Alteration of Gastric Functions and Candidate Genes Associated With Weight Reduction in Response to Sibutramine

CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2007;5:829 – 837

Alteration of Gastric Functions and Candidate Genes Associated With Weight Reduction in Response to Sibutramine MARIA I. VAZQUEZ ROQUE,* MICHAEL CAMILLERI,* MATTHEW M. CLARK,‡ DEBRA A. TEPOEL,* MICHAEL D. JENSEN,§ KAREN M. GRASZER,‡ SARAH A. KALSY,‡ DUANE D. BURTON,* KARI L. BAXTER,* and ALAN R. ZINSMEISTER㛳 *Clinical Enteric Neuroscience Translational and Epidemiological Research (C.E.N.T.E.R.) Group, Gastroenterology Research Unit, and ‡Department of Psychiatry and Psychology, and §Department of Medicine, Division of Endocrinology, and the 储Department of Health Sciences Research, Division of Biostatistics, Mayo Clinic College of Medicine, Rochester, Minnesota

Background & Aims: It is unclear whether weight loss with the noradrenergic (norepinephrine) and serotonergic (5-hydroxytryptamine) reuptake inhibitor, sibutramine, is associated with altered stomach functions and whether genetics influence treatment response. Methods: Forty-eight overweight and obese but otherwise healthy participants were randomized to placebo or sibutramine (15 mg/day for 12 weeks). At baseline and posttreatment we measured the following: gastric emptying for solids and liquids by scintigraphy, gastric volumes by single-photon emission computed tomography, maximum tolerated volume and 30minute postnutrient challenge symptoms, and selected gastrointestinal hormones. All participants received structured behavior therapy for weight management. The influence of candidate gene polymorphisms involved in norepinephrine and 5-hydroxytryptamine or receptor function (phenylethanolamine N-methyltransferase, guanine nucleotide binding protein ␤ polypeptide 3, ␣2A adrenoreceptor, and solute carrier family 6 [neurotransmitter transporter, serotonin] member 4 [homo sapiens] [SLC6A4]) on weight loss and gastric functions was evaluated. Results: The overall average weight loss posttreatment was 5.4 ⴞ 0.8 (SEM) kg with sibutramine and 0.9 ⴞ 0.9 kg with placebo (P < .001). The sibutramine group showed significant retardation in gastric emptying of solids (P ⴝ .03), reduced maximum tolerated volume (P ⴝ .03), and increased postprandial peptide YY compared with the placebo group. Obese females showed greater effects of sibutramine on weight loss and gastric emptying of solids and liquids. Gastric volumes and postchallenge symptoms were not significantly different in the 2 treatment groups. The LS/SS genotype of the promoter for SLC6A4 was associated with enhanced weight loss with sibutramine. Conclusions: Weight reduction with sibutramine is associated with altered gastric functions and increased peptide YY and is significantly associated with SLC6A4 genotype. The role of genetic variation in SLC6A4 on weight loss in response to sibutramine deserves further study.

G

obesity,1

iven the public health implications of there is a need to further elucidate the pathophysiology of obesity and to develop effective therapies for weight loss. Sibutramine, a serotonergic and adrenergic reuptake inhibitor approved for long-term treatment of obesity, acts by controlling energy intake and energy expenditure through serotonergic and adren-

ergic mechanisms in the hypothalamic nuclei that regulate appetite.2 The average weight loss after 1 year of sibutramine is typically about 5 kg3 and is enhanced when behavioral therapy is added to the treatment program.4 Serotonin inhibits food intake at a hypothalamic level5 and increases energy expenditure through effects on hypothalamic serotonergic neurons.6,7 The gastrointestinal tract controls energy intake through food consumption, digestion, and absorption of nutrients. The stomach signals satiation in response to ingestion of calories or volume.8,9 Stomach functions such as sensation, emptying, and accommodation of a meal also are influenced by serotonergic and adrenergic mechanisms.10,11 Genetic variation influences weight loss in response to sibutramine.12,13 Genetic variation in the conversion of norepinephrine to epinephrine (phenylethanolamine N-methyltransferase G-148A genetic variant) and function G proteins (guanine nucleotide binding protein ␤ polypeptide 3 [GN␤3] C825T) may influence weight loss in response to sibutramine.12,13 Several candidate genes modulate serotonin reuptake and the function of the ␣2 adrenoreceptors.14,15 Genetic variation in the serotonin transporter also influences food impulsivity16 and glycemic control.17 Gene transfection studies with solute carrier family 6 (neurotransmitter transporter, serotonin) member 4 (homo sapiens) (SLC6A4; the gene for the serotonin transporter) promoter show that homozygous short and heterozygous genotype are associated with impaired promoter function and synthesis of the protein compared with the wild-type homozygous long variant.18 The aims of this study were to compare gastric motor and sensory functions and fasting and postprandial changes in plasma glucagon-like peptide 1 (GLP-1), leptin, insulin, and peptide YY (PYY) levels in healthy overweight and obese individuals after randomized assignment to receiving either placebo or sibutramine (15 mg/day) for 12 weeks and to assess the influence of candidate norepinephrine and 5-hydroxytryptamine genes on the weight loss and gastric functions in response to sibutramine. Abbreviations used in this paper: BMI, body mass index; GLP-1, glucagon-like peptide 1; GN␤3, guanine nucleotide binding protein ␤ polypeptide 3; MTV, maximum tolerated volume; PYY, peptide YY; rs, Spearman R; SLC6A4, solute carrier family 6 (neurotransmitter transporter, serotonin) member 4 (homo sapiens). © 2007 by the AGA Institute 1542-3565/07/$32.00 doi:10.1016/j.cgh.2007.02.037

