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Trial registered on ANZCTR


Registration number
ACTRN12612000376842
Ethics application status
Approved
Date submitted
16/03/2012
Date registered
3/04/2012
Date last updated
4/02/2016
Type of registration
Prospectively registered

Titles & IDs
Public title
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.
Scientific title
Expression of fatty acid sensing receptors in human intestinal epithelial cells in response to duodenal fat infusion: relationships with gastrointestinal sensitivity to fat, habitual energy intake and body weight in healthy lean and obese adults.
Secondary ID [1] 280155 0
Nil
Universal Trial Number (UTN)
U1111-1129-6783
Trial acronym
N/A
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Obesity 286077 0
Healthy human physiology 286078 0
Condition category
Condition code
Diet and Nutrition 286273 286273 0 0
Obesity
Oral and Gastrointestinal 286429 286429 0 0
Normal oral and gastrointestinal development and function

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
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.
Intervention code [1] 284482 0
Other interventions
Comparator / control treatment
Uncontrolled
Control group
Uncontrolled

Outcomes
Primary outcome [1] 286737 0
Small intestinal expression of the fat sensing receptors CD36, GPR40, GPR119 and GPR120 during fasting and following 30 minute of lipid stimulation.

Absolute quantitative RT-PCR assays will measure the number of transcripts of fat sensing molecules. Biopsies stored in fixative will be used to confirm normal duodenal histology. In addition, we will perform dual label immunohistochemistry to assess the number, distribution and co-expression of targets in enterocytes and with enteroendocrine cells expressing the gastrointestinal peptides. Activation of GPRs will be assessed using phosphorylated extracellular signal-regulated kinase (pERK) immunohistochemistry.
Timepoint [1] 286737 0
Biopsies will be collected from the duodenum at baseline (fasting) and after 30 minutes of lipid exposure.
Primary outcome [2] 286738 0
Gastrointestinal motility and hormonal responses to intraduodenal fat infusion.
Timepoint [2] 286738 0
Blood samples will be collected at t = -15, 0, 15, 30, 45, 60, 75, 90, 105 and 120 minutes and assayed for blood glucose, insulin, glucagon, GIP, CCK, PYY, OEA, ghrelin and FFA and TG concentrations using well established techniques.
Gastrointestinal motility will be assessed using antropyloroduodenal manometry.
Primary outcome [3] 286739 0
Appetite perceptions
Timepoint [3] 286739 0
Visual analogue scale questionnaires will be completed at t = -15, 0, 15, 30, 45, 60, 75, 90, 105, 120 and 150 minutes
Secondary outcome [1] 296562 0
The relationships between the expression and sensitivity of fat sensing receptors and the gastrointestinal and appetite responses to fat, previous dietary intake and body weight.

Data will be analysed statistically using t-tests and repeated measures ANOVAs for variables measured over time, with group and time as factors. Post-hoc paired comparisons, corrected for multiple comparisons, will be performed if ANOVAs reveal significant effects. Statistical significance will be accepted at P < 0.05. Linear regression analysis will be performed to determine relationships between the expression of fat sensing molecules with GI and energy intake responses to fat.
Timepoint [1] 296562 0
Measurements will be collected at intervals throughout the interventions (e.g. as for blood samples above).
Secondary outcome [2] 296875 0
Energy intake
Timepoint [2] 296875 0
Assessed at a buffet-meal ingested between t = 120 - 150 minutes.

Eligibility
Key inclusion criteria
Thirty healthy lean (BMI: 19-25 kg/m2), and 30 obese (BMI: >30 kg/m2, waist circumference: males >100 cm; females >97 cm) Caucasian volunteers aged 18-55 years will be recruited. Participants will be age- and sex-matched, weight-stable (<5 % fluctuation) and not on a weight-loss diet for a minimum of 3 months before inclusion. Pre-menopausal female participants will habitually take a hormonal contraceptive. This will eliminate the effects of the menstrual cycle on GI function and energy intake.
Minimum age
18 Years
Maximum age
55 Years
Sex
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
-significant gastrointestinal symptoms; disease or surgery
-eating disorders
-current use of any prescribed or non-prescribed medications which affect energy metabolism, GI function, body weight or appetite
-history of any bleeding disorder, or use of anticoagulant or antiplatelet drugs (e.g. warfarin, aspirin, clopidogrel)
-diabetes mellitus (fasting blood glucose > 6.9 mmol/l and/or glycated haemoglobin > 6.2 %)
-epilepsy
-cardiovascular or respiratory disease
-any other significant illness
-allergy to local anaesthetic
-intake of > 20 g alcohol on a daily basis
-smokers (cigarettes, cigars, marijuana)
-use of fish oil
-restrained eaters, as determined by a score of > 12 on the eating restraint component of the Three Factor Eating Questionnaire (eating restraint will be assessed in all participants, but only used as an exclusion criterion in lean participants, as in our experience, the obese will have some degree of eating restraint)
-donation of blood in the 12 weeks prior to enrolment in the study. Participants will also be instructed to abstain from donating blood for 12 weeks after study completion. -A screening blood sample will be taken to ensure that only individuals with normal haemoglobin, iron and ferritin levels are included in the study.
-participation in any other research studies in the 12 weeks prior to enrolment in the study
-history of taste disorders
-consumption of a vegetarian diet
-high performance athletes
-inability to comprehend study protocol

