1

A role for

Glucagon-like

peptide-1 in the

Pathophysiology of

Irritable Bowel

Syndrome

2

Contents Non-Standard Abbreviations ............................................................................................. 3

Introduction ......................................................................................................................... 5

The Gastrointestinal Tract ................................................................................................. 5

The Enteric Nervous System (ENS) ................................................................................... 7

What is Irritable Bowel Syndrome? ................................................................................... 8

Irritable Bowel Syndrome Models ..................................................................................... 9

Wistar Kyoto Rat Irritable Bowel Syndrome Model ..................................................... 9

Maternal Separation Rat Irritable Bowel Syndrome Model........................................... 9

Glucagon-like Peptide-1 .................................................................................................. 10

Treating Irritable Bowel Syndrome ................................................................................. 11

The Brain Gut Axis .......................................................................................................... 11

The Hypothalamic-Pituitary-Adrenal Axis .................................................................. 12

Irritable Bowels Syndrome and the Immune System........................................................ 13

Interleukin-6 ................................................................................................................. 14

Aims of the Study .............................................................................................................. 15

Materials and Methods ..................................................................................................... 16

Animals ............................................................................................................................ 16

Tissue Preparation ........................................................................................................... 16

Whole mount preparation of Submucosal Plexus ........................................................ 16

Cross Section Preparation of the Distal Colon ............................................................. 17

Immunofluorescence ........................................................................................................ 17

Enzyme Linked Immunosorbent Assay (ELISA) ............................................................... 19

Statistical Analyses .......................................................................................................... 19

Results ................................................................................................................................. 20

Immunofluorescence labelling of GLP-1Rs in SMP preparations .................................. 20

Colonic Expression of L-cells and GLP-1R and IL-6 expressing cells ........................... 24

Plasma Concentrations of Glucagon-like peptide-1 ........................................................ 25

Discussion ........................................................................................................................... 27

Further Studies ................................................................................................................ 29

References........................................................................................................................... 31

3

Non-Standard Abbreviations

5-HT - 5-hydroxytryptamine

ACTH - adrenocorticotropic hormone

CaCl2 - calcium chloride

cm - centimetres

CRF - corticotropin releasing factor

DPP IV - dipeptidyl peptidase-4

ELISA - enzyme linked immunosorbant assay

ENS - enteric nervous system

FITC - fluorescein isothiocyanate

g - grams

GIT - gastrointestinal tract

GLP-1R - glucagon like peptide-1 receptor

h - hour

HPA - Hypothalamic Pituitary Axis

IBS - Irritable Bowel Syndrome IL-6,8,10,1β - interleukin-6,8,10,1β

KCl - potassium chloride

KH2PO4 - monopotassium phosphate

MgCl2 - magnesium chloride

ml - millilitre

mmol/L - millimoles per litre

MS - maternal separation

NaCl - sodium chloride

NaHCO3 - sodium bicaronate

NaH2PO4 - monosodium phosphate

Na2HPO4 - disodium hydrogen phosphate

PBS - phosphate buffered saline

pM - picomolar

TNF - tumour necrosis factor

µm - micrometres

WKY - Wistar Kyoto

4

Abstract

Introduction: Irritable bowel syndrome (IBS) symptoms include visceral hypersensivity

and altered gut motility. Additionally absorpto-secretory function is altered resulting in

constipation and/or diarrhoea. There is evidence which suggests altered immune cytokine

profiles may contribute to symptom flares. Interleukin-6 (IL-6) has been proposed as a

potential biomarker for IBS as it is elevated in IBS plasma. It also activates submucosal

neurons and stimulates colonic secretion. However other molecules may be important in a

specific characterisation of IBS, post-prandial exacerbated of symptoms. A possible

candidate for this is Glucagon-like peptide-1 (GLP-1), an incretin hormone which is

secreted following a meal and has antispasmodic effects on the gut. GLP-1 receptors are

also expressed in submucosal neurons and modulate neurally evoked chloride secretions.

Therefore the role of GLP-1 and the potential cross-talk between GLP-1 and IL-6 was

investigated further.

Methods: Whole mount preparations of the submucosal plexus from male Sprague

Dawley (SD) rats were fixed and stained with GLP-1R and dual labelled with IL-6,

calbindin, calretinin, nNOS, S100, Synapsin I or PSD-95. Cross sections were also

prepared from adult male SD and Wistar Kyoto rats and dual labelled with GLP1 and

GLP-1R or IL-6. GLP-1 levels were determined using an ELISA and results were

correlated with previously determined levels of circulating IL-6, TNF-α, IL-1β and IL-8.

Results: In immunohistochemistry studies GLP-1R were expressed in a punctate manner

in the neuronal fibres, cell bodies and glial cells. 26% (n=9/35) of calbindin neurons, 21%

(n=18/84) of calretinin neurons, 37.5% (n=48/128) of S100 cells and the majority, 63%

(n=27/43) of nNOS were GLP-1R positive. Dual labelling with GLP-1R and Synapsin I or

PSD-95 showed that GLP-1R is present in the synapses. GLP-1R co-localised with 30%

(n=26/87) of IL-6 positive neurons revealing potential crosstalk between the molecules. In

Elisa studies (n=9 samples per group) GLP-1 levels were significantly elevated in IBS-A

(p<0.05) plasma and there was a trend towards significance in IBS-D plasma samples.

Moreover plasma GLP-1 levels correlated with IL-6 levels.

Conclusion: These data demonstrate that GLP-1Rs are expressed on colonic submucosal

neurons, where GLP-1 induced cellular signalling is likely to be mediated by nitrergic

mechanisms. Moreover many of these cells co-express IL-6 revealing the potential cross-

talk between IL-6 and GLP-1. Correlation between IL-6 and GLP-1 levels in human IBS

plasma implicates this incretin hormone in the pathogenesis of IBS.

5

Introduction

The Gastrointestinal Tract

Fig 1:Schematic diagram of the gastrointestinal tract (Tharakan et al., 2010)

The gastrointestinal tract (GIT) provides the body with a continual supply of water,

nutrients and electrolytes. Food is propelled through the tract, where it is digested by

secreted digestive juices facilitating nutrient and water absorption. Regulation of this

fundamental process is both by extrinsic and intrinsic neuronal control and through

endocrine and paracrine processes. Figure 1 shows the various parts to the GIT and

digestive system. Each part of the GIT is adapted for specific functions: the oesophagus is

needed for the passage of food, the stomach functions as a temporary storage of food and

also digests food, the small intestine is needed to digest and absorb food and the large

intestine is needed for the absorption of water and electrolytes (Guyton and Hall, 2001).

