Effects of intragastric administration of L-tryptophan on the glycaemic response to a nutrient drink in men with type 2 diabetes — impacts on gastric emptying, glucoregulatory hormones and glucose absorption

Effects of intragastric administration of L-tryptophan on the glycaemic response to a nutrient drink in men with type 2 diabetes — impacts on gastric emptying, glucoregulatory hormones and glucose absorption

ParticipantsTwelve men with T2D (mean age: 63 ± 2 years, BMI: 30 ± 1 kg/m2, HbA1c: 49.7 ± 2.5 mmol/mol (6.7 ± 0.1%), duration of diabetes: 10 ± 2 years) participated in the study. Their diabetes was managed by metformin (500–2000 mg/day) alone (n = 7), or in combination with a DPP-4 inhibitor (n = 2) or a sodium-glucose transport inhibitor (n = 3). Participants were recruited by flyers placed at the Royal Adelaide Hospital and Diabetes South Australia, and through advertisements on online sites (University of Adelaide and Gumtree). Participants were excluded if they had significant gastrointestinal disorders or symptoms, cardiovascular or respiratory diseases or surgery, used medication known to affect gastrointestinal function and/or appetite, were lactose-intolerant, consumed protein supplements or >2 standard drinks (20 g) alcohol on >5 days a week, were vegetarians or smokers, or had an estimated glomerular filtration rate <45 mL/min, or low serum ferritin (<30 μg/L) or iron (<8 μmol/L) levels (a requirement by the Human Research Ethics Committee). In all cases, autonomic nerve function was evaluated using standardised cardiovascular reflex tests, and the presence of autonomic neuropathy (i.e. a score ≥3) represented an exclusion22. After inclusion, each participant was allocated to a treatment order of balanced randomisation (www.randomization.com) by a research officer who was not involved in data analysis. The Human Research Ethics Committee of the Central Adelaide Local Health Network approved the study protocol, and the study was performed in accordance with the Declaration of Helsinki and the National Health and Medical Research Council of Australia Statement on Ethical Conduct in Human Research. All participants provided written informed consent before inclusion. The study was registered as a clinical trial with the Australia and New Zealand Clinical Trials Registry (www.anzctr.org.au) (trial number: ACTRN12613000899741).Study designWe investigated the effects of intragastric administration of (i) 3 g (Trp-3) or (ii) 1.5 g (Trp-1.5) tryptophan, or (iii) control (0.9% saline) on plasma glucose (the primary outcome), C-peptide (a measure of insulin secretion), and glucagon concentrations and glucose absorption in response to, and gastric emptying of, a mixed-nutrient drink, consumed 30 min after the intragastric treatments.Control and tryptophan treatmentsTryptophan treatments were prepared by dissolving 1.5 g or 3 g food-grade tryptophan (PureBulk Inc., Roseburg, Oregon, USA), 58 mg CaCl2xH2O, and 1.75 g or 1.65 g NaCl, respectively, in 200-mL water for irrigation. Control solutions consisted of 58 mg CaCl2xH2O and 1.85 g NaCl in 200-mL water. The solutions were approximately iso-osmolar (control: 296, 1.5 g: 318, 3 g: 335 mOsm/kg) and had a pH of ~7 and a temperature of ~23 °C. Solutions were prepared by a research officer, who was not involved in the performance of the studies or data analysis, on the morning of each study and administered via a nasogastric catheter directly into the stomach. Syringes were covered, so that both study participants and the investigators performing the study were blinded to the nature of the solution. The doses of tryptophan were chosen based on our previous work and were well tolerated15.Study protocolParticipants were studied in a randomised, double-blind, cross-over fashion on three occasions each separated by 3–7 days. Participants were provided with a standardised meal (Beef lasagne, McCain Food, Wendouree, Victoria, Australia; energy content: 603 kcal), to be consumed between 6.30 p.m. and 7 p.m. on the night prior to each study. All participants were instructed to maintain their normal eating habits between study days and to discontinue their glucose-lowering medication for 48 h, and refrain from strenuous exercise and alcohol for 24 h, before each study. Participants were also asked to abstain from any solids or liquids, except water (which was allowed until 7 a.m.), after the evening meal until they attended the Clinical Research Facility at the University of Adelaide at 8.30 a.m. the next morning. An intravenous cannula was placed into a forearm vein, and the arm was kept warm with a heat pad for regular sampling of ‘arterialised’ blood. A baseline blood sample for measurement of plasma glucose and hormones, and a baseline antral area measurement, using two-dimensional (2D) ultrasound, with the participant seated in an upright position, were taken. Participants also completed a visual analogue scale (VAS) questionnaire to assess gastrointestinal symptoms. Each participant was then intubated with a soft-silicon feeding tube (outer diameter: 4 mm; Dentsleeve, Mississauga, Ontario, Canada), which was inserted through an anaesthetised nostril into the