Effect of fat on levels of fat-sensitive sensors in the small bowel and their relationship with gut function and body weight in healthy lean and obese humans.

Prior to commencement of the study, all participants will complete a 3-day (2 week days and 1 weekend day) weighed food diary to evaluate their habitual energy intake and the macronutrient composition of their diet, as well as their physical activity level. For this, electronic scales will be provided, and participants will be instructed to weigh and record all of the food they consume. Participants will also complete a Food Preference questionnaire to determine their preference for high-, or low-, fat foods. Participants will then be studied on two occasions following an overnight fast from 2200 h. On the first study day the effects of intraduodenal fat infusion on antropyloroduodenal motility, plasma insulin, CCK, GIP, GLP-1, PYY, ghrelin, OEA, insulin, glucagon, and FFA and triglyceride concentrations, appetite perceptions and energy intake will be evaluated. On the second study day, participants will undergo endoscopy where biopsies and blood samples will be collected while fasting, and following intraduodenal fat infusion. Study visits will be separated by a period of at least 7 days. All participants will be asked to refrain from vigorous exercise and alcohol intake for 24 hours before each study day. To further standardise conditions, subjects will be provided with a ready-to-eat meal (beef lasagne, McCain Foods (Aust) Pty. Ltd) for dinner the night before each study visit, which will be consumed at 1900 h with only water allowed as a drink. After this time subjects will be required to fast from all food and fluid (except for water). Part A: Effects of intraduodenal fat infusion on antropyloroduodenal motility, gut peptide secretion, appetite and energy intake. Participants will arrive at the Discipline of Medicine at ~0830 h after an overnight fast. A small diameter (~3.5 mm) manometric assembly will be inserted through an anaesthetised nostril and allowed to pass by peristalsis through the pylorus and into the duodenum. Its passage will be monitored by measuring the antroduodenal transmucosal potential difference (TMPD) using a red-dot monitoring electrode placed on the forearm as a reference. The manometric assembly will include 6 antral channels at 1.5 cm intervals, a 4.5 cm pyloric sleeve sensor, two equally spaced channels situated on the back of the sleeve, and seven duodenal channels, at 1.5 cm intervals, located on the distal end of the sleeve sensor. After correct positioning of the catheter, fasting motility will be monitored until the occurrence of a phase III of the migrating motor complex. An intravenous cannula will then be inserted into a forearm vein to obtain regular blood samples. At t = -15 min, a baseline blood sample (20 ml) will be taken and a visual analogue scale questionnaire (VAS) will be completed to assess perceptions of hunger, fullness, desire to eat, prospective consumption, as well as nausea and bloating. At t = 0 min, an intraduodenal infusion of 10% Intralipid (Fresenius Kabi Pty. Ltd., NSW) at 2 kcal/min, 1.8 ml/min (240 kcal), will commence, and be maintained for 120 minutes. Antropyloroduodenal pressures will be recorded between t = -15 and 120 min. The number of antral, duodenal and isolated pyloric pressure waves, and basal pyloric pressures will be calculated as described previously (1, 17, 25). During the infusions, 20 ml blood samples (1 * 5 ml serum tube, 1 * 5 ml Sodium Fluoride/EDTA tube and 1 * 10 ml EDTA treated tube) will be collected at t = -15, 0, 15, 30, 45, 60, 75, 90, 105 and 120 minutes for the determination of circulating glucose, insulin, glucagon, GIP, CCK, GLP-1, PYY, OEA, ghrelin, and FFA and triglyceride concentrations. VAS will also be completed, every 15 min between t = 0 - 120 min. At t = 120 min the infusion will be discontinued, and the participant extubated and offered a cold buffet-style meal (t = 120 - 150 min) for the assessment of energy intake. The meal will consist of bread, cold meats, cheese, lettuce, tomato, cucumber, mayonnaise, butter, apple, banana, yoghurt, chocolate custard, fruit salad, iced coffee, orange juice and water. The amount of food offered will be in excess of what the subjects are expected to consume, and subjects will be instructed to eat until they feel comfortably full. After ingestion of the meal, i.e. at t = 150 min, another blood sample will be taken and VAS completed. Oral fatty acid sensitivity One hour following the buffet-meal (i.e. at t = 210 min), participants will undergo sensory testing to determine their oral detection thresholds for oleic acid using 3-alternate forced choice methodology, an established sensory testing procedure. Test samples will be prepared by adding oleic acid (C18:1) (a fatty acid found commonly in the diet) at concentrations ranging from 0.02-12 mM to water. 