6

The large intestine consists of the cecum, colon, rectum and anal canal. The colon can be

divided into four parts; the ascending colon, the transverse colon, the descending colon and

the sigmoid colon. The ascending colon and proximal transverse colon are known as the

proximal colon and the distal transverse, descending and sigmoid colon are known as the

distal colon. The distal colon is the anatomical part under investigation in this study. The

wall of the colon as well as most of the GIT, typically includes, from outer to inner layer;

the serosa, a longitudinal muscle layer, a circular muscle layer, the submucosa and the

mucosal layer. As figure 2 shows, the mucosal lining of the large intestine consists of

simple columnar epithelium that has numerous crypts, known as crypts of Lieberkuhn.

These crypts contain three cell types; absorptive, goblet and granular, with goblet cells as

predominating cell type (Seeley et al., 2003).

Fig 2: Histology of the large intestine (Martini et al., 2012)

Goblet cells of the colon are responsible for the mucosal excretions. The function of the

mucous is to lubricate the wall of the lumen and to help the faecal matter stick together as

well as protecting the intestinal wall from bacterial activity that occurs within the faeces.

The mucous contains bicarbonate ions which is responsible for protecting the intestinal

wall from acids formed (Guyton and Hall, 2001). The colonic absorption of water occurs

by a molecular pump which exchanges HCO3- for Cl- in epithelial cells in response to acid

7

produced by colic bacteria. Also another pump exchanges Na+ for H+, water then crosses

the colon by osmosis due to the NaCl gradient.

Irritation to the wall of the colon causes an increase in mucous secretion by triggering local

enteric reflexes. The submucosal plexus and myenteric plexus are both part of the enteric

nervous system (ENS). As well as the enteric nervous system, the GIT is supplied with

parasympathetic and sympathetic innervations (Seeley et al., 2003). The submucosal

plexus which was under investigation in this study in relation to Irritable Bowel Syndrome

(IBS) only has parasympathetic input and controls mainly the secretion and absorption

functions. The myenteric plexus is responsible for contractility of the GIT and has both

sympathetic and parasympathetic input (Guyton and Hall, 2001).

The Enteric Nervous System (ENS)

The ENS is one of the main divisions of the autonomic nervous system and governs the

function of the gastrointestinal tract. The ENS is collected into two types of ganglia the

myenteric (Auerbach’s) plexus and the submucosal (Meissner’s) plexus. The myenteric

plexus is contained between the longitudinal and circular muscle layer. This plexus

contains many interconnecting neurons that extend the entire GIT. When this plexus is

stimulated there is increased tonic contraction of the gut wall, increased intensity of

rhythmical contractions, an increase in the rhythm of the contraction and increased velocity

of conduction of excitatory waves. The myenteric plexus has excitatory as well as

inhibitory neurons. The inhibitory neurotransmitter of this plexus is possibly vasoactive

intestinal polypeptide.

The submucosal plexus is contained within the submucosa. It controls the function within

each minute segment of the intestine. The plexus controls local intestinal secretion, local

absorption and local contraction of the submucosal muscle as the sensory signals that

originate in the epithelium are integrated into the submucosal plexus. The local contraction

causes various degrees of infoldings of the mucosa.

The different neurons within the submucosal and myenteric plexus release different types

of neurotransmitters. The neurotransmitters include acetylcholine, norepinephrine,

8

adenosine triphosphate, serotonin, dopamine, cholecystokinin, substance P, vasoactive

intestinal polypeptide, somatostatin, leu-enkephalin, met-enkephelin and bombesin

(Guyton and Hall, 2001). A colonic mucosal biopsy would contain the above mentioned

neurotransmitters as well as proteases and histamine. An increased activation of

submucosal neurons is seen in the presence of colonic mucosal biopsy supernatants from

Irritable Bowel Syndrome (IBS) patients (Buhner et al., 2012). Therefore it is important to

study the submucosal plexus further in relation to IBS.

What is Irritable Bowel Syndrome?

IBS is a functional gastrointestinal disorder. Symptoms include abdominal pain, discomfort

and altered bowel habits (Yale et al., 2008), affecting approximately 10% of the population

(Corsetti et al., 2004). IBS is a heterogeneous, multisystem, multidomain disorder as

opposed to a single disease entity (Crentsil, 2005) with several mechanisms being

proposed to explain the altered sensory responses observed (Yale et al., 2008). It is also a

polygenic disorder and the IBS phenotype can be determined by many common genetic

variants (Saito et al., 2010). To evaluate the role of genes in IBS, candidate genes are

studied that have an association with the intermediate phenotypes of IBS such as altered

colonic transit, colonic motility and compliance and decreased sensation thresholds and

ratings. Some of the candidate genes include genes involved in the alteration of

serotonergic mechanisms (Camilleri and Katzka, 2012).Studying genes involved in

serotonergic mechanisms show polymorphisms in the promoter region of serotonin

transporters in IBS (Murphy et al., 2004) .

Genetic abnormalities along with abnormalities in the enteric and the central nervous

system which lead to altered pain and motor functions are all thought to account for the

symptoms of IBS (Drossman, 2005). Also psychological stressors and ongoing enteric

infections have been implicated in the onset and maintenance of IBS (FitzGerald et al.,

2009). Indeed 7-31% of patients develop IBS after gastroenteritis (McKendrick, 1996) and

this is termed post infectious IBS, thus revealing the importance of immune activation in

this disorder.

9

As there is no biochemical marker or structural abnormality for IBS, diagnosis is based on

the presence of clinical symptoms according to Rome III criteria (Dorn et al., 2009).

However groups can be either distinguished as being IBS-D (diarrhoea predominant), IBS-

C (constipation predominant) or IBS-A (alternating between diarrhoea and constipation).

There is also a variability of symptoms between male and females and within each group

(Yale et al., 2008).

Irritable Bowel Syndrome Models

Wistar Kyoto Rat Irritable Bowel Syndrome Model

The Wistar Kyoto rat is a pre-clinical model of IBS. IBS is associated with an enhanced

visceral sensitivity in response to altered brain gut axis signalling such as that occurs

during stress. The WKY rats are a viscerally hypersensitive rat model of IBS as they are

genetically predisposed to pathological stress responses with alterations in colonic

accommodation and morphology (Martínez et al., 2007) which leads to enhanced colonic

motility and faecal output following a psychological stressor (O'Malley et al., 2010). Also

following stressful stimuli WKY rats display exaggerated corticosterone levels (O'Mahony

et al., 2010). A study has also shown that while the plasma Interleukin-6 (IL-6) levels are

similar, mucosal scrapings from WKY rats contain higher levels of IL-6 and excised

colons secrete more IL-6 than Sprague Dawley (SD) colons. The WKY secretions have

also been shown to excite submucosal neurons more so than SD secretions (O'Malley et

al., 2011a). O’Malley et al. also observed that under resting conditions the WKY rat in

comparison to the SD rat displays a pro-absorptive phenotype as was elicited by the

decrease in secretory currents. However this reversed in the presence of IL-6 where WKY

colonic tissues evoked a larger secretory current (O'Malley et al., 2012).

