Effect of L-Tryptophan and L-Leucine on Gut Hormone Secretion, Appetite Feelings and Gastric Emptying Rates in Lean and Non-Diabetic Obese Participants: A Randomized, Double-Blind, Parallel-Group Trial

Background/Objectives Gut hormones such as cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) play a role as satiation factors. Strategies to enhance satiation peptide secretion could provide a therapeutic approach for obesity. Carbohydrates and lipids have been extensively investigated in relation to peptide release. In contrast, the role of proteins or amino acids is less clear. Our aim was to compare the effects of the amino acids L-tryptophan (L-trp) and L-leucine (L-leu) separately on gastric emptying and gut peptide secretion. Participants/Methods The study was conducted as a randomized (balanced), double-blind, parallel-group trial. A total of 10 lean and 10 non-diabetic obese participants were included. Participants received intragastric loads of L-trp (0.52 g and 1.56 g) and L-leu (1.56 g), dissolved in 300 mL tap water; 75 g glucose and 300 mL tap water served as control treatments. Results Results of the study are: i) L-trp at the higher dose stimulates CCK release (p = 0.0018), and induces a significant retardation in gastric emptying (p = 0.0033); ii) L-trp at the higher dose induced a small increase in GLP-1 secretion (p = 0.0257); iii) neither of the amino acids modulated subjective appetite feelings; and iv) the two amino acids did not alter insulin or glucose concentrations. Conclusions L-trp is a luminal regulator of CCK release with effects on gastric emptying, an effect that could be mediated by CCK. L-trp’s effect on GLP-1 secretion is only minor. At the doses given, the two amino acids did not affect subjective appetite feelings. Trial Registration ClinicalTrials.gov NCT02563847


Introduction
Protein is currently believed to exert the greatest appetite-suppressing effect of the three macronutrients (carbohydrates, fats and proteins) in animals and humans (1). Highprotein diets have been extensively studied for their ability to reduce total energy intake and body weight (1). Mechanisms that have been suggested include stimulation of insulin release (2), postprandial thermogenesis (3), intestinal gluconeogenesis (4), and direct effects of amino acids in regions of the brain (5). In addition, it has been hypothesized that protein-induced satiation could be due to alterations in the release of gastrointestinal satiation peptides, such as cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) (5). Administration of different high-protein diets induced prolonged or higher concentrations of CCK, GLP-1 and PYY compared to normal-protein diets in normal or overweight patients (2,(6)(7)(8)(9). In addition, there is evidence that, like fat, protein digestion to dipeptides or tripeptides and free amino acids enhances gastrointestinal hormone release (10). Already in 1956, it was suggested that an elevated concentration of plasma amino acids serve as a satiation signal for food intake and thereby results in depressed food intake (11). To date the effect of amino acids on satiation peptide release is only rarely studied. Gannon et al. (12) studied the effect of different orally administered amino acids on plasma insulin, glucose and glucagon levels. The majority of amino acids did not significantly affect the glucose levels, however, induced an increase in both, circulating insulin and glucagon (12). These results were in line with a number of previous data showing increased insulin and glucagon release after administration of different amino acids (reviewed in (12)). GLP-1 is a well-known incretin hormone and it remains established whether the amino acid induced increase in insulin is mediated via increased secretion of GLP-1.
Greenfield et al. (13) studied the effect of glutamine on GLP-1, glucagon and insulin concentrations in human subjects. They found that glutamine increases circulating GLP-1, insulin and glucagon in lean, obese and type 2 diabetes subjects (13).
In our own pilot project we found, that the specific amino acid L-tryptophan is a potent stimulus of gallbladder contraction and that this effect is probably mediated through the dose-dependent increase in CCK levels. These results are in line with previous observations showing that L-tryptophan and L-phenylalanine stimulate CCK release in healthy humans (14)(15)(16)(17). A possible mechanism involved includes amino acids induced activation of CaR, which expression was found in CCK releasing I-cells of the gut (18,19). Details on specific amino acid stimulated secretion of other satiation peptides, such as GLP-1 and PYY, in humans are, to date, not available.

