The effect of different macronutrients on the ileal brake

Nutrient sensing can be defined as the ability to sense available nutrients and
to generate a physiologically adequate regulatory response to these
macronutrients involving adequate digestion and regulation of food intake.
Various physiological signals are involved in nutrient sensing, such as
humoral, neural, microbial and mechanical signals. It is clear that nutrient
sensing composes of a cascade of events. The densely innervated stomach
contributes to the initial feelings of satiation1. The subsequent release of
ingesta into the duodenum leads into a staggered release of hormones and other
substances, and direct or indirect neural activation all regulating meal
initiation, meal termination and digestion of nutrients.

The appearance of the food matrix into the duodenum, both during a meal and
during the postprandial phase results in a feed-back from different parts of
the intestine to the stomach, to the small intestine and to the central nervous
system. All these processes inhibit, in concert, food processing in the
gastrointestinal tract, satiation and appetite sensations and, consequently,
food intake. These processes are involved in the so-called intestinal brake.
The location at which the feedback process is initiated determines the severity
of the brake effect; the entry of nutrients into the duodenum and jejunum
activates the so-called duodenal and jejunal *brakes*: negative feedback
mechanisms that influence the function of more proximal parts of the
gastrointestinal tract. Activation of both of these feedback mechanisms results
in reduction of food intake and inhibition of hunger, probably partly by
inhibition of gastric emptying rate (thus contributing to enhanced and
prolonged gastric distension)2-6 and small intestinal transit time. More distal
in the small intestine, the ileal brake is a feedback mechanism that results in
inhibition of proximal gastrointestinal motility and secretion and increase
feelings of satiation and reduction of ad libitum food intake7-11.These results
all point to a potentially powerful role of the ileal brake in the regulation
of digestion, with direct or indirect impact upon eating behaviour and
satiation. The inhibitory effect of an intestinal brake, which can either be a
duodenal, jejunal or ileal brake, activation on satiation has been repeatedly
demonstrated, but it is uncertain whether this effect results from direct
stimulation of central satiation centres in the brain (directly due to CCK or
afferent signalling of the N. Vagus), or whether the brake effect on hunger and
satiation is achieved indirectly via the delay in gastric emptying. Also, the
sensing mechanisms of nutrients in the gut, which eventually lead to the
intestinal *brakes* are largely unknown, but involve a variety of mucosal
receptors. These receptors include for example, G-protein gustducin and
G-protein-coupled receptors, TRPV1, taste receptros and various other types
including melanocortin, opioid and PPAR

The current scientific data strongly suggest that activation of the ileal brake
provides the most powerful feedback mechanism to gastrointestinal transit and,
especially, satiety signals and food intake12, 13, 14 . Most studies have used
fat as macronutrient. The effects of several amounts, types and preparations of
fat on the ileal brake have previously been investigated and reported. We can
conclude that the ileal brake seems to induce stronger effects on gut function,
satiation and meal intake than the duodenal- and jejunal brake, when fat is the
brake substrate. Furthermore infusion of fat in the ileum induces stronger
effects when compared to oral ingestion. No dose-response effects were observed
in a human study to the effects of 3- and 9 g intra-ileal fat, respectively,
the higher dosage of fat seems to be appropriate to clearly establish an ileal
brake effect. Poly-unsaturated fat has stronger effects than saturated fat;
small fat droplets are more powerful to invoke the ileal brake than larger
droplets. The most potent fatty acid for ileal brake induction appears to be
C18:2 7, 8, 12.