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Methods Study Design and Participants This randomized, parallel-group, placebo-controlled, double-blind study evaluated the effect of sibutramine on gastric functions and weight loss in healthy overweight and obese participants. The study was approved by the Mayo Clinic Institutional Review Board. We evaluated 48 healthy men and women between the ages of 18 and 65 years who were overweight (body mass index [BMI] ⫽ 25–29.9 kg/m2) or obese (BMI ⬎ 30 kg/m2). They were recruited either by a contact letter mailed to a database of individuals who previously had expressed interest in participating in a clinical trial for obesity or by public advertisement. The randomization schedule was provided by the study statistician to the research pharmacy, but all investigators and patients were blinded. Allocation to treatment was concealed. Participants were not eligible to participate if they had cardiac (including hypertension), endocrine (other than hyperglycemia not requiring medical therapy), or unstable psychiatric disease; significant medical illness; or prior abdominal surgery (other than appendectomy, cholecystectomy, Caesarian section, or tubal ligation). They were excluded if their weight exceeded 137 kg (limit of the single-photon emission computed tomography camera) or if they used any medications that may alter gastrointestinal motility, appetite, or absorption (eg, orlistat [Xenical, Roche Pharmaceuticals, Nutley, NJ]). Participants completed the Hospital Anxiety and Depression Scale,19 a self-administered alcoholism screening test,20 and a questionnaire on eating and weight patterns21 to exclude psychiatric morbidity, alcoholism, and binge eating disorder and bulimia, respectively. Permissible medications were multivitamins, birth control pill, estrogen, and thyroxine replacement. After signing a written informed consent form and undergoing a screening evaluation and a pregnancy test (when applicable), subjects presented to the Mayo Clinic General Clinical Research Center. For 3 days, after overnight fasting, participants underwent baseline tests of gastric motor and sensory functions, dual-energy x-ray absorptiometry measurement of body composition, and blood samples. These tests were repeated after 12 weeks of treatment.

Gastric Volumes We used a previously validated, noninvasive method to measure gastric volume during fasting and 30 minutes after 300 mL of Ensure ([316 kcal], Ross Laboratories, Columbus, OH) using single-photon emission computed tomography.22 After an intravenous injection of 99mTcO4, which is taken up by the gastric mucosa, tomographic images of the gastric wall were obtained through the long axis of the stomach using a dualhead gamma camera (SMV Single-Photon Emission Computed Tomography System, SMV America, Twinsburg, OH) that rotates around the body. By using image processing libraries (AVW 3.0 Biomedical Imaging; Mayo Foundation, Rochester, MN), 3-dimensional rendering of the stomach permitted its volume (mL) to be calculated. There is high intraobserver reproducibility to measure gastric volume with this technique.23,24

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Gastric Emptying Gastric emptying of solids and liquids was measured using dual-isotope, planar scintigraphy and a meal (total calories, 296 kcal; 32% protein, 35% fat, and 33% carbohydrate) consisting of eggs labeled with 99mTc-sulfur colloid (0.75 mCi) during cooking, 1 slice of bread, and 240 mL of 1% milk labeled with 111In-DTPA (0.05 mCi). Anterior and posterior gamma camera images were obtained of the intragastric content immediately after meal ingestion, every 15 minutes for the first 2 hours, and then every 30 minutes for the next 2 hours. Data were analyzed as in previous studies25 to estimate gastric emptying.

Satiation and Postchallenge Symptoms A standardized nutrient drink (Ensure: 1 kcal/mL, 11% fat, 73% carbohydrate, and 16% protein) was used to measure satiation as the maximum tolerated volume (MTV) after ingestion at a rate of 30 mL/min, and symptoms 30 minutes postchallenge.26,27

Body Composition Total body fat and lean mass were measured by dualenergy x-ray absorptiometry (Lunar Corporation, Madison, WI) at baseline and after treatment.28

Fasting and 2-Hour Postprandial Hormonal Responses To measure levels of leptin, insulin, GLP-1, and PYY during the nutrient drink test, blood draws were obtained at ⫺15, 0 (beginning of nutrient drink test), 15, 30, 45, 60, and 120 minutes.

Immunochemical Assays of Plasma Leptin level was measured by the human leptin doubleantibody radioimmunoassay kit (Linco Research, Inc., St. Louis, MO). The separation of free and bound antigen is achieved by using a double-antibody system. Total ghrelin level was measured by a radioimmunoassay technique (Linco Research, Inc.). The assay uses 125I-labeled ghrelin and a ghrelin antiserum to determine the level of total ghrelin in plasma. Insulin was measured by a 2-site, 1-step (sandwich) immunoenzymatic assay performed on the Beckman UniCel DxI automated immunoassay system (Beckman Instruments, Chaska, MN). GLP-1 was measured as the biologically active GLP-1 (7-36, 7-37) using a 2-site noncompetitive immunoassay based on enzyme-labeled quantification of GLP-1 detected by a fluorogenic substrate. PYY was measured by radioimmunoassay (Linco Research, Inc.). PYY exists in at least 2 molecular forms, 1-36 and 3-36, both of which are physiologically active.