Hypotheses
Specific amino acids in the GI tract stimulate the release of satiation factors (GLP-1 and PYY) which is probably mediated through mechanisms involving the activation of the CaR and subsequent release of CCK.

Aim
To investigate the effect of intragastric (IG) amino acids on satiation peptide release and gastric emptying and appetite perception.

Overall design
The study will be performed as a randomized, double-blind, placebo-controlled, crossover study. A randomization list is prepared in advance by computer-randomization (Excel). Double-blind: the investigator (Dr. Anne Christin Meyer-Gerspach) who will administer the solution and the respective study subject are blinded as to the content of the solution. Only the principal investigator (Dr. B. Wölnerhanssen) has access to the randomization list and will prepare the solutions according to it in advance.
Effect of amino acids on gastric emptying and release of satiation peptides in normal weight and obese subjects: Version 2 Seite 6 von 13 Each subject will be studied on 5 occasions with at least three days apart. Sessions will take about 5 hours. Subject's food intake on the preceding day of each study day will be standardized: they will consume an identical meal before 8 p.m. and fast from 10 p.m. In addition, subjects will refrain from alcohol and strenuous exercise for 24 hours before each treatment. Subjects will be admitted to the Phase 1 Research Unit at 8:00 a.m. on each study day. The treatments of each part will be identical in design except for the intragastric perfusion.

Study subjects
Ten healthy normal weight and ten healthy obese volunteers will be recruited.
Recruitment is achieved by word-of-mouth. The screening procedure will include the following assessments: a medical interview and a full physical examination.
Anthropometric measurements including weight, height, BMI, as well as heart rate and blood pressure will be recorded for all participants. In women of child bearing age, a urine pregnancy test will be carried out. There is no safety risk in this study for pregnant women; however, pregnancy might influence results.
The following inclusion criteria are applied: • Healthy normal weight subjects with a body-mass index of 19.0-24.9 • Healthy obese subjects with a body-mass index of > 30 Committee of the University of Basel. All subjects will have to give written informed consent.

Effect of intragastric (IG) infusion of amino acids on satiation peptide release, gastric emptying rates and appetite perception
Subjects will swallow a feeding tube. The IG position of the feeding tube will be verified by rapid injection of 10 mL of air and auscultation of the upper abdomen. In addition, an antecubital vein catheter will be inserted for blood drawing. After taking two fasting blood samples (-15, -1 min), the test solutions will be infused intragastrically within 2 minutes.
The feeding tube will be removed immediately after the infusion is completed. Test solutions compounds are listed in table 1. The order of treatments will be randomized.
At regular time intervals (15,30,45, 60, 90, 120 and 180 min) blood samples will be collected on ice into tubes containing EDTA (6 µmol/L), a protease inhibitor cocktail (Complete ® , EDTA-free, 1 tablet/50 mL blood; Roche, Mannheim, Germany) and a dipeptidylpeptidase IV inhibitor (10 µL/mL; Millipore Corporation, St. Charles, Missouri, USA). Tubes will be centrifuged at 4 C° at 3 000 rpm for 10 min and plasma samples will be processed into different aliquots. All samples will be stored at -70 C° until analysis of metabolites including glucose and amino acid profiles as well as of hormonal responses including insulin, GLP-1, CCK, PYY and ghrelin. The total blood volume taken during one test day will be about 100 mL. Immediately after each blood collection, appetite perceptions, such as feelings of hunger, prospective food consumption, fullness and satiety will be recorded using visual analogue scales (VAS). For determination of the gastric emptying rates, end-expiratory breath samples will be taken at fixed time intervals after instillation of the test solution.

Materials
Effect of amino acids on gastric emptying and release of satiation peptides in normal weight and obese subjects: Version 2 Seite 8 von 13 L-tryptophan and L-leucine will be purchased from Sigma Aldrich Chemical Company, Germany (>97% pure). Glucose will be purchased from Hänseler, Switzerland and 13 Csodium acetate from ReseaChem, Switzerland.
Amino acids solutions will be administered in physiological amounts according to our pilot project. The IG infusions will be freshly prepared each morning of the study and will be at room temperature when administered.