Maternal Separation Rat Irritable Bowel Syndrome Model

The maternal separation (MS) rat model is another model of IBS. This model involves a

postnatal stress that induces alterations in the hypothalamic-pituitary-adrenal (HPA) axis of

the adult offspring (O'Malley et al., 2010). Like the WKY rat model the MS model exhibit

symptoms of anxiety and depression (O'Mahony et al., 2008). The MS rat also exhibits

10

exaggerated GI response to inflammatory stimuli and colorectal distension, showing

increased stress induced faecal output. This model shows an increased level of circulating

IL-6 following an immunological challenge as well as altered 5-HT levels, and visceral

hypersensitivity (O'Mahony et al., 2009).

The two rat models exhibit alterations in colonic motility (O' Mahony et al., 2011) which is

a prominent phenotype of IBS (Camilleri and Katzka, 2012). As gut motility is a major

function of glucagon-like peptide-1 studying GLP-1 in relation to IBS is of clinical interest

(Hellström, 2009).

Glucagon-like Peptide-1

GLP-1 is formed in the L cells following cleavage from a pro-glucagon precursor molecule

by post-translational processing and is a C-terminally amidated 7-37 amino acid peptide

(Hellström, 2011). GLP-1 regulates numerous gastrointestinal functions, it decreases

gastric acid secretions (Schjoldager et al., 1989), gastrointestinal transit, motility

(Hellström et al., 2008) and gastric wall tone (Schirra et al., 2002).

The MMC are waves of activity that sweep through the intestine during the fasting state

and they help trigger peristaltic waves which facilitate transport of the bolus through the

GIT (Tolessa et al., 1998b). Helstrom et al. showed that GLP-1 inhibits the migrating

myoelectric complex (MMC) during fasting in healthy and IBS patients (Hellström et al.,

2008).

Along with regulating gastrointestinal motility functions GLP-1 has anti-hyperglycaemic

actions. GLP-1 is released from L-cells in the ileum and colon following intake of

carbohydrates and fat and is considered an important regulator of postprandial insulin

secretion from the pancreas in response to a meal (Hellström et al., 2008). GLP-1 acts on

β-cells of the pancreas to cause an increase in insulin secretion even before blood sugar

levels become elevated (Hellström, 2009). GLP-1 has also been shown to suppress

glucagon release from the pancreas and to enhance the sensitivity of pancreatic B-cells to

glucose (Gutniak et al., 1994).

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GLP-1 has a half-life of about 1-2 minute and is degraded by depiptidyl peptidase IV

(DPP- IV) which is an enzyme present in the plasma (Hellström, 2011). The fact that it has

such a short half-life together with the fact that GLP-1 has no effects on intestinal muscle

strips in vitro concludes that direct endocrine or autocrine actions are unlikely. Therefore

neurocrine actions of GLP-1 are most likely (Tolessa et al., 1998a).

GLP-1 has also been has also been shown to be implicated in stress induced alteration in

colonic transit via central CRF pathways and peripheral cholinergic pathways in rats

(Nakade et al., 2007). IBS symptoms are exacerbated following a meal as lipids in the

intraduodenum are shown to increase visceral sensitivity (Caldarella et al., 2005) and as

GLP-1 is released following intake of carbohydrates and fat (Hellström et al., 2008) it is

important to investigate GLP-1 in relation to IBS.

Treating Irritable Bowel Syndrome Despite the prevalance and economic burden of IBS treatment options remain limited.

Current treatment options include dietary adjustment, for example increasing the amount of

fiber in the diet, the use of antidiarroheals and the use of laxatives (Dalrymple and Bullock,

2008). IBS pain is due to visceral hypersensitivity and patients who have this pain as their

most common complaint are most impacted by IBS and and there are currently no

treatments targeting this pain (Olden, 2003).

As previously described GLP-1 plays a role in the regulation of gut motility and therefore

could be important clinically in treating pain that is associated with abnormal motor

activity in the gut. A clinical trial was formulated where the GLP-1 hormone analogue

ROSE-010 was administered to suitable IBS patients. The results found that the GLP-1

analogue ROSE-010 caused meaningful pain relieve in a significant proportion of the

patients and delayed gastric emptying of food without retarding small bowel or colonic

transit (Hellström et al., 2009).

The Brain Gut Axis The Brain gut axis includes the parasympathetic innervation and the sympathetic

innervation, which supplies nerve fibres from the brain and spinal cord to the GIT. The

brain gut axis also includes the previously described enteric nervous system (Saulnier et

12

al., 2013). A system which has a major influence on the brain gut axis is the HPA axis

(Dinan, 1994).

The Hypothalamic-Pituitary-Adrenal Axis

Fig 3: The HPA axis and central and peripheral role of CRF pathways influencing immune

processes (Kiank et al., 2010).

Figure 3 outlines the HPA axis. The HPA axis includes the hypothalamus, the pituitary

gland and the adrenal gland. Corticotropin releasing factor (CRF) is released from the

paraventricular nucleus of the hypothalamus at the median emminance into the primary

capillary plexus of the hypothalamo-hypophyseal portal system. It is then carried to the

13

anterior lobe of the pituitary where it stimulates corticotropes to release

adrenocorticotropic hormone (ACTH). ACTH stimulates the synthesis of cortisol,

glucocorticoids and mineralocorticoids (Gillespie and Nemeroff, 2005). CRF containing

neurons are widely distributed throughout extrahypothalmic brain areas and acts as a

neuromodulatory agent in coordinating behaviour, endocrine, autocrine and immune

responses to stress (Corsetti et al., 2004). CRF release is also controlled by

neurotransmitters such as 5-HT and norepinephrine in response to stress (Dinan, 1996)

Patients with IBS show an exaggerated stress response when administering CRF and this

could be seen by the enhanced levels of ACTH and cortisol (Dinan et al., 2006, FitzGerald

et al., 2009). The increase in ACTH could be due to the exogenous CRF acting

synergically with the pro-inflammatory cytokines and other secretogogues to bring about

ACTH release. The increase could also be due to the CRF1 receptor on the anterior

pituitary being up-regulated in IBS (Gillespie and Nemeroff, 2005). The enhanced level of

ACTH and pro-inflammatory cytokines were shown not to be due to a decreased

sensitivity of the glucocorticoid receptor (Dinan et al., 2006). There is also evidence of a

similar upregulation of CRF1 receptor in the gut as a result of stress. This upregulation of

CRF1 receptors warrants further investigation of CRF1 receptor antagonists as a treatment

option for IBS (Fukudo et al., 1998, Gillespie and Nemeroff, 2005).

Irritable Bowels Syndrome and the Immune System

It is well established that acute gastrointestinal infections initiate the onset of IBS

symptoms in at least a sub-group of patients (McKendrick and Read, 1994). While some

IBS patients show persistent levels of low grade inflammation with increased levels of pro-

inflammatory cytokines and activated T lymphocytes and mast cells (van der Veek et al.,

2005). It is also shown that IBS patients have altered GI permeability (Camilleri et al.,

2012) and most pro-inflammatory cytokines have the capacity to alter intestinal epithelial

permeability (Al-Sadi et al., 2009).

