Effect of Gastric Bypass and Gastric Banding on Proneurotensin ...

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The Journal of Clinical Endocrinology & Metabolism 91(9):3544 –3547 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2006-0256

Effect of Gastric Bypass and Gastric Banding on Proneurotensin Levels in Morbidly Obese Patients Mirjam Christ-Crain,* Rolf Stoeckli,* Andrea Ernst, Nils G. Morgenthaler, Stefan Bilz, Ma´rta Korbonits, Joachim Struck, Andreas Bergmann, Beat Mu¨ller, and Ulrich Keller Division of Endocrinology, Diabetes, and Clinical Nutrition (M.C.-C., R.S., S.B., B.M.,U.K.), University Hospital, CH-4031 Basel, Switzerland; SphingoTec GmbH (A.E.), D-16556 Borgsdorf, Germany; Research Department (N.G.M., J.S., A.B.), B.R.A.H.M.S. AG, D-16761 Hennigsdorf/Berlin, Germany; and Department of Endocrinology (M.C.-C., M.K.), St. Bartholomews Hospital, London EC1M 6BQ, United Kingdom Context: Neurotensin is produced mainly in the N cells of the ileum and has a role in appetite regulation; levels are decreased in obese subjects and increase after bariatric surgery. Mature neurotensin is very unstable, with a short half-life. Objective: The objective of this study was to compare baseline and postoperative levels of the more stable neurotensin precursor, proneurotensin/neuromedin (pro-NT/NMN), in patients after gastric banding, gastric bypass, and nonoperated controls, respectively, during long-term follow-up. Design and Setting: This was a prospective observational study in a university hospital. Participants and Main Outcome Measures: Overnight fasting plasma pro-NT/NMN concentrations were measured with a new sandwich immunoassay in morbidly obese subjects at baseline and 6, 12, and 24 months after gastric banding (n ⫽ 8), Roux-en-Y gastric bypass (n ⫽ 5), and in nonoperated controls (n ⫽ 7).

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ARIATRIC SURGICAL PROCEDURES are increasingly performed in morbid obesity and currently represent the most effective treatment modality (1). Circulating gastrointestinal peptides have emerged as important regulators of appetite and body weight and appear to be partly responsible for the weight loss after bariatric surgery. Neurotensin is a recently described gastrointestinal peptide stored in specific cells (N cells) of the ileal and jejunal mucosa, implicated in the regulation of gastrointestinal function and food intake (2, 3). A dose-related increase in neurotensin plasma concentrations is found after fat ingestion, whereas glucose and amino acids produce only a minor or no effect (4). Major physiological functions of neurotensin are the stimulation of pancreatic secretion, inhibition of gastric and small bowel motility, and gastric acid secretion, as well as facilitating fatty acid translocation from the intestinal lumen (5– 8). There is also evidence for a central effect of neurotensin because intracerebroventricular injections of neurotensin led to a dosedependent decrease of food intake in rats (3). Interestingly, First Published Online June 20, 2006 * M.C.-C. and R.S. contributed equally to this work. Abbreviations: BMI, Body mass index; IQ, interquartile; pro-NT/ NMN, proneurotensin/neuromedin; RYGB, Roux-en-Y gastric bypass. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Results: After gastric bypass and banding, body weight decreased by (mean ⫾ SD) 29.5 ⫾ 5.5 and 22.8 ⫾ 5.9 kg, respectively. The decrease after 3 and 6 months was more pronounced after gastric bypass compared with gastric banding (P ⬍ 0.05). Plasma pro-NT/NMN levels in patients after gastric bypass increased from 246.3 ⫾ 174.3 pmol/liter on admission to 748.3 ⫾ 429.6 pmol/liter after 24 months (P ⬍ 0.01). In contrast, in patients with gastric banding, pro-NT/NMN concentrations remained stable (207.3 ⫾ 60.5 pmol/liter at admission, 226.6 ⫾ 116.8 pmol/liter after 24 months). Neither body weight nor plasma pro-NT/NMN levels changed in nonoperated controls. Conclusion: Plasma pro-NT/NMN levels show a more pronounced increase after gastric bypass compared with gastric banding, suggesting that specific bariatric surgical procedures result in distinct alterations of gastrointestinal hormone metabolism. The more stable precursor pro-NT/NMN provides a new tool to quantify neurotensin levels in clinical practice. (J Clin Endocrinol Metab 91: 3544 –3547, 2006)