Assessment of appetite
Appetite perceptions (feelings of hunger, fullness, satiation and prospective food consumption) will be scored at regular intervals using visual analogue scales (VAS).
These scores are based on validated data described by Blundell  have previously been designed and described in more detail (25).

Assessment of gastric emptying rates
Gastric emptying rates will be assessed using the 13 C-sodium acetate breath test. This test is an accurate, non-invasive, simple method without radiation exposure, and represents a reliable alternative to scintigraphy, the gold standard for measuring gastric emptying (26,27). The test solutions will be labelled with 50 mg 13 C-sodium acetate; the substrate is rapidly absorbed in the proximal small intestine, metabolized in the liver with the production of 13 CO 2 which is exhaled rapidly, thus, reflecting gastric emptying of nutrients (26,27). Subjects will be asked to exhale through a mouth-piece to collect an end-expiratory breath sample into a foil bag at certain time intervals. The 13 CO 2 breath content will be determined by non-dispersive infrared spectroscopy using an isotope ratio mass spectrophotometer (IRIS; Wagner Analysen Technik, Bremen, Germany). 13 C-abundance in breath is expressed as relative difference (δ ‰) from the universal reference standard (carbon from Pee Dee Belemnite limestone). 13 C-enrichment is defined as the difference between preprandial 13 C-abundance in breath and 13  Cholecystokinin (CCK) will be measured with a commercially available radioimmunoassay kit (Euro-Diagnostica, Malmö, Sweden). This kit is for assay of CCK in plasma. The intra-and interassay coefficient of variation for this assay is below 5.5% and 13.7%, respectively.
Total ghrelin will be measured with a commercially available radioimmunoassay kit (Millipore Corporation, Billerica, Massachusetts, USA). This kit is for quantitative determination of total ghrelin in serum, plasma or tissue culture media. It is highly specific for ghrelin and shows no cross-reactivity with other hormones, like glucagon, leptin or insulin. The intra-and inter-assay coefficient of variation for this assay is below 10.0 % and 14.7 %, respectively.
Insulin will be measured with a commercially available ELISA kits (Abnova, Taipei City, Taiwan). This kit is for quantitative determination of insulin in human plasma (EDTA). It is highly specific for insulin and shows no cross-reactivity with other peptides, e.g. cpeptide or glucagon. The intra-and inter-assay coefficient of variation is below 8.1% and 8.5%, respectively.
Plasma glucose concentration will be measured by a commercially available glucoseoxidase-method (Bayer Consumer Care AG, Basel, Switzerland). This method is highly specific for measurement of glucose in serum or plasma.

Statistical analysis
Descriptive statistics will be used for demographic variables such as age, weight, height, and BMI. Measures of task performance, appetite ratings, physiological data, hormone levels and gastric emptying rates will be analysed using repeated measures ANOVAs.
When significant differences will be found, the Tukey's test for pairwise comparisons will be applied. However, if due to the limited sample size for the behavioral part precludes a parametric approach, a nonparametric statistical test procedure will be applied AUC, etc.) will be obtained using PK Functions for Excel®. AUC values will be calculated by the trapezoidal rule. Differences between treatments will be assessed using either the non-parametric Friedman-test (in case of significant differences followed by pairwise comparison using the Wilcoxon signed ranks test and Bonferroni's correction to account for multiple of comparisons); or the General linear model procedure of repeated-measures ANOVA using simple contrast and Bonferroni correction of P values for multiplicity of comparison. VAS will be analysed by calculating AUC and return to baseline values (interception with y-axis using linear interpolation). Differences will be assessed using the non-parametric Friedman-test or the General linear model procedure of repeated-measures ANOVA. Transformations will be performed before analysis, if response variables are non-normally distributed. All statistical analysis will be done using SPSS for windows software (version 19.0; SPSS Inc, Chicago, Illinois). Values will be reported as mean ± SEM. Differences will be considered to be significant at P ≤ 0.05.
Based on past experience, a sample size of 12-16 subjects per protocol should allow detecting differences of 15 to 25% in the means of our endpoint parameters with a statistical power of 80%.