Candidate Genes All participants in the study (men and women, obese and overweight, sibutramine and placebo-treated, except for 1 man who was randomized to sibutramine) underwent venous blood sampling for analysis for the candidate genes. Venous blood drawn from a forearm vein was stored and submitted for genetic analysis as de-identified samples. Genomic DNA was

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Table 1. Gastric Parameters by Treatment Assignment in Overweight Participants Overweight Placebo (n ⫽ 12)

GE solid t1/2, mina GE liquid t1/2, min Gastric volume fasting, mL Gastric volume postmeal, mL MTV, mLa Aggregate symptom score, mm (visual analogue scale) Weight, kga BMI, kg/m2 Fasting leptin level, ng/mL Fasting ghrelin level Fasting insulin level, ␮U/mL Fasting GLP-1 level,pmol/L Fasting PYY level, pg/mL ⌬ postprandial-baseline leptin level, ng/mL ⌬ postprandial-baseline ghrelin level, ng/mL ⌬ postprandial-baseline insulin level, ␮U/mLa ⌬ postprandial-baseline GLP-1 level, pmol/L ⌬ postprandial-baseline PYY level, pg/mLa

Sibutramine (n ⫽ 12)

Pretreatment

Posttreatment

Pretreatment

Posttreatment

119.5 ⫾ 6.4 17.9 ⫾ 1.7 202 ⫾ 17 700 ⫾ 26 1114 ⫾ 89 141 ⫾ 21 78.4 ⫾ 1.7 27.5 ⫾ 0.4 14.3 ⫾ 2.1 793 ⫾ 59 10.2 ⫾ 2.9 3.2 ⫾ 1.0 90.9 ⫾ 6.3 ⫺0.9 ⫾ 0.23 ⫺164 ⫾ 22 71.2 ⫾ 9.1 4.9 ⫾ 1.3 71.6 ⫾ 16.5

121.7 ⫾ 8.6 19.2 ⫾ 2.6 222 ⫾ 13 690 ⫾ 30 1059 ⫾ 111 117 ⫾ 19 76.3 ⫾ 2.6 26.8 ⫾ 0.5 13.0 ⫾ 2.0 818 ⫾ 73 7.6 ⫾ 1.5 3.9 ⫾ 1.1 90.8 ⫾ 8.0 ⫺1.1 ⫾ 0.23 ⫺198 ⫾ 31 79.2 ⫾ 15.2 3.5 ⫾ 1.6 57.9 ⫾ 11.1

114.7 ⫾ 7.1 21.6 ⫾ 3.2 229 ⫾ 27 720 ⫾ 36 1266 ⫾ 97 147 ⫾ 22 80.9 ⫾ 2.6 28.4 ⫾ 0.3 20.5 ⫾ 2.7 606 ⫾ 41 7.9 ⫾ 0.7 6.6 ⫾ 2.8 108 ⫾ 7.9 ⫺1.4 ⫾ 0.56 ⫺97 ⫾ 20 89.9 ⫾ 13.6 3.8 ⫾ 1.9 86.2 ⫾ 10.9

131.2 ⫾ 8.5 22.8 ⫾ 4.4 221 ⫾ 21 711 ⫾ 40 1077 ⫾ 84 170 ⫾ 27 74.6 ⫾ 2.5 26.5 ⫾ 0.5 13.4 ⫾ 2.5 755 ⫾ 81 6.2 ⫾ 0.9 5.0 ⫾ 1.7 99.4 ⫾ 8.6 ⫺1.2 ⫾ 0.44 ⫺151 ⫾ 36 58.9 ⫾ 10.0 7.3 ⫾ 1.6 114.3 ⫾ 17.6

NOTE. All values are mean ⫾ SEM unless otherwise specified. Observed data without imputation, minimum number was 9 subjects over all the subgroups. aP ⬍ .05 for effect of sibutramine vs placebo.

isolated from whole blood using the Purgene DNA purification performed on the Autopure LS (Gentra Systems, Minneapolis, MN). GN␤3 genotypes were determined by methods in the literature using real-time polymerase chain reaction based on TaqMan chemistries and TaqMan primer-probe assays for GN␤3 SNP C825T (rs6489738) amplification and detection by fluorescence. The distribution of GN␤3 SNP C825T genotypes in 152 healthy controls in our prior studies from the same community was CC 50.7%, TC 40.8%, and TT 8.6%.29 Molecular assays to genotype the SERT-P (SLC6A4) promoter and ␣2-1291C/G polymorphisms that respectively influence serotonergic and ␣2 adrenergic functions were performed as described earlier.14,30 ␣2-1291C/G also was analyzed by denaturing high-performance liquid chromatography using the Varian Helix System (Varian Inc., Palo Alto, CA). The distribution of SLC6A4 SNP genotypes in 120 healthy controls in our prior studies from the same community was LL 31%, LS 49%, and SS 20%.14 The genotype distribution for ␣2A-1291C/G was CC 57%, CG 36%, and GG 7%.14 All PNMT data analyzed for G148A single-nucleotide polymorphism, as well as random samples in which polymorphisms were detected on the assays described earlier, were confirmed by dye terminator sequencing performed at the Mayo Molecular Biology Core Facility using an ABI 3730xl sequencer (PerkinElmer Applied Biosystems, Foster City, CA). Primers are available on request. In Caucasians, the reported distributions of GG, GA, and AA PNMT genotypes (G148A) are 39%, 36%, and 25%, respectively.12 After the genotypes were analyzed, results were submitted to the study coordinator (D.A.I.) to re-associate with the patient identifiers and subsequent analysis with the rest of the data by the study statistician (A.R.Z.).

Treatment and Follow-up Evaluation After completion of baseline studies, participants were randomized (stratified by sex and BMI group) to receive either placebo or sibutramine, 15 mg/day, for 12 weeks. All participants were provided with the LEARN manual,31 a commercially available self-help behavioral manual for weight loss. They also received brief, structured, behavior therapy sessions (10 –15 min) delivered by trained master’s- or doctoral-level behavioral therapists (M.M.C., S.A.K., K.M.G.) every 4 weeks. To enhance consistency of the behavioral visits, each session had 3 content areas to review, participants were encouraged to read corresponding sessions in the LEARN manual, and the interventionists met monthly to ensure adherence to the study protocol. During these study visits, vital signs, compliance, and side effects were evaluated. During the last 3 days of treatment, participants underwent the same studies as performed at baseline.