Figure 3 outlines CRF pathways and the influence they have on immune processes.

Immune cells such as macrophages, T lymphocytes, mucosal mast cells, and dendritic cells

14

respond to CRF receptor signalling mainly by promoting inflammation. However central

CRF evoke an anti-inflammatory response, which may counter-regulate inflammatory

responses in the gut, by increasing glucocorticoids and catecholamines and decreasing

parasympathetic activity (Kiank et al., 2010). In response to infection CRF containing

neurons also respond to pro-inflammatory cytokines (Chrousos, 1995).

Cytokines are small cell signalling protein molecules secreted by leukocytes and various

other cells. Fundamentally cytokines influence innate and adaptive immunity (Ortiz-Lucas

et al., 2010). Specifically pro-inflammatory cytokines such as IL-6 and IL-8 are shown to

be increased in patients with IBS and are shown to activate the HPA axis (Dinan et al.,

2006, FitzGerald et al., 2009). Analysis has also shown that HPA hyperresponsivity is

related to enhanced IL-6 levels (Dinan et al., 2006). In turn activating the HPA axis causes

an increased release of glucocorticoids which depresses inflammatory processes of the gut

(Turnbull and Rivier, 1999). It is unclear whether the increase in IL-6 and IL-8 are

biological markers for a trait of the syndrome or IBS itself (Dinan et al., 2006).

Interleukin-6

Interleukin-6 (IL-6) is a pro-inflammatory and anti-inflammatory cytokine (Scheller et al.,

2011) and stimulates secretion into the colonic lumen by modulating submucosal neurons.

IL-6 is elevated in the mucosa and plasma of the pre-clinical model of IBS, the WKY rat

(O'Malley et al., 2012, O'Malley et al., 2011a). IL-6 levels are also increased in the plasma

of IBS patients which may reflect an important mechanism which leads to symptom flares

(Dinan et al., 2006).

Following administration of cholinesterase inhibitor there is an increase in IL-6 synthesis

which correlates to an increase in abdominal bloating (Dinan et al., 2008). IL-6 along with

IL-1 β was shown to act as excitatory neuromodulators of myenteric neurons via

presynaptic inhibition by inhibiting acetylcholine release from pre-synaptic neurons

(Keller et al., 2001). It has also been shown that IL-6 inhibits nicotinic and noradrenergic

neurotransmitter release in guinea pig submucosal neurons (Xia et al., 1999).IL-6 receptors

are expressed on rat submucosal neurons and following exposure to recombinant IL-6 there

is an increase in extracellular calcium and colonic excretion that overrides the pro-

15

absorptive phenotype, which is regulated by epithelial cholinergic activity at rest

(O'Malley et al., 2011b).

Adipose tissue is a major source of IL-6 concentration and therefore contributes to the

induction of insulin resistance. Contracting skeletal muscles during exercise also increases

circulating IL-6 concentrations. It is also proposed to promote nutrient availability and

improves whole body insulin sensitivity. Pancreatic alpha cells are also a primary target of

IL-6 action. IL-6 promotes alpha cell proliferation and inhibits apoptosis. In a high fat diet

alpha cells mass expands in an IL 6 dependent manner. IL-6 was also shown to promote

GLP-1 secretion and production from intestinal L cells and pancreatic alpha cells

(Ellingsgaard et al., 2011)

Other cytokines such as interferon γ, tumour necrosis factor (TNF), transforming growth

factor and interleukin-10 (IL-10) regulate inflammatory and immune responses

(Gonsalkorale et al., 2003a). A UK study showed that frequency of the high IL-10

producer genotype for IL-10 (an anti-inflammatory cytokine) were significantly reduced in

patients with IBS as compared with healthy controls (Gonsalkorale et al., 2003b).

Aims of the Study

The aims of the study were to indentify the location of GLP-1R and IL-6 within the

submucosal ganglia of the distal colon. After getting location of GLP-1R and IL-6 within

the submucosal ganglia we wanted to observe the quantity and location of GLP-1, IL-6 and

GLP-1R across the entire cross section of the distal colon. To relate this to the human

condition we investigated levels of GLP-1 in IBS patients and compared them to healthy

controls and aimed to correlate these values with levels of other pro-inflammatory

cytokines.

16

Materials and Methods

Animals Male Sprague Dawley rats were purchased from Harlan UK weighing approximately 250-

350 g. The rats were housed in groups of four to six per cage. They were maintained on a

12/12 h dark–light cycle and were given food and water ad libitum and kept at a room

temperature of 21-23oC. Principles of laboratory animal care were followed, and all

procedures were carried out in accordance with EU directive 89/609/EEC and approved by

the Animal Experimentation and Ethics Committee.

Tissue Preparation

Whole mount preparation of the Submucosal Plexus

A section of the colon was excised from each rat, cut 8cm proximal from the anus. The

excised distal colon was stored in ice cold Krebs (pH7.4) solution at 4oC; containing in

mmol/L: 117 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4 and 11 D-

glucose. The pellets were then expunged using a 10 ml syringe filled with Krebs. Using a

forceps the section of colon was pulled over a glass rod allowing for the mesenteric blood

vessels to be removed. The colon was lightly scored along where the mesentery was

removed, using a blade.

Fig4: Layers of the colon. JD Wood et al

Figure 4 shows the various layers of the colon. The longitudinal and circular muscle

containing the myenteric plexus were removed using a cotton swab. A scissors was used to

17

cut all the way through the mesentery and the remaining submucosal layer was pinned out

mucosal side up on a sylgard lined plate filled with Krebs saline. Figure 4 also shows that

the submucosal ganglion is contained within a separate layer from the mucosa. Using

forceps the mucosal layer was removed and the remaining submucosal plexus was

prepared for Immunofluorescent Imaging as detailed below.

Cross Section Preparation of the Distal Colon

Sections of colon from four separate Sprague Dawley rats (the control model) and four

separate Wistar Kyoto rats (the pre-clinical IBS model) had been fixed and frozen from a

previous study. Short sections of the colons were cut and brought up to -250C in the

cryostat (Leica, Nussloch, Germany; CM1900 UV). Using the cryostat, sections were

prepared. Firstly Tissue-Tek® O.C.T. (optimum cutting temperature) compound

embedding medium was applied to the stage and left to freeze for ten minutes. The colon

was then mounted vertically onto the stage and more mounting medium was applied over

the colon, again the medium was left to freeze for another 10 minutes. 10 μm sections were

taken with 30 μm between sections. Eight sections were taken from each colon and applied

to two separate Fisherbrand glass slides, four sections were applied to a slide to be stained

for anti-GLP-1R and anti-GLP-1 and four sections were applied to a slide to be stained

with anti-IL-6 and anti-GLP-1.