in obese human subjects, lower neurotensin levels have been observed compared with lean controls (9). After gastric banding and bypass procedures, respectively, neurotensin levels are increased (9 –11). However, direct comparisons of neurotensin levels after gastric banding and gastric bypass, respectively, have never been performed. Unfortunately, the reliable measurement of mature neurotensin is limited due to its instability and its rapid clearance from the circulation with a half-life of 2– 6 min, often leading to an underestimation of the actual neurotensin levels (12, 13). Recently, the more stable precursor peptide proneurotensin/neuromedin (pro-NT/NMN) has been identified, which is produced in stoichiometric amounts to mature neurotensin (14). This study compared the baseline and postoperative levels of proneurotensin in patients after gastric banding, gastric bypass, and nonoperated controls, respectively, during longterm follow-up. Subjects and Methods Study subjects Twenty patients with morbid obesity [body mass index (BMI) ⬎ 37 kg/m2] evaluated for bariatric surgery were investigated. In five subjects, the treatment was gastric bypass [proximal Roux-en-Y gastric bypass (RYGB), open procedure, Roux limb 75 cm], and in eight patients, the treatment was a laparoscopically placed adjustable silicone gastric

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Christ-Crain et al. • Proneurotensin in Bariatric Surgery

J Clin Endocrinol Metab, September 2006, 91(9):3544 –3547

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banding (Swedish gastric banding; Obtech Medical AG, Baar, Switzerland). Treatment decision was based on clinical grounds. Seven obese subjects did not receive operations and served as controls. Before surgical intervention, all patients were interviewed and examined by a physician, a psychologist, and a nutritionist. All patients were followed by the Division of Endocrinology, Diabetes, and Clinical Nutrition, University Hospital Basel, at 3, 6, 12, and 24 months after surgery. The characteristics of the study population have been described in detail elsewhere (15). The study protocol had prior approval by the hospital Institute Ethical Review Board, and written informed consent was obtained from all patients.

and four were male. The mean age of the patients was 44.9 ⫾ 8.4 yr, and the mean BMI was 42.0 ⫾ 3.0 with a mean weight of 114.7 ⫾ 8.6 kg. Baseline pro-NT/NMN levels were (median, IQ range) 145.3 (111.0 –265.2) pmol/liter. The baseline characteristics were similar in the three groups with respect to age, BMI, and pro-NT/NMN levels (Table 1). Similarly, plasma ghrelin and leptin levels were similar at baseline (15). Median pro-NT/NMN levels were not significantly different in female [166.2 (111.0 –276.1) pmol/liter] compared with male [118.0 (99.0 –190.0) pmol/liter] patients P ⫽ 0.34).

Hormone measurement

Body weight during follow-up

At each visit, plasma samples were collected in the fasting state, processed within 30 min, and frozen at ⫺70 C. pro-NT/NMN was measured by a new sandwich immunoassay, as described (14). Briefly, pro-NT/NMN was measured in a sandwich immunoassay employing polyclonal rabbit antibodies against the pro-NT/NMN-peptides 44 – 62 and 98 –117. Antibodies were purified from rabbit antisera by affinity chromatography. Antibodies to amino acids 44 – 62 were coated on polystyrene tubes (2 ␮g/tube) as solid phase, and antibodies to amino acids 98 –117 were labeled with acridiniumester-N-hydroxy-succinimid for chemiluminescence detection. The assay was calibrated using a human reference serum pool (995.1 pmol/liter pro-NT/MNM) and serial dilutions of this pool in horse serum, resulting in calibrators between 6.0 and 995.1 pmol/liter. The analytical assay sensitivity as determined with normal horse serum was 9.95 pmol/liter. Serum samples with pro-NT/ NMN concentrations above the highest calibrator were prediluted in normal horse serum and measured again, and the concentration was subsequently extrapolated. Pro-NT/NMN concentrations in 124 healthy blood donors (sample collection was not standardized for nutritional state or daytime of sampling) gave a median concentration of 338.9 pmol/liter (range, 53.6 –1473 pmol/liter). Plasma ghrelin and leptin were measured as previously described (15).