Statistical Analysis For the primary end points (weight, gastric emptying), the changes (postdrug ⫺ predrug value) were analyzed using an analysis of covariance with sex and BMI group (overweight vs obese) as covariates. A similar approach was used to assess treatment effects on gastric volumes, satiation (MTV and aggregate symptom score), and the fasting and hormone responses to the meal (ie, mean postmeal value ⫺ average fasting in each subject) using sex, BMI group, and the corresponding predrug value as covariates. Thus, for example, we compared the effects of treatment (data appear in Tables 1 and 2 in posttreatment columns) on the change in PYY level after the meal (ie, mean postprandial ⫺ fasting) using as a covariate the corresponding change mea-

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Table 2. Gastric Parameters by Treatment Assignment in Obese Participants Obese Placebo (n ⫽ 11)

GE solid t1/2, mina GE liquid t1/2, min Gastric volume fasting, mL Gastric volume postmeal,mL MTV, mLa Aggregate symptom score, mm (visual analogue scale) Weight, kga BMI, kg/m2 Fasting leptin level, ng/mL Fasting ghrelin level Fasting insulin level, ␮U/mL Fasting GLP-1 level, pmol/L Fasting PYY level, pg/mL ⌬ postprandial-baseline leptin level, ng/mL ⌬ postprandial-baseline ghrelin level, ng/mL ⌬ postprandial-baseline insulin level, ␮U/mLa ⌬ postprandial-baseline GLP-1 level, pmol/L ⌬ postprandial-baseline PYY level, pg/mLa

Sibutramine (n ⫽ 13)

Pretreatment

Posttreatment

Pretreatment

Posttreatment

119.4 ⫾ 16.3 20.8 ⫾ 3.6 215 ⫾ 15 669 ⫾ 34 1289 ⫾ 133 158 ⫾ 22 100.8 ⫾ 3.6 35.7 ⫾ 0.9 29.4 ⫾ 6.3 586 ⫾ 66 13.6 ⫾ 2.7 3.7 ⫾ 1.0 114 ⫾ 9.9 ⫺2.2 ⫾ 0.8 ⫺85 ⫾ 22 127.6 ⫾ 24.6 6.8 ⫾ 1.3 104.8 ⫾ 13.7

113.9 ⫾ 10.7 16.7 ⫾ 2.1 236 ⫾ 18 676 ⫾ 27 1328 ⫾ 131 187 ⫾ 18 100.5 ⫾ 3.8 35.6 ⫾ 1.1 24.9 ⫾ 4.8 555 ⫾ 41 13.8 ⫾ 2.8 4.3 ⫾ 1.3 97.1 ⫾ 8.2 ⫺1.0 ⫾ 0.7 ⫺85 ⫾ 20 128.9 ⫾ 18.6 6.3 ⫾ 1.9 94.3 ⫾ 15.4

116.7 ⫾ 11.1 20.4 ⫾ 2.6 255 ⫾ 19 729 ⫾ 29 1211 ⫾ 66 123 ⫾ 13 96.0 ⫾ 4.0 34.0 ⫾ 1.0 20.4 ⫾ 2.8 641 ⫾ 50 13.1 ⫾ 1.8 3.2 ⫾ 0.6 104.0 ⫾ 10.9 ⫺1.6 ⫾ 0.4 ⫺103 ⫾ 20 109.8 ⫾ 13.4 4.8 ⫾ 0.8 60.2 ⫾ 10.4

119.6 ⫾ 10.3 26.5 ⫾ 4.7 233 ⫾ 17 678 ⫾ 25 1044 ⫾ 119 171 ⫾ 16 90.1 ⫾ 4.6 32.1 ⫾ 1.2 17.4 ⫾ 2.5 660 ⫾ 48 11.3 ⫾ 1.9 1.9 ⫾ 0.4 86.3 ⫾ 7.1 ⫺1.1 ⫾ 0.2 ⫺146 ⫾ 18 103.2 ⫾ 13.1 5.7 ⫾ 0.9 73.6 ⫾ 13.5

NOTE. All values are mean ⫾ SEM unless otherwise specified. Observed data without imputation, minimum number was 9 subjects over all the subgroups. aP ⬍ .05 for effect of sibutramine vs placebo.

sured before treatment (value appears in Table in pretreatment columns). For all end points, in addition to the main effects of sex, BMI group, and treatment group, three 2-way interaction terms and a single 3-way interaction term were included in these models. To examine the potential influence of candidate genes on treatment effects, separate analysis of covariance models were used for each of the end points noted earlier for each single candidate gene (included in the model as a categoric predictor with 2 specific genotypes, eg, LL vs LS/SS for SLC6A4). Sex, actual BMI value, and, for some responses, the corresponding predrug value were included as covariates, and a gene category by treatment interaction term was included in the model. To enable intent-to-treat analyses, the few missing (postdrug) response values were imputed using the corresponding overall (subjects with nonmissing data) mean value for that response. The error degrees of freedom in the respective analysis of covariance models were reduced by 1 for each missing value imputed.

Results Demographics, Baseline Gastric Function, and Genotype Distributions Participants were recruited from January to December 2005. The trial flow is summarized in Figure 1. Compliance with study medication was checked by pill count at the end of treatment and, for all participants, less than 1% of assigned medication was returned at the end of study. Three subjects withdrew from the sibutramine group: 2 because of side effects (hypertension) and another withdrew for personal reasons. Two subjects in the placebo group did not continue because of noncompliance with study procedures.

Figure 1. Trial flow.

