The ex vivo effect of bitter, sweet, salt, umami and sour tastants on the release of gastrointestinal satiety peptides

The gastrointestinal tract generates many signals that play a role in the
regulation of eating behavior, most importantly satiety signals. The gut is
therefore an appealing target for food products to induce satiety and reduce
food intake. Gut peptides, such as cholecystokinin (CCK) and glucagon-like
peptide-1 (GLP-1), have been demonstrated to reduce food intake and hunger
after intravenous administration1-3. However, because of incoherent results
between studies there is still controversy about the effect of the release of
satiety peptides on food intake. On the other hand, long-term use of a GLP-1
agonist has been shown to reduce body weight in obese individuals4. Together
these results illustrate the potential of targeting the gastrointestinal tract
in weight management and weight loss strategies.

In order for different nutrients to influence hunger/satiety, the presence of
these nutrients in the small intestine has to be sensed. There appear to be two
major principles of nutrient sensing in the gastrointestinal tract5. Firstly,
nutrients or their direct breakdown products can interact with receptors on the
microvilli of enteroendocrine cells. These enteroendocrine cells respond by
secreting mediators including cholecystokinin (CCK), Peptide YY (PYY) and
Glucagon Like Peptide-1 (GLP-1) which are either transported through the blood
stream or activate their receptors on vagal nerve endings. In parallel, a
second mechanism is operating. Nutrients (lipids, amino acids) are taken up by
enterocytes, which for example form lipid-derived mediators. These mediators
also interact with receptors on vagal nerve endings. In other words, nutrient
sensing is primarily mediated by enteroendocrine cells individually scattered
in the lining of the epithelium.

Enteroendocrine cells express various specific luminal receptors. These
receptors may activate intracellular pathways through direct gating of ion
channels, but recently, there has been a growing interest in the G-protein
coupled receptors (GPCR*s) such as the G-protein gustducin. These recently
discovered receptors include GPR119, GPR93, GPR55, GPR120, GPR40, TRPV1 and
various other types6, 7. Different nutrients such as free fatty acids and
peptides can be recognized by these receptors. Activation of these receptors
leads to an intracellular signaling cascade which results in the release of
gastrointestinal mediators.

Taste refers to five basic oral perceptions: sweet, salty, sour, bitter and
umami. Nutrients are initially detected in the mouth by interacting with
different transduction elements, including ion channels and GPCRs that are
expressed in the apical membranes of specialized epithelial cells, known as
taste receptor cells. Recently the sensors detecting sweet and bitter
sensations and amino acids have been identified and characterized as unrelated
GPCR families: the T1R (sweet and amino acids) and T2R (bitter) receptor
families. Several investigators reported the expression of bitter, sweet and
umami taste receptors in the gut. Bezencon et al showed that T1R1, T1R2, T1R3,
α-gustducin and TRPM5 are expressed in the stomach, small intestine and colon
of humans8. Activation of the taste receptor-signalling molecules, which are
expressed in entero-endocrine cells (EEC), by luminal content induces an
increase in intracellular Ca2+, which triggers the release of peptides like
PYY, GLP-1 or CCK. If this could be confirmed in humans, tastants possibly
could be used in weight management and weight loss strategies

Chen et al showed that addition of the bitter stimulus denatonium benzoate (DB)
increased the intracellular Ca2+ in STC-1 cells, leading to CCK release9. This
fits with the strong CCK-stimulating ability of proteins, which are generally
perceived as having a bitter compound. Geraedts et al showed that bitter, sour
and sweet tastants induce the largest effect on CCK release in the STC-1 cell
line10. In humans, the precise effect of stimulating taste receptors in the
small intestine is unknown and the release of gut derived satiety peptides in
response to bitter and sour stimuli could play a role in protecting against
potentially toxic (bitter) substances.

It is known that the appearance of certain nutrients in the small intestine
results in a negative feedback mechanism from different parts of the intestine
to the stomach, the small intestine and to the central nervous system. These
processes inhibit food processing, appetite sensations and food intake, and
furthermore they increase feelings of satiety and satiation. It was shown that
the ileal brake induced stronger effects on gut function than the duodenal or
jejunal brake when fat was used as a brake substrate. Since taste receptors
were found in the small intestine, infusion of different tastants directly into
the small intestine could result in the activation of an intestinal brake or
the release of satiety peptides. Therefore we would like to investigate the
effects of representatives of the five tastants (sweet, salty, sour, bitter and
umami) on the ex vivo release of the gut satiety peptide CCK and GLP-1. First
we will use duodenal tissue to confirm the release of gut satiety peptides. The
duodenum is the first small bowel part to sense nutrients upon oral ingestion.
When positive results with tastants have been obtained (release of gut
peptides) in the duodenum, the procedure will be extended to distal ileum
tissue. We anticipate, based on the strong inhibitory effect of the ileal brake
on satiety in humans in vivo, that this effect is even more pronounced than in
the duodenum.