Both gastric bypass surgery and banding resulted in a significant weight loss during the follow-up period of 2 yr (Table 1 and Fig. 1). The decrease of body weight from baseline in the bariatric surgery groups was significantly more pronounced at all time points compared with nonoperated controls (P ⬍ 0.01). The decrease of body weight from baseline at 3 and 6 months in the gastric bypass group was greater compared with the gastric banding group (P ⬍ 0.05). After follow-up of 24 months, the difference in changes in body weight was not statistically significant between gastric bypass and gastric banding patients. In the control subjects, body weight did not change significantly.

Statistical analysis Data in the text are shown as means ⫾ sd or median and interquartile (IQ) ranges in case of not normally distributed data, respectively, and in figures as means ⫾ 95% confidence intervals. Frequency comparison was performed by ␹2 test. Two-group comparison of normally distributed data was performed by Student’s t test. For multigroup comparisons, one-way ANOVA with least square difference for post hoc comparison was applied. For data not normally distributed, the MannWhitney U test was used if only two groups were compared, and the Kruskal-Wallis one-way ANOVA was used if more than two groups were compared. Repeated-measures ANOVA was performed for serial measurements. All testing was two-tailed, and P ⬍ 0.05 was considered to indicate statistical significance. Correlation analyses were performed by calculating Spearman rank correlations.

Results Baseline characteristics

Baseline characteristics of the 20 patients in the three groups are shown in Table 1. Sixteen patients were female,

Hormone levels during follow-up

Plasma pro-NT/NMN levels showed a significant increase after gastric bypass (from 246.3 ⫾ 174.3 pmol/liter at admission to 865.9 ⫾ 599.8 after 6 months, 850.4 ⫾ 316.3 after 12 months, and 748.3 ⫾ 429.6 after 24 months; P ⬍ 0.01). This increase was significantly more pronounced at 6 (P ⬍ 0.05), 12 (P ⬍ 0.01), and 24 (P ⬍ 0.05) months compared with the gastric banding and the control group. At 3 months, proNT/NMN levels also tended to be higher in the gastric bypass group compared with the gastric banding group (P ⫽ 0.09). After gastric banding and in obese controls, there was no significant change of pro-NT/NMN levels (Table 1 and Fig. 2). The changes of plasma pro-NT/NMN levels in all patients after 24 months did not correlate with the changes in body weight (r ⫽ ⫺0.23, P ⬎ 0.05) or with the changes in plasma ghrelin (r ⫽ 0.06, P ⬎ 0.05) or in circulating leptin levels (r ⫽ 0.21, P ⬎ 0.05). In the gastric bypass group, the correlation of changes in plasma pro-NT/NMN with changes of body weight was r ⫽ 0.40, with changes in circulating ghrelin r ⫽ 0.40 and leptin r ⫽ ⫺0.80 (P for all correlations not significant). In the gastric banding group, the respective correlations were r ⫽ 0.02, 0.07, and ⫺0.32.

TABLE 1. Age, BMI, and pro-NT/NMN levels at baseline and 24 months after treatment Controls (n ⫽ 7)

Age (yr) BMI (kg/m2) Pro-NT/NMN (pmol/liter)

Baseline

24 Months

49.9 ⫾ 6.9 41.1 ⫾ 2.6 112 (85–160)

41.0 ⫾ 3.4b 141 (103–184)b

Pa

NS NS

ASGB (n ⫽ 8) Baseline

24 Months

41.1 ⫾ 7.3 41.7 ⫾ 2.8 212 (151–265)

33.2 ⫾ 4.7 187 (157–277)

Pa

⬍0.01 NS

RYGB (n ⫽ 5) Baseline

24 Months

43.8 ⫾ 9.8 43.6 ⫾ 4.4 124 (104 – 432)

32.9 ⫾ 6.7 664 (351– 866)

Pa

⬍0.01 ⬍0.01

ASGB, Adjustable silicone gastric banding; NS, not significant. Data are shown as mean ⫾ SD in normally distributed data and as median (IQ range) in not normally distributed data, respectively. a Statistical results within the group from baseline to 24 months (repeated measure ANOVA). b P ⬍ 0.01. Statistical analysis for the comparison of baseline values and values at 24 months, respectively, among the three groups.

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FIG. 1. Changes of body weight from baseline to 24 months postoperatively. Points, Mean values; whiskers, 95% confidence intervals. P represents the result of the repeated measure ANOVA.