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Table 3. Baseline Characteristics of Participants

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been provided by BMI category, sex, and treatment assigned in Table 4.

Overweight (n ⫽ 24)

Obese (n ⫽ 24)

36.7 ⫾ 1.7 71

44.2 ⫾ 2.6 54

87.5 4.2 8.3 79.6 ⫾ 1.6 27.9 ⫾ 0.3 34.0 ⫾ 0.5 44,007 ⫾ 1846 33,202 ⫾ 1261 117.1 ⫾ 4.7

95.8 4.2 0 98.2 ⫾ 2.7 34.8 ⫾ 0.7 40.4 ⫾ 0.9 50,131 ⫾ 2813 45,477 ⫾ 1864 117.9 ⫾ 0.4

19.8 ⫾ 1.8

20.6 ⫾ 2.1

216.2 ⫾ 16.2

236.6 ⫾ 12.7

494.0 ⫾ 15.4

464.5 ⫾ 18.3

1190.1 ⫾ 66.6 144.0 ⫾ 15.2

1247.1 ⫾ 69.6 138.6 ⫾ 12.7

17.4 ⫾ 1.8

24.5 ⫾ 3.3

9.08 ⫾ 1.5

13.3 ⫾ 1.5

Satiation, Postchallenge Symptoms, and Gastric Volumes

4.9 ⫾ 1.5

3.4 ⫾ 0.6

99.4 ⫾ 5.2

108.6 ⫾ 7.4

There was a significant overall treatment effect of sibutramine on MTV (P ⫽ .032) and a borderline significant overall effect of sex on MTV (P ⫽ .077), but no significant interaction effects were detected. More specifically, a significant treatment effect on MTV was observed in obese women, with lower volume tolerated on sibutramine compared with placebo (P ⫽ .034). No overall treatment, sex, or group differences were observed in fasting and postprandial gastric volumes.

Age, y Sex, females, % Ethnicity, % White African American Hispanic Weight, kg BMI, kg/m2 Waist circumference, in Lean body mass, g Fat body mass, g Gastric emptying solids, t½, min Gastric emptying liquids, t½, min Fasting gastric volume, mL Postprandial gastric volume, mL MTV, mL Aggregate symptom score, mm Fasting leptin level, ng/mL Fasting insulin level, ␮U/mL Fasting GLP-1 level, pmol/L Fasting PYY level, pg/mL

NOTE. All values are mean ⫾ SEM unless otherwise specified.

The demographics are shown in Table 3. Tables 1 and 2 summarize the gastric function and hormone data in the overweight and obese participants by treatment assignment. Table 4 summarizes the percentage genotype distributions for the 4 candidate genes evaluated in 47 of the 48 participants (a DNA sample from 1 patient was not available because of technical difficulties). These distributions have

Gastric Emptying There was a significant overall treatment effect of sibutramine on gastric emptying of solids (P ⫽ .036, Table 1), but not liquids (Figure 2). A significant treatment by sex interaction was detected (P ⫽ .030). In particular, a significant reduction in gastric emptying of solids (P ⫽ .020) and liquids (P ⫽ .002) was observed in obese women treated with sibutramine compared with placebo.

Weight Change After 12 weeks of treatment, subjects treated with sibutramine had a significant weight reduction of 5.4 ⫾ .8 kg compared with .9 ⫾ .9 kg with placebo (P ⬍ .001). Overweight subjects on sibutramine lost 6.4 ⫾ 1.3 kg compared with 1.5 ⫾ 1.3 kg in the placebo group. The obese subjects on sibutramine showed a 4.5 ⫾ 1.0 kg decrease compared with .3 ⫾ 1.2 kg in the placebo group. Obese women treated with sibutramine had a significant weight loss (P ⫽ .002 vs placebo). In obese men, the treatment effect was rather modest (Figure 3), although treatment by sex according to the BMI group interaction was not significant (P ⫽ .122).

Fasting and Postnutrient Hormonal Responses No significant effect of treatment was observed for any hormone tested during fasting (except ghrelin).

Table 4. Percentage Genotype Distribution for the 4 Candidate Genes of Interest by BMI Category, Sex, and Treatment Gene SLC6A4

␣2A1291 C/G GN␤3 C825T

PNMT G148A

Genotype

Laboratory controls*

Total

Obese

Overweight

Male

Female

Sib

Pla

LL LS SS CC CG GG CC TC TT AA GA GG

31 49 20 57 36 7 51 40 9

31 46 21 48 36 15 50 48 — 35 52 8

21 46 33 33 46 21 50 50 — 38 62 0

42 46 8 63 25 9 50 46 — 33 42 17

22 44 28 33 50 12 61 33 — 44 39 6

37 47 18 57 27 16 43 57 — 30 60 10

32 44 20 44 40 12 40 56 — 36 52 8

30 48 22 53 30 17 61 39 — 35 52 9

NOTE. Total count for patients does not reach 100% because DNA was not available from 1 male patient who was randomized to sibutramine. Sib, sibutramine; Pla, placebo. Data from studies by Kim et al14 and Andresen et al.29 *Data from control groups in previous studies in our laboratory.

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There was a significant correlation between postnutrient change in leptin and weight loss (Spearman R [rs] ⫽ 0.47; P ⫽ .002) and between postprandial change in ghrelin and weight loss (rs ⫽ ⫺0.39; P ⫽ .010). Other possible associations with degree of weight loss were observed for change in baseline leptin (rs ⫽ 0.39; P ⫽ .009), baseline ghrelin (rs ⫽ ⫺0.37; P ⫽ .014), and the total calories ingested at the ad libitum meal (rs ⫽ 0.38; P ⫽ .011). There were no significant associations of the change in weight with the change in gastric emptying of solids or liquids, the change in MTV, or the change in the average postprandial PYY, GLP-1, or insulin levels, nor with the baseline changes in PYY, GLP-1, and insulin levels.