5% (w/v) gum acacia and liquid paraffin will be added to produce perceptually identical textural attributes, including lubricity and viscosity, between control and FA samples (45). 0.01% (w/v) EDTA will be added to prevent fatty acid oxidation. The test involves choosing an “odd” sample from a group of three samples based on taste (subjects wear nose-clips to ensure detection of differences is not due to smell). Each participant will receive a tray containing 3 sets of 3 * 10 ml samples (2 water, 1 FA) randomly sorted and labelled with 3-digit blinding codes. Subjects will be instructed to move back and forward through the samples from one set and to identify the “odd” sample. The subject then completes sets 2 and 3, i.e. 3 triangle tests in total. If they correctly identify the “odd” sample on three consecutive tests, then this concentration will be recorded as their detection threshold. If they are incorrect, the next increasing concentration will be presented. There is a 0.036% chance of correctly guessing 3 triangle tests in succession by luck. Part B: Evaluation of expression of the fat sensing molecules CD36, GPR40, GPR119 and GPR120 during fasting and in response to intraduodenal fat infusion. On the second day, endoscopies will be performed by a gastroenterologist in the Gastrointestinal Investigation Unit (Ward Q7), Royal Adelaide Hospital. An intravenous cannula will be inserted into a forearm vein to allow for blood sampling, and then local anaesthetic spray (Co-phenylcaine Forte) and 2 % lignocaine gel will be applied to the nose and pharynx, and a small-diameter (5.3 mm) fibre-optic endoscope will be passed through the nose into the second part of the duodenum, with mimimal insufflation of air. As per current practice, no sedation is required in volunteers during the insertion of the endoscope. Four mucosal biopsies (2 for RT-PCR and 2 for immunohistochemistry) will be taken from the duodenum using standard endoscopic biopsy forceps (t = -5 min). The tip of the endoscope will remain in the duodenum until t = 30 min. At t = 0 min, intraduodenal fat infusion (as described above) will begin via the endoscopic biopsy channel at a rate of 2 kcal/min. After 30 min of fat infusion, 4 duodenal biopsies (as above) will be collected. The endoscope will then be withdrawn, and a further biopsy taken from the gastric body as a reference. Biopsies will be stored immediately in RNAlater or fixative (4% paraformaldehyde) prior to further processing for RT-PCR or IHC, respectively. RNA will be extracted using commercial extraction kits (QIAGEN RNAeasy micro kit). Samples will be excluded if diffuse mucosal disease involving the upper gastrointestinal tract, including Crohn’s disease, coeliac disease, or ischaemic changes, is evident either macroscopically or on histopathological examination of mucosal biopsies, when the latter is clinically indicated. Blood samples (20 ml) for the assessment of gut peptides will be collected at t = -15, 0, 10, 20 and 30 minutes. Absolute quantitative RT-PCR assays will be performed using Quantitect SYBR green assays (Qiagen). These assays will measure the number of transcripts of fat sensing molecules (including CD36, GPR119, GPR40 and GPR120). Biopsies stored in fixative will be used to confirm normal duodenal histology. In addition, we will perform dual label immunohistochemistry slides for CD36, GPR40, GPR119, GPR120, to assess the number, distribution and co-expression of targets in enterocytes and with enteroendocrine cells expressing the gastrointestinal peptides CCK, GLP-1 and PYY. Activation of GPRs will be assessed using phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry.