Study procedures

1. Screening: Before inclusion in the study, potential subjects will complete a screening questionnaire Subjects will then be informed about their eligibility. Also the procedure and risks will be explained. If they decide to participate, they will fill in and sign the standard MRI-screening form and the informed consent form. Also a training session date will be planed.
2. Training session: When subjects want to participate and match to the criteria a training session will be planned. During this training session a naso-gastric tube will be inserted by a qualified nurse (according to the "maagsonde-protocol" UMC Utrecht). After insertion of the tube subjects rest to allow water used to assist insertion of the tube to leave the stomach and to become comfortable with the tube. During this insertion the nurse and the subject can see if a subject can handle the naso-gastric tube and can or will participate in the study. Subjects will rate hunger, fullness, thirst, desire to eat, prospective consumption, nauseous, and anxiety on a VAS scale. To simulate the position in the MR scanner, subjects will be asked to lie down on a doctors table. A towel will be placed under need there right side, in this way the stomach will be more in the position when standing/sitting, this is to simulate normal gastric filling. A tube will be placed between the teeth of the subject (like a straw) after which an oral load of 500 ml will be ingested through the tube in the month. This ingestion will be driven by a pomp (100ml/min), which simulates a sip systematically. First 200 ml will be ingested, then there will be a pause of 30 seconds, second the next 150 ml will be pumped, following again a 30 second rest period, then the last 150 ml will be ingested. The subjects know the rate and speed of the sips and can stop the pomp on command when necessary. The subject will also hear a tone just before the sip is ingested. Here a subject learns to drink with a naso-gastric tube placed and when lying down. After ingestion the subjects will fill in the same questionnaire again. The naso-gastric tube will be removed after the rating. This training session will take about 40 minutes.
3. Assessments and procedures on the study days: All subjects will be asked to arrive in the morning after an overnight fast of at least 10 hours. The evening before the scan subjects will be ask to eat a standardized meal given by the researchers (AH kant \& klaar). An appetite questionnaire will be filled in (hunger, fullness, thirst, desire to eat, quantity to eat, desire to eat something sweet or savory, nauseous, and anxiety). An intravenous cannula will be placed by a qualified person. Next, the naso-gastric tube will be placed (same procedure as training session). After the subject has been placed in the scanner (as in the training session with a towel under need there right side); one blood draw will be done for baseline measurements (e.g. glucose, insulin, cholecystokinin and ghrelin for every time point). The appetite questionnaire will be filled in again in the scanner. A reference and anatomic scan will be done. Now the fmri session begins; after five minutes of baseline scanning, food administration will start (t = 0). This consists of administration of the intra gastric load through the naso-gastric tube (caloric or non-caloric) or of ingestion of the caloric load through a tube (oral). Blood will be drawn at t = 2.5, t = 5, t = 10, t = 15 and at t = 30 min. Before every blood sample subjects will give fullness and nausea ratings. One of the researchers will stand next to the subject/scanner during the whole experiment. If aspiration occurs a researcher will be there to help immediately. The researchers also make sure a subject knows how to get out of the scanner as quick as possible if aspiration occurs. At t = 30 min the subject will be taken out the scanner and fill in the appetite questionnaire. The naso-gastric tube and intravenous cannula will be removed. Subsequently subjects will be provided with an ad libitum breakfast buffet at t = 45 min. Food weight and macro-nutrient intake will be recorded. After breakfast the appetite questionnaire is filled in once more.
4. Blood sampling and storage: On the study day, a 10 ml blood sample will be drawn by certified experienced medical personnel. Blood samples will be put on ice immediately and then centrifuged at 1730 × g for 10 min at 4 ˚C. Blood material from the collection tubes will be split into two portions and stored, one portion for the analyses and one portion as a backup. Blood serum will be stored at -80 ˚C at the Rudolf Magnus Institute until sample collection is complete. Only RMI laboratory personnel and the investigators will have access to the samples. Blood samples will be coded as follows: P01, P02, etc. The key to this code will only be known by the principal and medical investigators and not by the personnel performing the blood analyses. Samples will be kept for 10 years and then destroyed.
5. Naso-gastric tube: A qualified nurse will place the naso-gastric tube. The placement will go according to the 'maagsonde -protocol' (UMC Utrecht). After insertion of the tube subjects rest to allow water used to assist insertion of the tube to leave the stomach and to become comfortable with the tube.
6. Withdrawal of individual subjects: Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons. Each subject who does not complete the study for any reason will have a post-study screening by the end of the particular study day to ensure that the drop-out is not the result of a structural problem. Data of subjects who withdrew prematurely will be used for statistical analyses, as far as possible.
7. Replacement of individual subjects after withdrawal: In case subjects withdraw during the study (fMRI sessions), they will not be replaced. If a subject decides not to participate during the training session a new subject will be recruited.
8. Follow-up of subjects withdrawn from treatment: Subjects who withdraw from the study will not be followed up, unless they withdraw because of an adverse event.
9. Post-study screening: Subjects will be asked how they experienced the study procedures. This information, if the subject is willing to share it, will be used to evaluate (the burden of) the study protocol.