Influence of Candidate Genes on Weight Loss and Gastric Functions The SLC6A4 LS/SS genotype had a significant influence on the effect of treatment for the change in weight (P ⫽ .024). Subjects with SLC6A4 LS/SS genotype on sibutramine had an average weight loss of 6.1 ⫾ 1.0 kg compared with a 0.1 ⫾ 0.9 kg average weight increase on placebo; in contrast, for subjects with the homozygous long (SLC6A4 LL) genotype, changes in weight were similar in both treatment groups, with an average weight loss of 3.3 ⫾ 1.8 kg in placebo compared with a 3.9 ⫾ 1.6 kg weight loss in the sibutramine group. No effects on weight response to treatment were found with the ␣2A-1291 C/G, PNMT, and GN␤3 genotypes. None of the candidate genes significantly influenced the gastric functions evaluated.

Discussion

Figure 2. Gastric emptying of solids and liquids in obese or overweight individuals treated with placebo or sibutramine.

The effects of treatment on postnutrient values of leptin or GLP-1 also were not significant. Review of the data for postprandial delta PYY shows that, for both the overweight and obese groups, there was a higher delta PYY with sibutramine during posttreatment compared with pretreatment with sibutramine, whereas the delta PYY with placebo was lower posttreatment compared with pretreatment. The converse applies for postprandial delta insulin levels, which were lower postsibutramine compared with presibutramine, particularly in the overweight group, and were essentially unchanged with placebo. The statistical analysis shows that, postnutrient challenge, the PYY levels were higher (P ⫽ .035) and insulin levels were lower (P ⫽ .009) in the sibutramine group compared with placebo. There was no significant effect of treatment by BMI group or by sex on the PYY or insulin levels. The analysis of covariance indicated treatment effects on the fasting ghrelin levels (P ⫽ .038) and no interactions of treatment with sex or BMI group. No treatment effects were detected for the postprandial change in ghrelin response (P ⫽ .15), nor any treatment interactions with sex or BMI group.

In this study, overweight and obese subjects treated with sibutramine had an expected significant weight reduction (average, 5.4 ⫾ 0.8 kg). We found that sibutramine caused a significant delay in gastric emptying of solids relative to placebo treatment, with a greater effect being shown in obese women, in whom there was also a delay in gastric emptying of liquids. There was also a significant effect of sibutramine on the maximum volume that could be ingested compared with placebo. This reduction in the volume of meal tolerated in response to sibutramine treatment also was significant among obese female participants. We also found that individuals with the SLC6A4 LS/SS genotype had greater weight loss with sibutramine treatment. The observed effects of sibutramine on gastric emptying have not been reported previously. There are only 2 animal studies in the literature on the effects of sibutramine on gastrointestinal function. In a recent study, Xu and Chen32 showed that sibutramine, 5 mg/kg, increased fasting tone and decreased postprandial accommodation in the proximal stomach in a study of 7 dogs. In another study by Kim et al,33 sibutramine mesylate decreased gastric volume and total acidity when administered to beagle dogs at doses of 3.45 and 11.50 mg/kg. These dose levels are far higher than the total doses (10 and 15 mg) approved for use in human beings in the treatment of obesity. Our study in human beings provides observation of the effect of sibutramine (15 mg/day) on gastric emptying, documents the effect on the volume of food tolerated in a satiation test, but does not confirm the negative effects on gastric accommodation observed in dogs at dose levels at least 20-fold higher than those permissible in human beings. The observations in our study are consistent with the observations that early satiation and weight loss are associated with

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Figure 3. Change in body weight in overweight and obese males and females treated with placebo and sibutramine (A). Note the overall significant effects of sibutramine on weight loss are most clearly demonstrable in obese females. Changes in gastric emptying of solids (B) and liquids (C) in overweight and obese males and females treated with placebo and sibutramine. Note that overall significant effects of sibutramine on gastric emptying are most clearly demonstrable in obese females and overweight males.

functional dyspepsia26 and that gastric emptying delay is one of the factors that has been proven to be associated with postprandial symptoms such as satiation and bloating in dyspepsia and in obese individuals.34,35 An alternative hypothesis is that greater weight loss may cause the earlier satiation. However, based on the regression parameters from a prior study,27 one could account for an average reduction of postprandial gastric volume of 12 mL and of MTV of less than 30 mL based on the approximate 2 kg/m2 reduction in BMI with sibutramine. Because sibutramine treatment was associated with a mean reduction in MTV of at least 150 mL, weight loss per se cannot explain the change in satiation. As expected, fasting levels of leptin decreased with weight loss.36 We also observed an increased response of PYY to a challenge liquid meal with sibutramine. This would appear to be counterintuitive given the delay in gastric emptying of nutrients and the release of PYY from ileal mucosa. However, it is well known that ␣and ␤-adrenergic mechanisms regulate PYY secretion37– 40; therefore, we believe that the increase in PYY postprandially reflects the direct effect of the sibutramine on the release of PYY. Batterham et

al41 suggested that exogenous PYY3–36 is associated with reduced nutrient intake. It is conceivable that the increased PYY released in response to sibutramine may in part mediate the effect of sibutramine on gastric emptying and weight loss, given the known effects of PYY as part of the ileal brake mechanism,42 retarding gastric emptying and reducing upper-gastrointestinal motility. Although the present studies do not allow determination of whether the effect of sibutramine on gastric emptying is direct through the action on serotonergic or adrenergic mechanisms or indirect through the release of PYY, the data suggest that the role of these different mechanisms and their contribution to weight loss deserve further study. We observed lower postnutrient plasma insulin concentrations with sibutramine treatment. This may reflect the greater weight loss and expected coincident improvement in insulin action, the delayed gastric emptying of solid and liquid nutrient, or, conceivably, a direct inhibition of insulin secretion by the sibutramine. In view of the evidence that norepinephrine inhibits insulin release by isolated pancreatic islet cells,43 the latter possibility cannot be excluded.