This will be investigated by using the Ussing chamber. The Ussing chamber was
first described in 1951 by the Danish physiologists Ussing and Zerhan. This
technique enables to study vital tissue outside of the body for several hours,
and has many applications. It is mostly used to study ion transport, drug and
protein absorption, and several pathophysiological processes both in animals
and humans. Additionally, it provides a suitable model to study effects of a
variety of compounds on intestinal tissue secretions. The Ussing chamber can be
used to (simultaneously) study different types of substances and this makes it
a fast, non-invasive and low cost screening method. Our group showed
previously that different types of proteins exert different effects on the
release of CCK and GLP-1 by human intestinal mucosa11. We also validated the
use of the Ussing chamber to study satiety hormone release8 by intestinal
mucosa. The results of this study will be used to design a follow-up human in
vivo experiment, targeting the ileal brake with tastants.

Hypotheses:
- The five tastants (sour, sweet, salt, bitter and umami) each induce GI
peptide release by human duodenal mucosa ex vivo.
- The five tastants (sour, sweet, salt, bitter and umami) each induce GI
peptide release by human ileal mucosa ex vivo.
- Denatonium benzoate (bitter) induces the largest effects on the release of
gut derived satiety peptide hormones.
- The five tastants induce different larger effects (higher release of
CCK, GLP-1 and PYY) on proximal distal (duodenumileum) versus than distal
proximal (ileumduodenum) parts of the human small intestine. of the human

Primary objective:
• To assess the effect of sour, sweet, salt, bitter and umami tastants on the
release of satiety hormones (CCK, GLP-1 and PYY) in human duodenal mucosa ex
vivo

• To assess the effect of sour, sweet, salt, bitter and umami tastants on the
release of satiety hormones (CCK, GLP-1 and PYY) in human ileal mucosa ex vivo

Secondary objective:
• To investigate the effect of the five tastants on different parts (duodenum
and ileum) of the human small intestine

This study is designed as an observational study with invasive measurements

Patients undergo gastroduodenoscopy/colonoscopy for a medical reason. If a
patient decides to participate in this study, 6-8 biopsies of the duodenom or
ileum will be taken if no relevant abnormalities are found during endoscopy.
This is the only difference with the regular procedure.

All patients undergo gastroduodenoscopy or colonoscopy for a medical reason.
Only difference with regular procedure is taking 6-8 extra biopsies at the end
of the procedure. This will extend the duration of the endoscopies with 2-3
minutes.

Gastroduodenoscopy
The gastroduodenoscopy, performed by a gastroenterologist, is a standard
procedure that takes 10 to 20 minutes. These patients have a medical indication
to undergo a gastroduodenoscopy. The only difference, with the standard
procedure, is that 6-8 extra biopsies will be taken (with a standard biopsy
forceps). Diagnostic upper GI endoscopy is a remarkably safe procedure. One
large US study estimated an overall complication rate (including mucosal
biopsy) of 0.13% and an associated mortality of 0.004%. Taking the additional
biopsies will be the only extra risk for the patient. We would like to include
patients who already need to undergo a gastroduodenoscopy for a medical reason.
By this mean we can diminish the risk of a gastroduodenoscopy for healthy
volunteers.

Colonoscopy
In patients undergoing colonoscopies for a medical reason (which is the case in
the patients used in this study), there is a very small risk (ranging from
0.016-%0.2%) of bowel perforation. Shiffman et al conducted a study on the risk
of bleeding after endoscopic biopsy or polypectomy. They found that 4.6% of all
patients (32 of 694) reported bleeding, 28 had a minor and self-limited,
clinically insignificant bleeding and 4 (0.58%) had a major bleeding which
required hospitalization or treatment. All 4 of these patients had undergone
colonic polypectomy. Since the colonoscopy in these patients is performed
because of medical reasons (not for research reasons), the patients will be
informed about these risks by the gastroenterologist. We are taking a few
extra small biopsies (with standard forceps), and therefore we expect that the
risk of adverse events because of these extra biopsies would be much lower than
0.58%