As shown above, fat is rather extensively been studied in humans, but much less
is known about proteins and carbohydrates. As such, fat may serve as a positive
control in ileal brake studies. Until present, the effects of the other
macronutrients to induce the ileal brake remain largely unknown. There is
evidence that carbohydrates induce the ileal brake13-15. Infusion of glucose
into the small intestine, at a rate mimicking the overall gastric emptying rate
of 2-3 kcal/min, reduces hunger and desire to eat, increases fullness and
reduces subsequent food intake16. Furthermore, infusion of glucose also
inhibits gastric emptying and increases pancreatic and intestinal secretion16,
17.
Proteins may also exert effects, although data are scarce and not convincing15,
18. However, it becomes more and more accepted that proteins may induce
stronger effects on satiation and food intake than fat or carbohydrates.
Recently, researcher at Wageningen University conducted an in vivo study in
pigs (manusscript in preparation). They infused different nutrients into the
pig ileum and collected portal blood for CCK, GLP-1 and PYY analysis and
measured food intake. They found that infusion of the protein casein and the
carbohydrate sucrose induced the highest release of satiety hormones (CCK,
GLP-1) and reduced food intake in pigs. Different studies, in both humans and
animals showed that oral ingestion of casein and sucrose induced satiety
hormone release reduced daily food intake, acid secretion and duodenal motor
activity. These new data validate our choise of using casein and sucrose in
this human in vivo study.

Hypotheses:
- Ileal delivery of casein and/or sucrose induces stronger effects on satiation
compared to placebo
- Ileal delivery of casein induces stronger effects on satiation than ileal fat
delivery
- Ileal delivery of sucrose induces stronger effects on satiation than ileal
fat delivery



Primary objective:
• To assess the effect of casein and sucrose delivered to the ileum on satiation



Secondary objectives:
• To investigate the effect of ileal delivery of casein and sucrose on ad
libitum meal intake.
• To asses the effect of casein and sucrose delivered to the ileum on gastric
emptying rate
• To compare the different effects of casein, sucrose and safflower oil on
small bowel transit time
• To asses the effect of casein and sucrose delivered to the ileum on
gallbladder volumes

This study is designed as a double-blind randomized cross-over study with six
different treatments (casein (high and low dose) / sucrose (high and low dose)
/ safflower oil (positive control)/ saline (placebo)).

Healthy volunteers will compleet 6 test days in total. Every test day another
substance will be infused into the ileum via the nasoileal catheter:
casein (high and low dose), sucrose (high and low dose), safflower oil
(positive control) and saline (placebo)
Infusion is via the nasoileal catheter which will be inserted on monday ( test
days are on tuesday, wednesday and thursday). Every morning starts with
checking the position of the catheter. If the position in the ileum is
confirmed, the test day can start. After the third test day (thursday) the
nasoileal catheter will be removed. The same volunteer will undergo the similar
tests a week later (after wash-out period of 4 days). In week 2, casein will be
switched for sucrose and the negative control (placebo) will be switched for a
positive control (safflower oil). Randomisation determines the order of these
infused solutions.

3 Short visits (1 hour each): screening, study day 1 and study day 2 (both
insertion of nasoileal catheter)
6 Longer visits (4 hours each): test day 1,2,3 and test day 4,5,6


Blood sampling
On each test day (test day 1-6), after the position of the ileal catheter has
been confirmed by fluoroscopy, a flexible intravenous cannula (Biovalve 1,0mm)
is inserted into an antecubital vein in the fore-arm for blood sampling. Per
time point 8ml of blood is drawn, totalling 88ml per day (total of 264 mL for
the 3 test days in week 1). Before the start of test week 2, haemoglobin and
haematocrit are determined to ensure normal values of both substances. Test
week 2 can start if both parameters are in the normal range. After collection,
one K2EDTA tube will be centrifuged at 2600 rpm for 20 min at 20°C. The other
K2EDTA will be centrifuged at 2500 rpm for 15 min at 4°C, the supernatant will
be collected and this will be centrifuged again at 4000 rpm for 10 min at 4°C.
Plasma will be collected in 1-mL aliquots and stored at -80°C until analysis.
The blood samples that are not used during the analysis of this study will be
stored and kept for a maximum period of ten years. The tubes will be coded, and
the code will be kept by prof. dr. A.A.M Masclee, the principal investigator.
This enables us to do further analysis within the aim of this protocol.