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A third observation is that subjects with SLC6A4 LS/SS genotypes had a greater weight loss with sibutramine compared with placebo. This may suggest that people with partial functioning of the serotonin transporter have an increased availability of serotonin for receptor-ligand interaction, leading to a more effective action of sibutramine, which also functions to prevent the re-uptake of serotonin. It is intriguing that overall weight loss with placebo was 0.9 ⫾ 0.9 kg, whereas patients with LL SLC6A4 genotype on placebo lost 3.3 ⫾ 1.8 kg. This may suggest the hypothesis that the LL SLC6A4 genotype may enhance the response to placebo. There was some evidence that the TT/TC genotype of GN␤3 was associated with greater weight loss in response to placebo in a prior study comparing weight loss in response to sibutramine vs placebo.44 The mechanism whereby such genetic variation in SLC6A4 leads to a greater response to placebo is unclear and requires further study. We were unable to confirm the reports in the literature on the association of weight loss with the genotype polymorphisms of the phenylethanolamine N-methyltransferase12 and GN␤3.13 Further studies are required to evaluate the role of pharmacogenetics in sibutramine therapy and to determine whether the genetic influence on the weight response is mediated through a change in gastric function. In summary, our data suggest that the stomach may play an important role in the induction of satiation and reduced calorie intake in patients receiving sibutramine for weight loss. However, we regard these results as exploratory, and further studies are required to confirm the effects of sibutramine on gastric emptying and satiation. Moreover, the role of genetic variation on weight loss in response to sibutramine deserves further study; however, it appears that genetic modulation of endogenous serotonin may be a factor that may explain, at least in part, the wide variation in the weight loss observed in clinical trials with this medication. Understanding such genetic variation may facilitate the selection of candidates or individualizing the dose of medication to achieve the desired effect. References 1. Flegal KM, Carroll MD, Ogden CL, et al. Prevalence and trends in obesity among US adults, 1999-2000. JAMA 2002;288:1723– 1727. 2. Yanovski SZ, Yanovski JA. Obesity. N Engl J Med 2002;346:591– 602. 3. Arterburn DE, Crane PK, Veenstra DL. The efficacy and safety of sibutramine for weight loss: a systematic review. Arch Intern Med 2004;164:994 –1003. 4. Wadden TA, Berkowitz RI, Womble LG, et al. Randomized trial of lifestyle modification and pharmacotherapy for obesity. N Engl J Med 2005;353:2111–2120. 5. Meguid MM, Fetissov SO, Varma M, et al. Hypothalamic dopamine and serotonin in the regulation of food intake. Nutrition 2000;16:843– 857. 6. Weintraub M, Sundaresan PR, Madan M, et al. Long-term weight control study. I (weeks 0 to 34). The enhancement of behavior modification, caloric restriction, and exercise by fenfluramine plus phentermine versus placebo. Clin Pharmacol Ther 1992;51: 586 –594. 7. Weintraub M, Sundaresan PR, Schuster B, et al. Long-term weight control study. IV (weeks 156 to 190). The second double-blind phase. Clin Pharmacol Ther 1992;51:608 – 614. 8. Deutsch JA, Young WG, Kalogeris TJ. The stomach signals satiety. Science 1978;201:165–167.