Immunofluorescence

Immunofluorescence of whole mount SMP preparations

Using 96-well plates seven separate sections of the submucosal plexus were stained from

each of the four Sprague Dawley rats. The whole mount preparations were first fixed

overnight in Zamboni’s fixative [paraformaldehyde (from 16% stock solution), picric acid

(saturated aqueous), 10M NaOH, 0.1M phostphate buffer] at 4oC. The sections were

washed three times for 15 minutes in phosphate buffered saline (PBS) (containing in

mmol/L: 137 NaCl, 2.7 KCl, 10 Na2HPO4.2H2O, 2.0 KH2PO4) and agitated. The

preparations were then permeabilised for 1 hour in 0.1% Triton X-100 in PBS at room

temperature (R.T.). Again the sections were washed three times (X3) for 15 minutes in

PBS. For 1 hour the sections were incubated in blocking solution (1% donkey serum in

PBS) at R.T. All sections were incubated overnight at 4oC with a 1:250 dilution of anti-

18

GLP-1 receptor (GLP-1R) antibody (affinity purified rabbit polyclonal antibody a synthetic

peptide conjugated to KLH derived from within 250-350 of Human GLP1R; Abcam,

Cambridge, UK). The sections were washed again in PBS for 15 minutes X3. The GLP-1R

antibody labelled tissues, were incubated with a 1:250 dilution of Texas Red conjugated

donkey anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA,

USA). The sections were washed for 10 minutes in PBS X3. For dual labelling the sections

were incubated overnight with a separate primary antibody including neuronal markers for

calbindin (1:300; mouse, Swant, Bellinzona, Switzerland) calretinin (1:300, goat; Swant)

and neuronal nitric oxide synthase (nNOS, 1:300; goat, Abcam), the glial cell marker S100

(1:300, mouse; Sigma-Aldrich, St. Louis, MO) and the anti-rat IL-6 antibody (affinity

purified goat antibody immunised E.coli-derived rIL-6, 1:300; R&D Systems, Abingdon,

UK). Sections were also stained with the pre-synaptic marker Synapsin I (1:250, goat;

Santa Cruz Biotechnology, Santa Cruz, CA) and the post-synaptic marker PSD-95 (1:200;

mouse, Santa Cruz Biotechnology). Slides were again washed for 10 minutes in PBS X3.

The sections were then incubated for 2 hours with Fluorescein isothiocyanate (FITC)

conjugated antibody (1:250 goat anti mouse Jackson ImmunoResearch) and (1:250 donkey

anti-goat; Milipore). Dako fluorescent mounting medium was used to mount the sections

onto glass slides and a cover slip was placed over them.

Immunofluorescence of Cross Sections of Colon

The cross sections were prepared for immunofluorescent imaging. Two slides containing

four sections were prepared for each rat. One of the two slides from each rat was incubated

with a 1:200 dilution of GLP-1 antibody (mouse antibody; Antibody Shop). A 1:300

dilution goat anti-mouse rhodamine conjugated secondary antibody was used as a

fluorophore (Jackson ImmunoResearch). This slide was then dual labelled with a 1:250

dilution of GLP-1R antibody (Abcam) with FITC conjugated donkey anti-rabbit secondary

antibody as the fluorophore. The second slide from each rat was incubated with a 1:200

dilution of GLP-1 antibody and with a 1:300 dilution of the anti-mouse rhodamine

conjugated secondary antibody. The second slide was also incubated with the anti-rat IL-6

antibody (1:300; R&D Systems) and with a FITC conjugated anti-goat secondary acting as

the fluorophore.

19

Image analyses

Neurons in submucosal ganglia were imaged using Cell-F software (Soft Imaging

Solutions) and an Olympus BX51 fluorescent microscope [Olympus America, Inc,

Melville, NY, USA] with an Olympus DP71 digital camera [Optronics, Goleta, CA,USA]

with filter sets for TRITC [excitation (557nm), emission (576nm)] and FITC [excitation

(470nm), emission (525nm)]. Images were also obtained from the FLUOVIEW FVlOi-

Olympus-confocal. Three ganglia per tissue were imaged from four separate rats. With the

Cell-F software co-localisation between the cell markers (calbindin, calretinin, nNOS and

S100) and GLP-1R was determined by counting the number of cells positive for the cell

markers and calculating the percentage of these cells that co-localised with GLP-1R. Co-

localisation between the synaptic markers (PSD-95 and Synapsin I) and GLP-1R was

determined using the confocal. Co-localisation between and location of GLP-1R and GLP-

1 was also determined using confocal microscopy as was co-localisation between and

location of GLP-1 and IL6 on the colon cross sections.

Enzyme Linked Immunosorbent Assay (ELISA)

A sandwich ELISA was carried out to determine the GLP-1 concentration in plasma

samples from 9 healthy, 9 IBS-C, 9 IBS-D and 9 IBS-A patients following manufacturer’s

guidelines (Milipore; Cat. EGLP-35K). The assay was run in duplicate. DPP-IV

(dipeptidyl peptidase-IV) was added to the samples to inhibit GLP-1 degradation. A multi-

pipette was used to ensure accurate measurements of solutions were made. The plates were

read on a Synergy HT fluorescent microplate reader with an excitation/emission

wavelength of 355 nm and 460 nm respectively and the lowest level of GLP-1 that could

be detected was 2 pM. Having solutions of known GLP-1 concentrations, from 0 to 100

pM, a standard curve was created. From this curve the GLP-1 concentrations of the plasma

samples were determined.

Statistical Analyses

Experiments were conducted in at least four different animals and in the case of the ELISA

at least 9 patients per group. ELISA data was analysed using GraphPad prism for windows

(version 7). Paired or unpaired two-tailed Student’s t-test were used where appropriate,

P<0.05 was considered significant.

20

Results

Immunofluorescence labelling of GLP-1Rs in SMP

preparations

Immunohistochemistry techniques were carried out on whole mount preparations taken

from the distal colon of male, adult SD rats. To determine the neuronal cell type that

expressed GLP-1R co-localisation, dual labelling studies were conducted with calbindin

antibodies to label cholinergic sensory neurons, anti-calretinin for cholinergic motor

neurons and anti-nNOS for nitrergic neurons. Anti-S100 labelled glial cells in the

submucosal ganglia.

In Figure 5 the images show GLP-1R (red stain) dual labelled with calbindin (Fig 5(a)

n=9/35), calretinin (Fig 5(b) n=18/84), nNOS (Fig 5(c) n=27/43) and S100 (Fig 5(c)

n=48/128) (green stain), (Fig 5; n= 3 ganglia per tissue from 4 different rats). GLP-1R

expression is found in the cell bodies but is also prominent in the neuron fibres and glial

cells. The prominent staining in the neuronal cell fibres is also quite punctate indicating

that GLP-1Rs are clustered together.

