. Currently available pharmacological agents licensed for weight reduction therapy have limited efficacy (20). Several drugs now in development affect widely distributed central neurotransmitter systems and may therefore have a broad spectrum of side effects. Mimicking postprandial satiety by administering a natural postprandial hormone such as oxyntomodulin may provide a more specific treatment for obesity.
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ACKNOWLEDGMENTS |
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The study was conceived by K.W., A.J.P., and S.R.B. The study protocol and ethical application was formulated by K.W., A.J.P., C.J.S., M.P., S.M.E., K.G.M., A.M.W., G.S.F., K.M., M.A.G., and S.R.B. K.W. and A.J.P. enrolled patients and performed the study interventions, hormone assays, and data analysis. Dietetic support and data analysis were performed by S.M.E. in collaboration with G.S.F. K.W. and A.J.P. drafted the manuscript, and all authors were involved in its critical appraisal and final approval.
The use of oxyntomodulin for the treatment of obesity is the subject of two pending patent applications (WO 2003/022304 and WO 2004/06285) in the name of Imperial College Innovations, which have been exclusively licensed to Thiakis Limited.
We thank the volunteers for their invaluable help with the study. We also thank Dr. David Stephens for statistical advice and the Department of Biochemistry, Charing Cross Hospital, for its assistance with the measurement of glucose and lipids.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address correspondence and reprint requests to Prof. Stephen R. Bloom, Endocrine Unit, Imperial College Faculty of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. E-mail: s.bloom{at}imperial.ac.uk
Received for publication April 15, 2005 and accepted in revised form May 17, 2005
OLI, oxyntomodulin-like activity
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REFERENCES |
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