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9. Deutsch JA. The role of the stomach in eating. Am J Clin Nutr 1985;42:1040 –1043. 10. Thumshirn M, Camilleri M, Choi MG, et al. Modulation of gastric sensory and motor functions by nitrergic and alpha2-adrenergic agents in humans. Gastroenterology 1999;116:573–585. 11. Tack J, Sarnelli G. Serotonergic modulation of visceral sensation: upper gastrointestinal tract. Gut 2002;51:i77–i80. 12. Peters WR, MacMurry JP, Walker J, et al. Phenylethanolamine N-methyltransferase G-148A genetic variant and weight loss in obese women. Obes Res 2003;11:415– 419. 13. Hauner H, Meier M, Jockel KH, et al. Prediction of successful weight reduction under sibutramine therapy through genotyping of the G-protein beta3 subunit gene (GNB3) C825T polymorphism. Pharmacogenetics 2003;13:453– 459. 14. Kim HJ, Camilleri M, Carlson PJ, et al. Association of distinct alpha(2) adrenoceptor and serotonin transporter polymorphisms with constipation and somatic symptoms in functional gastrointestinal disorders. Gut 2004;53:829 – 837. 15. Yeo A, Boyd P, Lumsden S, et al. Association between a functional polymorphism in the serotonin transporter gene and diarrhoea predominant irritable bowel syndrome in women. Gut 2004;53:1452–1458. 16. Camarena B, Ruvinskis E, Santiago H, et al. Serotonin transporter gene and obese females with impulsivity. Mol Psychiatry 2002;7:829 – 830. 17. Yamakawa M, Fukushima A, Sakuma K, et al. Serotonin transporter polymorphisms affect human blood glucose control. Biochem Biophys Res Commun 2005;334:1165–1171. 18. Lesch KP, Bengel D, Heils A, et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996;274:1527–1531. 19. Zigmond AS, Snaith RP. The hospital anxiety and depression scale. Acta Psychiatr Scand 1983;67:361–370. 20. Swenson WM, Morse RM. The use of a self-administered alcoholism screening test (SAAST) in a medical center. Mayo Clin Proc 1975;50:204 –208. 21. Yanovski SZ. Binge eating disorder: current knowledge and future directions. Obes Res 1993;1:306 –324. 22. Bouras EP, Delgado-Aros S, Camilleri M, et al. SPECT imaging of the stomach: comparison with barostat, and effects of sex, age, body mass index, and fundoplication. Single photon emission computed tomography. Gut 2002;51:781–786. 23. Delgado-Aros S, Burton D, Brinkmann BH, et al. Reliability of a semi automated analysis to measure gastric accommodation using SPECT in humans. Gastroenterology 2001;120:A-287. 24. De Schepper H, Camilleri M, Cremonini F, et al. Comparison of gastric volumes in response to isocaloric liquid and mixed meals in humans. Neurogastroenterol Motil 2004;16:567–573. 25. Camilleri M, Zinsmeister AR, Greydanus MP, et al. Towards a less costly but accurate test of gastric emptying and small bowel transit. Dig Dis Sci 1991;36:609 – 615. 26. Tack J, Piessevaux H, Coulie B, et al. Role of impaired gastric accommodation to a meal in functional dyspepsia. Gastroenterology 1998;115:1346 –1352. 27. Delgado-Aros S, Cremonini F, Castillo JE, et al. Independent influences of body mass and gastric volumes on satiation in humans. Gastroenterology 2004;126:432– 440. 28. Jensen MD, Kanaley JA, Roust LR, et al. Assessment of body composition with use of dual-energy x-ray absorptiometry: evaluation and comparison with other methods. Mayo Clin Proc 1993; 68:867– 873. 29. Andresen V, Camilleri M, Kim HJ, et al. Is there an association between GN␤3 C825T genotype and lower functional gastrointestinal disorders? Gastroenterology 2006;130:1985–1994. 30. Camilleri M, Atanasova E, Carlson PJ, et al. Serotonin-transporter polymorphism pharmacogenetics in diarrhea-predominant irritable bowel syndrome. Gastroenterology 2002;123:425– 432.

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31. Brownell K. The LEARN program for weight management. 10th ed. Dallas, TX: American Health Publishing Company, 2004. 32. Xu J, Chen JD. Peripheral mechanisms of sibutramine involving proximal gastric motility in dogs. Obesity 2006;14:1363–1370. 33. Kim EJ, Park EK, Suh KH. Safety pharmacology of sibutramine mesylate, an anti-obesity drug. Hum Exp Toxicol 2005;24: 109 –119. 34. Delgado-Aros S, Camilleri M, Cremonini F, et al. Contributions of gastric volumes and gastric emptying to meal size and postmeal symptoms in functional dyspepsia. Gastroenterology 2004;127: 1685–1694. 35. Delgado-Aros S, Camilleri M, Castillo EJ, et al. Effect of gastric volume or emptying on meal-related symptoms after liquid nutrients in obesity: a pharmacologic study. Clin Gastroenterol Hepatol 2005;3:997–1006. 36. Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292–295. 37. Zhang T, Brubaker PL, Thompson JC, et al. Characterization of peptide-YY release in response to intracolonic infusion of amino acids. Endocrinology 1993;132:553–557. 38. Brechet S, Plaisancie P, Dumoulin V, et al. Involvement of beta1and beta2- but not beta3-adrenoceptor activation in adrenergic PYY secretion from the isolated colon. J Endocrinol 2001;168: 177–183. 39. Lin HC, Neevel C, Chen JH. Slowing intestinal transit by PYY depends on serotonergic and opioid pathways. Am J Physiol 2004;286:G558 –G563. 40. Lin HC, Neevel C, Chen PS, et al. Slowing of intestinal transit by fat or peptide YY depends on beta-adrenergic pathway. Am J Physiol 2003;285:G1310 –G1316. 41. Batterham RL, Cohen MA, Ellis SM, et al. Inhibition of food intake

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in obese subjects by peptide YY3-36. N Engl J Med 2003;349:941–948. 42. Spiller RC, Trotman IF, Adrian TE, et al. Further characterisation of the ’ileal brake’ reflex in man— effect of ileal infusion of partial digests of fat, protein, and starch on jejunal motility and release of neurotensin, enteroglucagon, and peptide YY. Gut 1988;29: 1042–1051. 43. Yajima H, Komatsu M, Sato Y, et al. Norepinephrine inhibits glucose-stimulated, Ca2⫹-independent insulin release independently from its action on adenylyl cyclase. Endocr J 2001;48: 647– 654. 44. Hauner H, Meier M, Jockel KH, Frey UH, Siffert W. Prediction of successful weight reduction under sibutramine therapy through genotyping of the G-protein beta3 subunit gene (GNB3) C825T polymorphism. Pharmacogenetics 2003;13:453– 459.

Address requests for reprints to: Michael Camilleri, MD, Mayo Clinic, Charlton 8-110, 200 First Street SW, Rochester, Minnesota 55905. e-mail: [email protected]. Supported by a General Clinical Research Center (grant M01RR00585) from the National Institutes of Health. Dr Camilleri is supported by grants R01 DK67071 and K24 DK02638 and Dr Jensen was supported in part by the Minnesota Obesity Center grant DK50456 from the National Institutes of Health. Dr Camilleri participates as a member of a Mayo Clinic advisory group for the development of devices for the treatment of obesity. The Mayo Clinic owns equity in the company Enteromedics, and Dr Camilleri first received an annual royalty in 2005. There is no individual or institutional financial interest in the drug under study, subutramine. The authors thank Mrs Cindy Stanislav for excellent secretarial support.