stomach. Immediately thereafter (t = −31 min, ~8.45 a.m.), participants received the 200-mL intragastric bolus of one of the two doses of tryptophan, or control, within 1 min. The tube was then removed. Further blood samples and VAS ratings were collected at t = −20, −10 and −1 min. At t = −1 min, each participant consumed, within 1 min, a mixed-nutrient drink (350 mL, 500 kcal, 74 g carbohydrates, including maltodextrin and sucrose, 18 g protein and 15 g fat) consisting of 325-mL Nestle Resource Plus® vanilla (Nestle Healthcare Nutrition, Tongala, Victoria, Australia) plus 25-mL water to make up the final volume, containing 3 g 3-OMG (Sigma-Aldrich, Milwaukee, Wisconsin, USA). Blood samples and VAS ratings were collected subsequently at 15-min intervals from t = 15 to 60 min and at 30-min intervals from t = 60 to 120 min. Measurements of antral area were obtained immediately after the drink (t = 0 min), at 5-min intervals from t = 0 to 60 min, and at 15-min intervals from t = 60 to 120 min. At t = 120 min, the cannula was removed, and participants were provided with a light lunch, after which they were free to leave the laboratory.MeasurementsPlasma glucose, C-peptide, glucagon and serum 3-OMG concentrationsFor glucose and hormones, venous blood samples (7 mL) were collected into ice-chilled ethylenediaminetetraacetic acid-containing tubes. For serum 3-OMG, 3-mL venous samples were collected into untreated tubes and allowed to clot. Plasma and serum were each separated by centrifugation at 3200 rpm for 15 min at 4 °C within 15 min of collection and stored at −80 °C until subsequent analysis.Plasma glucose concentrations (mmol/L) were quantified by the glucose oxidase method using a glucose analyser (YSI 2300 Stat Plus, Yellow Springs Instruments, Yellow Springs, Ohio, USA). Intra- and inter-assay coefficient variations (CVs) were ≤2%.Plasma C-peptide concentrations (pmol/L) were measured by ELISA immunoassay (10-1136-01, Mercodia, Uppsala, Sweden). The minimum detectable limit was 15 pmol/L and the inter- and intra-assay CVs were 8.3% and 2.9%, respectively. C-peptide reflects endogenous insulin secretion, since it is not extracted by the liver and its half-life is longer than that of insulin23.Plasma glucagon concentrations (pg/mL) were measured by radioimmunoassay (GL-32K, Millipore, Billerica, Massachusetts, USA). The minimum detectable limit was 15 pg/mL, and inter- and intra-assay CVs were 6.9% and 4.2%, respectively.Serum 3-OMG concentrations (mmol/L) were measured by liquid chromatography and mass spectrometry, with a sensitivity of 0.0103 mmol/L24.Gastric emptyingGastric emptying was evaluated by measuring antral area using 2D ultrasound (Aloka SSD-650 CL; ALOKA Co. Ltd., Tokyo, Japan). Imaging was performed with the transducer positioned vertically to obtain a sagittal image of the antrum, with the superior mesenteric vein and the abdominal aorta in a longitudinal section25. Images were taken at the end of inspiration to minimise the effects of the motion of the stomach that occurs with regular breathing26. A region-of-interest was drawn around the cross-section of the antrum using in-built software. The intragastric retention of the meal, expressed as a percentage of the meal at t = 0 min, at any given time25, and the gastric half-emptying time (T50), defined as the time taken for 50% of the meal to empty from the stomach27, were calculated.GI symptomsNausea and bloating were quantified using a VAS questionnaire28. The strength of each sensation was rated on a 100-mm horizontal line, where 0 mm represented ‘sensation not felt at all’ and 100 mm ‘sensation felt the greatest’. Participants were asked to indicate how they were feeling at each time point by placing a vertical mark on the 100-mm line.Data and statistical analysisThe number of participants included was determined by power calculations based on our previous studies15,16. We calculated that n = 12 participants would allow detection of a 1.0 mmol/L reduction in blood glucose at α = 0.05 with a power of 80%15.Areas under the curve (AUCs) for plasma glucose, C-peptide, glucagon and VAS ratings were calculated using the trapezoidal rule, from t = −31 to −1 min to assess the effects of tryptophan alone, and from t = −1 to 120 min (t = 0 to 120 min for gastric emptying and serum 3-OMG) to assess the responses to the mixed-nutrient drink. AUCs were analysed using general linear model mixed-model analysis, with baseline values as a covariate. C-peptide and glucagon concentrations at t = −1 min (i.e. immediately pre-drink), which we reported previously to correlate with subsequent glycaemic control15, peak plasma glucose, time to peak glucose and gastric half-emptying time (T50) were analysed using repeated-measures analysis of variance (ANOVA). Post-hoc comparisons, adjusted for multiple comparisons by Bonferroni correction, using parametric tests, were performed when significant ANOVA effects were found. Statistical analysis was performed using SPSS software (version 25, IBM, Chicago, Illinois, USA). Differences were considered statistically significant at P ≤ 0.05, and 0.05 < P ≤ 0.1 as trend. All data are reported as mean ± SEM.

Via Source link