In Figure 7 it shows the percentage of total neurons that are positive for the different cell

markers which co-localises with GLP1R. Dual labelling with GLP-1R and calbindin

antibodies show a 26% (n=9/35) co-localisation in the cell bodies of neurons. Dual

labelling with GLP-1R and calretinin shows a 21% (n=18/84) co-localisation in the cell

body of neurons. Dual labelling with GLP-1R and nNOS shows a 63% (n=27/43) co-

localisation in the cell body of neurons. There was also GLP-1R staining of glial cells as

dual labelling with GLP-1R and S100 shows a 37.5% (n=48/128) co-localisation with the

glial cells of neurons.

21

Fig 5(a): GLP1R (Texas Red) Fig 5(b): GLP1R (Texas Red) and nNOS

and Calbindin (FITC), (FITC)

Fig 5(c): GLP1R (Texas Red) Fig 5(d): GLP1R (Texas Red) and

and calretinin (FITC) and S100 (FITC)

Fig 5: Scale bars= 20 μm

The punctate nature of the GLP1-R staining suggested that the receptor was potentially

expressed at synapses. To investigate this further, tissues were dual labelled with GLP1-R

antibody and the presynaptic marker Synapsin I and also dual labelled with GLP-1R

antibody and the postsynaptic marker PSD-95. GLP-1R expression (red stain) and

Synapsin I or PSD-95 (green stain) dual labelling can be seen in the confocal images in

Figure 6. Some co-localisation was seen between GLP-1R and the presynaptic marker

Synapsin I (Fig 6(a)) and the postsynaptic marker PSD-95 (fig 6(b)).

Cell Body

22

To determine cross-talk, co-localisation studies were conducted between IL-6 and GLP-

1R. Dual labelling with GLP-1R and IL-6, shows that co-localisation is present in 30%

(n=26/87) of neuronal cell bodies. In Figure 6(c) one neuronal body (indicated by arrow)

shows co-localisation between GLP-1R and IL-6. GLP-1R is the red stain and IL-6 which

is conjugated to the FITC secondary antibody is the green stain.

Fig 6(a): GLP1R (Texas Red) and Synapsin I (FITC). The area within the white box has

been magnified. Regions of co-localisation (yellow staining) are indicated by the arrows.

Scale bar=20 μm

Fig 6(b): GLP-1R (Texas Red) and PSD-95 (FITC). The area within the white box has

been magnified. Regions of co-localisation (yellow staining) are indicated by the arrows.

Scale bar =20 μm

23

Fig 6(c): GLP-1R (Texas Red) and IL-6 (FITC). The area within the white box has been

magnified. A cell co-expressing and IL-6 and GLP-1Rs is indicated by the arrow (n=3

ganglia per tissue from 3 different rats). Scale bar=20 μm

The histogram below (Figure 7) shows the percentage of cells expressing GLP-1Rs. GLP-

1R co-localised with a greater percentage of nNos positive neurons in comparison to other

cell markers. From Figure 7 you can also see that GLP-1R co-localises with a lesser

percentage of calretinin positive neurons in comparison to the other cell markers.

C e ll M a rk e rs

%

Calb

ind

in

Calr

et i

nin

nN

os

S100

IL-6

0

2 0

4 0

6 0

8 0

C a lb in d in

C a lre tin in

n N os

S 1 0 0

IL -6

Fig 7: Graph showing percentage of cell marker positive cells that co-localise with GLP-

1R

Nerve

Fibre Cell

Body

24

Colonic Expression of L-cells and GLP-1R and IL-6

expressing cells To determine whether GLP-1 secreting L-cells were in close proximity to GLP-1R

expressing submucosal neurons transverse sections were immunolabelled with GLP-1 and

GLP-1R and sections were also labelled with GLP-1 and IL-6 antibody. The

immunohistochemistry techniques were also used to compare the GLP-1, IL-6 and GLP-

1R staining between the IBS pre-clinical model the WKY rat, which has altered GI

morphology and the control model the SD rat. Co-localisation can be seen in Figure 8(a).

The co-localisation is between GLP-1 (red staining) and GLP-1 receptor (green staining)

and shows a couple of L-cells (indicated by arrows) in the mucosal layer of the WKY rat.

The co-localisation between IL-6 (green staining) and GLP-1 (red staining) can be seen in

Figure 8(b). A GLP-1 expressing L-cell that does not co-localise with IL-6 staining is

indicated by the arrowhead and two L-cells co-expressing GLP-1 and IL-6 are indicated by

the arrows.

Fig 8(a): Transverse section of GLP-1 and GLP-1R staining from distal colon of a WKY

rat. Scale bar= 5μm

25

Fig 8(b): Transverse Section of GLP-1 and IL-6 staining from distal colon of a SD rat.

Scale bar= 5μm

Plasma Concentrations of Glucagon-like peptide-1

Fig 9: Standard curve of GLP-1 Concentrations

An ELISA was used to calculate the GLP-1 concentrations in plasma samples from healthy

controls and from each subtype of IBS. Figure 9 is a standard curve of GLP-1

concentrations and the GLP-1 concentrations of plasma samples were determined from this

curve. A Student’s t-test showed that there was a significant difference between plasma

GLP-1 content in IBS-A patients in comparison to controls (n=9 samples, p<0.09, figure

10). The Student’s t-test did not show a significant difference between IBS-C patients and

0 25 50 75 100 1250

250

500

750

1000

Flu

ore

scen

ce (

nm

)

GLP-1 concentration (pM)

26

healthy controls (n=9 samples, p>0.05), however there was a trend toward a significant

difference between IBS-D patients and healthy patients (n=9 samples, p=0.09, figure 10).

Fig 10: GLP-1 concentrations (pM) in healthy controls, and IBS patients. A significant

elevation in GLP-1 levels in IBS-A (n=9) in comparison to healthy controls (p<0.05, t-

test).

Interestingly when compared to previously published data on the levels of cytokines in IBS

plasma (McKernan et al., 2011) a significant correlation was found between the levels of

IL-6 and GLP-1 in healthy controls, IBS-C, IBS-D and IBS-A (r2=0.12, p<0.05, Figure

11). No correlation was detected between GLP-1 and IL-1β, TNF-α or IL-8.

Linear regresson GLP-1 IL-6

6.5 7.0 7.5 8.0 8.50

1

2

3

4

5

GLP-1 (pg/ml)

IL-6

(p

g/m

l)

Figure 11: Linear regression between levels of IL-6 and GLP-1 in healthy controls, IBS-C,

IBS-D and IBS-A.

Healthy IBS-C IBS-D IBS-A0

2

4

6G

LP

-1 (

pM

)

N=9 N=9 N=9 N=9

*

p=0.09

27

Discussion

The findings from this study show that GLP-1 concentrations are elevated in IBS-A plasma

and this is correlated with raised IL-6 levels previously reported (Dinan et al., 2006).

