Stratifying low-calorie sweeteners for type 2 diabetes risk (“SWEET n SOUR”).

At the screen visit, investigators will explain the study protocol and answer any questions the volunteers may have about what is involved, before the volunteers provide written, informed consent. An intravenous cannula will be inserted and 10 mL fasting venous blood will be collected for blood picture, iron studies, HbA1c, glucose, and liver and kidney function. An oral glucose tolerance test will be performed (75g glucose in 300 mL with addition of 150mg 13C-acetate) with 5mL blood collection at 30, 60, 120, and 180 min for measurement of plasma glucose concentrations. Breath samples will also be collected every 15 min to measure baseline gastric emptying. A urine pregnancy test will be performed in female participants during the screening and prior to each study visit. Volunteers who pass the screening visit will be randomised in a schedule determined by the Investigational Drugs Pharmacy, RAH, in a double-blind, parallel design, to one of 6 intervention groups (35 participants per group), which will receive the following supplements (each of equivalent sweetness other than placebo) in 3 divided doses to be taken orally in gelatin capsules with each meal for 4 weeks: (i) acesulfame potassium (Ace-K) (total dose 420mg daily or 140mg three times per day), (ii) aspartame (420mg daily or 140mg three times per day), (iii) saccharin (168mg daily or 56mg three times per day), (iv) stevia (rebaudioside A) (279mg daily or 93mg three times per day) (v) sucralose (141mg daily or 47mg three times per day), or (vi) placebo (cellulose only) The contents of each capsule will be made up to 140mg by the addition of cellulose to the specified dose of each LCS. Compliance with taking capsules will be reinforced by daily mobile phone messages, weekly telephone calls and emails, and evaluated by counting the numbers of capsules remaining on the final study visit. Each participant will attend for two tube studies, immediately before and at the end of the 4-week intervention. Participants will complete a 3-day diet diary prior to each study day to document their energy, macronutrient and LCS intake. Late on the day preceding each study visit, or on the morning of the visit, subjects will be asked to collect two stool samples, one in a low head-space tube for future microbial transplant, stored at 4 degrees, and one in a custom tube designed for collection point homogenisation and stabilisation at ambient temperature (Norgen tube, for metagenomic assessment). We will provide non-allergenic gloves, a cooler box and ice pack for transport of the former, and will provide non-allergenic gloves and written instructions on proper collection technique for the latter. On the evening preceding the study day (~1900h), participants will be given a standardised evening meal (McCain’s frozen beef lasagne (McCain Foods Proprietary Ltd, Victoria, Australia); 2472kJ (or vegetarian lasagne for participants who are vegetarians)) to consume with water. Following this meal, participants will be asked to fast from solids and liquids (water may be consumed until 10 pm) until the following morning, when they will attend the CRF of the AHMS building at 0800h. This will be reinforced by a telephone call from one of the investigators. On each tube study day, the participant’s body weight will be recorded, and a silicone rubber catheter (Dentsleeve International Ltd., Mui Scientific, Ontario, Canada) will be inserted through an anaesthetised nostril into the stomach and allowed to pass into the duodenum by peristalsis. The catheter will be positioned with the infusion port located 12 cm below to the pylorus (in the duodenum). The correct positioning of the catheter will be monitored continuously by measurement of the transmucosal potential difference in the stomach (~ -40 mV) and the duodenum (~ 0 mV). For this purpose, an intravenous cannula will be placed subcutaneously in the left forearm and filled with sterile saline as a reference electrode. An intravenous cannula will be placed into a forearm vein for blood sampling. After the correct positioning of the intraduodenal catheter, the participant will be asked to empty his or her bladder. A 150 mL solution containing 30g glucose together with 3g of the glucose absorption marker 3-O-methyl glucose, 3-OMG) will then be infused intraduodenally at 5 mL/min (4 kcal/min) from t = 0 to 30 min, after which the intraduodenal catheter will be removed. Blood will be sampled from the IV cannula, with the arm kept warm with a heat pad, at t = -5, 10, 20, 30, 40, 50, 60, 90, and 120 min (25 mL per time point, total 225 mL per study day). Gastrointestinal symptoms will be monitored using 100mm visual analog scales (VAS) at the same intervals. Plasma will be separated and stored for subsequent measurement of glucose, 3-OMG, insulin, GLP-1, and GIP concentrations. Urine will also be collected at t = 60 and 120 min for measurement of glucose and 3-OMG concentrations. At t = 120 min, a meal will be provided and the participant will be allowed to leave the facility once blood glucose concentrations are stable. Subjects will also attend the facility 3 weeks into the intervention after an overnight fast, for a follow-up oral glucose tolerance test (75g glucose in 300 mL plus 150mg 13C-acetate), with breath testing for measurement of gastric emptying as at the screening visit, to ascertain whether LCS exposure can modify intestinal feedback so as to accelerate gastric emptying of glucose.