During blood sampling, the volunteers will remain seated in a comfortable
chair, with an adjustable back. No side effects are expected when sampling
blood in this manner. Before the start of test week 1 and test week 2,
haemoglobin and haematocrit are determined to ensure that these parameters are
in normal range


Gastric emptying rate
Gastric emptying of the test meal will be determined by using a 13C stable
isotope breath test.
13C-octanoic acid (100 mg, Campro Scientific bv, Veenendaal the Netherlands)
will be mixed into the standardized breakfast test meal ingested at t=0. Breath
samples of 13CO2 will be collected from volunteers by breathing into a
re-usable plastified aluminium bag at baseline (immediately before consumption
of the test product, t = -15 minutes) and between 15 and 240 min after start of
the treatment. Actual time at which the breath sample is collected will be
registered and will be used in the kinetic evaluation. Samples will be
collected, stored and afterwards analysed using Isotope Ratio Mass Spectometry
(Finnigan MAT 252).

Gastric emptying rate will be determined by using a 13C stable isotope breath
test. The 13C octanoid acid breath test is a reliable and safe test for
measuring the gastric emptying rate of solid meals. It is widely used and even
possible to use in children and pregnant women. Therefore we don*t expect any
side effects.


Small bowel transit time
Duodeno-caecal transit measurement will be performed by lactulose hydrogen
breath analysis, as described by Ledeboer et al. Via an opening of the catheter
located in the duodenum 6 g of lactulose (Legendal, Inpharzam, Amersfoort) is
administered at t=30 min together with the start of ileal infusion. Samples of
end-expiratory breath are taken under basal conditions and at 10 min intervals
during the first hour and at 15-30 min intervals during the second, third and
fourth hour after meal ingestion with lactulose administration. The samples are
directly analysed using a handheld hydrogen breath test unit. Small bowel
transit time is defined as the time between lactulose administration and the
onset of a sustained rise in breath hydrogen concentration of at least 10 parts
per million (ppm) above basal level.

Together with the start of the ileal infusion, 6g of lactulose will be infused
in the duodenum to measure small bowel transit time. This method, first
described by Bond et al, appears to provide a simple, safe and non-invasive
means of studying small bowel transit time in healthy humans.


VAS scores for satiety and GI symptoms
Scores for satiety feelings (e.g., satiety, fullness, hunger, prospective
feeding, desire to eat, desire to snack) and gastrointestinal symptoms
(burning, bloating, belching, cramps, colics, warm sensation, sensation of
abdominal fullness, nausea and pain) will be measured using Visual Analogue
Scales (VAS, 0 to 100 mm) anchored at the low end with the most negative or
lowest intensity feelings (e.g., extremely unpleasant, not at all), and with
opposing terms at the high end (e.g., extremely pleasant, very high, extreme).
Volunteers will be asked to indicate on a line which place on the scale best
reflects their feeling at that moment. The scoring forms will be collected
immediately so that they cannot be used as a reference for later scorings.


Gallbladder ultrasound
Gallbladder (GB) volumes will be measured in volunteers by real-time
ultrasonography (Technos, 3.5 MHz transducer) (see study design for measurement
frequency). GB volume will be calculated by the sum of cylinders method using a
computerized system. In this method, the longitudinal image of the gallbladder
is divided into series of equal height, with diameter perpendicular to the
longitudinal axis of the gallbladder image. The uncorrected volume is the sum
of volumes of these separate cylinders. To correct for the displacement of the
longitudinal image of the gallbladder from the central axis, a correction
factor is calculated from the longitudinal and transversal scans of the
gallbladder. Gallbladder volume is calculated by multiplication of the
uncorrected volume with the square of the correction factor. This is done
automatically by the computer connected to the echoscope. The mean of the two
measurements will be used for analysis. The assumptions and the mathematical
formula used to calculate gallbladder volume have been described and validated
previously

Echoscopy of the gallbladder is not associated with any risks.


Catheter placing and fluoroscopy
The subjects will perceive mild discomfort during the placement of the
catheter. The radiation exposure during the positioning of the feeding tube is
minimal (0.06 mSv). Each test day starts with checking the position of the
tube. The radiation exposure of this procedure is minimal as well (0.01 mSv).
The total exposure to radiation (during all test days) will be approximately
0.18 mSv (0.06 mSv + 0.06 mSv + 0.06 mSv) , which equals the radiation, which
is received during a three-hr flight in an aeroplane at a 4-km altitude
(www.nrg-nl.com).



All participants are healthy volunteers and we don't expect any health benefits
or disadvantages.