Discussion

According to our results, pro-NT/NMN levels show a more pronounced increase after gastric bypass surgery compared with gastric banding. Plasma pro-NT/NMN concentrations remained increased during the whole follow-up period of 24 months, without correlation to changes in body weight and plasma leptin or ghrelin levels, respectively. The mechanism behind the reduced food intake after gastric bypass surgery is not fully understood. Changes in gastrointestinal peptide concentrations have been suggested to be at least partly responsible (16, 17). In our study, neurotensin levels after gastric bypass surgery were markedly elevated, suggesting that neurotensin might represent a further endocrine candidate to explain the decreased appetite and energy intake after gastric bypass despite weight loss, which usually causes compensatory hyperphagia. Possible mechanisms may act either centrally or peripherally because there is a rapid transit of ingested hypercaloric food to the ileum, leading to changes in gastric emptying and intestinal

FIG. 2. Changes of pro-NT/NMN levels from baseline to 24 months in the three groups of patients. Points, Mean values; whiskers, 95% confidence intervals. P represents the result of the repeated measure ANOVA.


transit (10). Furthermore, neurotensin decreases food intake if injected intracerebroventricularly in rats (18). Plasma concentrations of several gastrointestinal hormones are differentially regulated by various bariatric surgical procedures (10). Previous studies showed increased plasma fasting and/or postprandial neurotensin levels after jejunoileal bypass surgery, both within 6 months after the surgical procedure (11) and in the long-term follow-up (19), respectively. In contrast, only a modest increase in fasting neurotensin levels has been reported after gastric banding after an almost 2-yr follow-up (9). A direct comparison of circulating neurotensin levels in patients after gastric bypass and gastric banding surgery has not yet been performed. Our study did not reveal significant changes of pro-NT/ NMN levels after gastric banding, and no correlation was observed between pro-NT/NMN levels and changes in body weight. Thus, alterations in gastrointestinal hormone regulation associated with specific surgical techniques, rather than weight loss per se, seem to determine plasma neurotensin concentrations. It might be tempting to speculate that the increased plasma neurotensin levels may contribute, at least in part, to the more pronounced reduction in satiety after RYGB compared with a purely restrictive bariatric operation. In view of the recent evidence supporting the fact that neurotensin is another target for leptin action (20) and is down-regulated in leptin-deficient models like the ob/ob mouse (21), the lack of a significant correlation between the two neuropeptides may be surprising. The sample size may have been too small to reach a statistically significant correlation. Our study has some limitations. First, plasma neurotensin levels were only measured in the fasting and not in the postprandial state. However, postprandial results most likely would render even much higher neurotensin levels (19). Second, the number of subjects studied was relatively small, and we cannot exclude that a larger sample size would have revealed more subtle differences. Third, because it was an observational study, we cannot draw any conclusions about a causal role of pro-NT/NMN levels in the regulation of body weight and appetite. Pro-NT/NMN could also reflect the changes in appetite induced by other mechanisms and gut hormones [such as polypeptide YY (PYY) and glucagon-like peptide-1 (GLP-1)] (22, 23) and thereby be a new useful marker for changes in satiety. Fourth, we did not monitor food intake, which is difficult to perform in freeliving individuals, during the study period; thus, a possible effect of pro-NT/NMN on appetite and satiety remains speculative. Strengths of our study are that previous studies investigated neurotensin levels in patients after jejunoileal bypass, a procedure that has largely been abandoned for the treatment of morbid obesity (10, 11, 19, 24), whereas our study investigates pro-NT/NMN levels in patients treated with the currently accepted RYGB. It is a prospective study, which compares pro-NT/NMN concentrations with and without different gastric restriction procedures, and measurements were started before surgical treatment. Measuring the more stable precursor pro-NT/NMN, we provide a new alternative tool to quantify neurotensin levels in clinical practice.


In conclusion, herein we report markedly increased concentrations of the precursor peptide of neurotensin in patients after RYGB surgery with no changes observed after gastric banding. Thus, specific bariatric surgical procedures result in distinct alterations of gastrointestinal hormone metabolism with subsequently differential effects on satiety and food intake. Acknowledgments Received February 3, 2006. Accepted June 13, 2006. Address all correspondence and requests for reprints to: Mirjam Christ-Crain, M.D., Department of Endocrinology, Charterhouse Square, St. Bartholomews Hospital, London EC1M 6BQ, United Kingdom. E-mail: [email protected]. M.C.-C., R.S., N.G.M., J.S., A.B., S.B., M.K., B.M., and U.K have nothing to declare. A.E. is an employee of SphingoTec GmbH, the manufacturer of the Pro-NT/NMN assay.

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