However it is likely that GLP-1 is more than just a biomarker of IBS as GLP-1 receptor

staining is expansive and punctate among submucosal neuronal fibres and neurons where it

may contribute to changes in colonic absorption and/or secretion. The data also show that

IL-6 staining was in neuronal bodies among submucosal neurons and co-localised with

GLP-1R in a number of cases, thereby revealing potential for cross-talk between the

molecules.

GLP-1 has been shown to inhibit the MMC of the antrum of the stomach which leads to

inhibition of gastric emptying and also inhibits MMC in the duodenum and jejunum of the

small intestine in both healthy subjects and in IBS subjects. GLP-1 is also shown to inhibit

gastrointestinal motility following a meal (Hellström et al., 2008). GLP-1 analogue ROSE-

010 has also been shown in clinical trials to alleviate abdominal pain and inhibit gastric

emptying without slowing colonic transit in IBS-C patients (Hellström et al., 2009). What

is not clear however is what underlying mechanism GLP-1 is mediating these alleviating

effects and to what extent does it differ among IBS subtypes. GLP-1 and GLP-1R have

been shown to be present in the stomach and in the small intestine (Eissele et al., 1992),

(Hellström et al., 2008) and distal colon (Eissele et al., 1992, Amato et al., 2010). While

submucosal expression of GLP-1Rs has been reported in colonic submucosal neurons

(Baldassano et al., 2012), our findings have shown the type of ganglionic cells expressing

the receptor and co-localisation with the pro-inflammatory cytokine, IL-6.

The GLP-1 receptor co-localises with 63% of nNOS positive neurons. Neuronal nitric

oxide synthase synthesises nitric oxide which is a free radical signalling molecule and acts

as a smooth muscle cell relaxant in the GIT (Shah et al., 2004). GLP-1 causes gastric

accommodation (Delgado-Aros et al., 2002) and this accommodation depends on a nitregic

link (Barragán et al., 1994, Tolessa et al., 1998a). Indeed applying a NO synthase inhibitor

leads to a blockage in postprandial augmentation of gastric volume by GLP-1(Andrews et

al., 2007). nNOS has been found to co-localise with GLP-1R in the myenteric neurons of

the duodenum and proximal colon indicating that nitrergic pathways play a role in the

28

inhibitory effects of GLP-1 on muscle activity (Amato et al., 2010). nNOS has also been

implicated in the pathogenesis of IBS (Reinders et al., 2005, Tjong et al., 2011), with

elevated nNOS expression and NO production from the distal colon of MS rodents (Tjong

et al., 2011) . Clinical studies have also shown elevated rectal mucosal and plasma NO

levels in IBS patients (Reinders et al., 2005, Yazar et al., 2005). Our data suggests the

effects of GLP-1 on submucosal neurons in the distal colon may also be mediated by

nitrergic pathways.

GLP-1R co-localises with 21% of calretinin neurons and 26% of calbindin neurons.

Calretinin is a calcium binding protein and is predominantly in motor neurons. Calbindin is

also a calcium binding protein and is predominantly in sensory neurons (Abalo et al.,

2009). Our data showed the co-localisation between calbindin and GLP-1R was lower than

the co-localisation between nNOS and GLP-1R. Like-wise co-localisation between

calretinin and GLP-1R was lower than the co-localisation between nNOS and GLP-1R.

Our data therefore suggests that the effects of GLP-1R on submucosal neurons of the distal

colon is mediated to a greater extent by nitrergic pathways than motor or sensory

pathways.

A previous study has shown that IL-6 and IL-6R staining was primarily cytosolic and co-

localised with the neuronal cell markers calretinin, calbindin and the glial cell maker S100

(O'Malley et al., 2011b). We have demonstrated that GLP-1R is primarily expressed in

nNOS positive neurons. Interestingly IL-6 receptor has been shown to co-localise with a

similar percentage of nNOS labelled cells as does GLP-1R (O'Malley et al., 2011b,

O'Malley et al., 2011a). In this study IL-6 is shown to be in the neuronal bodies showing

cytosolic expression. Dual labelling with IL-6 and GLP-1R showed that co-localisation is

present in 30% of neuronal bodies revealing a potential area of cross talk. Further studies

to investigate if IL-6 receptors and GLP-1 receptors are in close proximity would also

provide valuable information.

The GLP-1R staining was punctate along the neuronal bodies and fibres. To determine if

these receptor clusters were at synapses neurons were dual labelled with the pre-synaptic

marker, Synapsin I and the post-synaptic marker, PSD-95. GLP-1R clusters appeared to

co-localise with both Synapsin I and PSD-95 indicating that GLP-1 may be important in

29

the synaptic excitation of submucosal neurons and may directly influence the secretion of a

neurotransmitter or act as a neurotransmitter itself.

Cross Sections were obtained in his study to demonstrate the location and quantity of GLP-

1, GLP-1R and IL-6 across the entire cross section of the distal colon. GLP-1 and IL-6

were shown to co-localise within L-cells in the colon. L- cells within the gut secrete GLP-

1 and a study has shown that IL-6 receptors are also expressed on L-cells. IL-6 has also

been shown to cause an increase in GLP-1 secretion from L-cells (Ellingsgaard et al.,

2011), hence with elevations in IL-6 in IBS patients this may result in increased secretion

of GLP-1. Indeed the correlation seen in our study between IL-6 and GLP-1 levels in the

IBS samples would substantiate this theory.

In summary GLP-1R co-localised with calbindin, calretinin, nNOS and S100 and to the

greatest extent with nNOS, therefore concluding that GLP-1 could be exerting its effects

through a nitrergic pathway. Co-localisation studies also allowed us to conclude that a

certain level of cross talk may exist between GLP1-R and IL-6 and this may result in

functional changes in the gut such as altered secretion and absorption. In this way, GLP-1

may contribute to the pathogenesis of IBS.

Further Studies As the ELISA data of IBS-C and IBS-D plasma samples were trending towards

significance further experiments could be carried out with more plasma samples.

The functional role of GLP-1 in IBS may be further characterised by carrying out calcium

imaging studies. Exposure of submucosal neurons to GLP-1 would determine whether

GLP-1 excites or inhibits submucosal neurons. Using an animal model of IBS, the

sensitivity of submucosal neurons in a model of altered GI function could be assessed, as

has been carried out for IL-6 (O'Malley et al., 2011b). Moreover supernatants pooled from

WKY rats could be added to the submucosal preparation from both SD and WKY rats.

Following neuronal excitation by the added supernatant, an anti-GLP-1 neutralising

antibody could then be added to the supernatant. If there is a reduced calcium response it

would show whether GLP-1 in secretions are also involved in exciting submucosal

neurons.