Study design
Purpose of the study
Prevention
Allocation to intervention
Non-randomised trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
All participants will be studied on two occasions in a non-randomised, non-blinded fashion.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Masking / blinding
Who is / are masked / blinded?



Intervention assignment
Other design features
Phase
Not Applicable
Type of endpoint/s
Statistical methods / analysis

Recruitment
Recruitment status
Completed
Date of first participant enrolment
Anticipated
2/05/2012
Actual
21/05/2012
Date of last participant enrolment
Anticipated
Actual
11/08/2015
Date of last data collection
Anticipated
Actual
Sample size
Target
60
Accrual to date
Final
60
Recruitment in Australia
Recruitment state(s)

Funding & Sponsors
Funding source category [1] 284903 0
University
Name [1] 284903 0
University of Adelaide
Country [1] 284903 0
Australia
Funding source category [2] 284908 0
Hospital
Name [2] 284908 0
Royal Adelaide Hospital Clinical Project Grant
Country [2] 284908 0
Australia
Primary sponsor type
Individual
Name
Dr Tanya Little
Address
Discipline of Medicine
Level 6, Eleanor Harrald Building
Royal Adelaide Hospital
North Terrace
Adelaide, SA, 5000
Country
Australia
Secondary sponsor category [1] 283784 0
Individual
Name [1] 283784 0
Professor Christine Feinle-Bisset
Address [1] 283784 0
Discipline of Medicine
Level 6, Eleanor Harrald Building
Royal Adelaide Hospital
North Terrace
Adelaide, SA, 5000
Country [1] 283784 0
Australia

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 286901 0
Royal Adelaide Hospital Research Ethics Committee
Ethics committee address [1] 286901 0
Ethics committee country [1] 286901 0
Australia
Date submitted for ethics approval [1] 286901 0
25/11/2011
Approval date [1] 286901 0
19/12/2011
Ethics approval number [1] 286901 0
111212

Summary
Brief summary
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 33933 0
Dr Tanya LIttle
Address 33933 0
University of Adelaide Discipline of Medicine
Level 6, Eleanor Harrald Building
Royal Adelaide Hospital
North Terrace
Adelaide, SA, 5000
Country 33933 0
Australia
Phone 33933 0
+61 8 8313 2999
Fax 33933 0
Email 33933 0
[email protected]
Contact person for public queries
Name 17180 0
Dr Tanya Little
Address 17180 0
Discipline of Medicine
University of Adelaide
Level 6, Eleanor Harrald Building
Frome Road
Royal Adelaide Hospital
Adelaide SA 5000
Country 17180 0
Australia
Phone 17180 0
+61 8 8222 0724
Fax 17180 0
+61 8 8223 3870
Email 17180 0
[email protected]
Contact person for scientific queries
Name 8108 0
Dr Tanya Little
Address 8108 0
Discipline of Medicine
University of Adelaide
Level 6, Eleanor Harrald Building
Frome Road
Royal Adelaide Hospital
Adelaide SA 5000
Country 8108 0
Australia
Phone 8108 0
+61 8 8222 0724
Fax 8108 0
+61 8 8223 3870
Email 8108 0
[email protected]

No information has been provided regarding IPD availability


What supporting documents are/will be available?

No Supporting Document Provided



Results publications and other study-related documents

Documents added manually
No documents have been uploaded by study researchers.

Documents added automatically
SourceTitleYear of PublicationDOI
EmbaseDuodenal fatty acid sensor and transporter expression following acute fat exposure in healthy lean humans.2017https://dx.doi.org/10.1016/j.clnu.2016.02.005
EmbaseEndocannabinoids and cannabinoid receptors as regulators of endocrine functions and tissue metabolism: Plasma endocannabinoid levels in lean, overweight, and obese humans: Relationships to intestinal permeability markers, inflammation, and incretin secretion.2018https://dx.doi.org/10.1152/ajpendo.00355.2017
N.B. These documents automatically identified may not have been verified by the study sponsor.