30

To analyse effects of GLP-1 on absorption and secretion across the colonic epithelial

membrane Ussing Chamber electrophysiology experiments could be conducted using SD

and WKY rats. The effects of GLP-1 levels on secretion would be compared between both

rat models. The Ussing chambers design could be exploited to investigate the capacity that

IL-6 has on GLP-1 secretion and also to investigate the capacity that GLP-1 has on IL-6

secretion.

Also as CRF expression is seen to be prominent in the submucosal plexus of the proximal

and distal colon (Yuan et al., 2010) and GLP-1 has been shown to be mediated in stress

induced alteration of colonic transit via CRF mediated pathways (Nakade et al., 2007),

further studies could be carried out to determine potential cross-talk between GLP-1 and

CRF within the submucosal ganglia. Immunofluorescent techniques could be carried out to

determine co-localisation between CRF and GLP-1. Also Ussing chamber experiments

could be carried out to investigate the capacity that CRF has on GLP-1 secretion.

The Immunofluorescent results in this study could be further investigated by carrying out

Western Blot Analysis. Western Blot Analysis would be carried out on colonic human

biopsies to determine the concentration of IL-6 and GLP-1 in the submucosal plexus and

other layers of the colon. It would allow for a comparison of concentrations of IL-6 and

GLP-1 between the different layers of the colon.

31

References ABALO, R., VERA, G., RIVERA, A. J., MORO-RODRÍGUEZ, E. & MARTÍN-

FONTELLES, M. I. 2009. Postnatal maturation of the gastrointestinal tract: a

functional and immunohistochemical study in the guinea-pig ileum at weaning.

Neurosci Lett, 467, 105-10.

AL-SADI, R., BOIVIN, M. & MA, T. 2009. Mechanism of cytokine modulation of

epithelial tight junction barrier. Front Biosci, 14, 2765-78.

AMATO, A., CINCI, L., ROTONDO, A., SERIO, R., FAUSSONE-PELLEGRINI, M. S.,

VANNUCCHI, M. G. & MULÈ, F. 2010. Peripheral motor action of glucagon-like

peptide-1 through enteric neuronal receptors. Neurogastroenterol Motil, 22, 664-

e203.

ANDREWS, C. N., BHARUCHA, A. E., CAMILLERI, M., LOW, P. A., SEIDE, B.,

BURTON, D., BAXTER, K. & ZINSMEISTER, A. R. 2007. Nitrergic contribution

to gastric relaxation induced by glucagon-like peptide-1 (GLP-1) in healthy adults.

Am J Physiol Gastrointest Liver Physiol, 292, G1359-65.

BALDASSANO, S., WANG, G. D., MULÈ, F. & WOOD, J. D. 2012. Glucagon-like

peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig

small intestine in vitro. Am J Physiol Gastrointest Liver Physiol, 302, G352-8.

BARRAGÁN, J. M., RODRÍGUEZ, R. E. & BLÁZQUEZ, E. 1994. Changes in arterial

blood pressure and heart rate induced by glucagon-like peptide-1-(7-36) amide in

rats. Am J Physiol, 266, E459-66.

BUHNER, S., LI, Q., BERGER, T., VIGNALI, S., BARBARA, G., DE GIORGIO, R.,

STANGHELLINI, V. & SCHEMANN, M. 2012. Submucous rather than myenteric

neurons are activated by mucosal biopsy supernatants from irritable bowel

syndrome patients. Neurogastroenterol Motil, 24, 1134-e572.

CALDARELLA, M. P., MILANO, A., LATERZA, F., SACCO, F., BALATSINOU, C.,

LAPENNA, D., PIERDOMENICO, S. D., CUCCURULLO, F. & NERI, M. 2005.

Visceral sensitivity and symptoms in patients with constipation- or diarrhea-

predominant irritable bowel syndrome (IBS): effect of a low-fat intraduodenal

infusion. Am J Gastroenterol, 100, 383-9.

CAMILLERI, M. & KATZKA, D. A. 2012. Irritable bowel syndrome: methods,

mechanisms, and pathophysiology. Genetic epidemiology and pharmacogenetics in

irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol, 302, G1075-84.

CAMILLERI, M., MADSEN, K., SPILLER, R., GREENWOOD-VAN MEERVELD, B.,

VAN MEERVELD, B. G. & VERNE, G. N. 2012. Intestinal barrier function in

health and gastrointestinal disease. Neurogastroenterol Motil, 24, 503-12.

CHROUSOS, G. P. 1995. The hypothalamic-pituitary-adrenal axis and immune-mediated

inflammation. N Engl J Med, 332, 1351-62.

32

CORSETTI, M., CAENEPEEL, P., FISCHLER, B., JANSSENS, J. & TACK, J. 2004.

Impact of coexisting irritable bowel syndrome on symptoms and

pathophysiological mechanisms in functional dyspepsia. Am J Gastroenterol, 99,

1152-9.

CRENTSIL, V. 2005. Will corticosteroids and other anti-inflammatory agents be effective

for diarrhea-predominant irritable bowel syndrome? Med Hypotheses, 65, 97-102.

DALRYMPLE, J. & BULLOCK, I. 2008. Diagnosis and management of irritable bowel

syndrome in adults in primary care: summary of NICE guidance. BMJ, 336, 556-8.

DELGADO-AROS, S., KIM, D. Y., BURTON, D. D., THOMFORDE, G. M.,

STEPHENS, D., BRINKMANN, B. H., VELLA, A. & CAMILLERI, M. 2002.

Effect of GLP-1 on gastric volume, emptying, maximum volume ingested, and

postprandial symptoms in humans. Am J Physiol Gastrointest Liver Physiol, 282,

G424-31.

DINAN, T. G. 1994. Glucocorticoids and the genesis of depressive illness. A

psychobiological model. Br J Psychiatry, 164, 365-71.

DINAN, T. G. 1996. Serotonin and the regulation of hypothalamic-pituitary-adrenal axis

function. Life Sci, 58, 1683-94.

DINAN, T. G., CLARKE, G., QUIGLEY, E. M., SCOTT, L. V., SHANAHAN, F.,

CRYAN, J., COONEY, J. & KEELING, P. W. 2008. Enhanced cholinergic-

mediated increase in the pro-inflammatory cytokine IL-6 in irritable bowel

syndrome: role of muscarinic receptors. Am J Gastroenterol, 103, 2570-6.

DINAN, T. G., QUIGLEY, E. M., AHMED, S. M., SCULLY, P., O'BRIEN, S.,

O'MAHONY, L., O'MAHONY, S., SHANAHAN, F. & KEELING, P. W. 2006.

Hypothalamic-pituitary-gut axis dysregulation in irritable bowel syndrome: plasma

cytokines as a potential biomarker? Gastroenterology, 130, 304-11.

DORN, S. D., MORRIS, C. B., HU, Y., TONER, B. B., DIAMANT, N., WHITEHEAD,

W. E., BANGDIWALA, S. I. & DROSSMAN, D. A. 2009. Irritable bowel

syndrome subtypes defined by Rome II and Rome III criteria are similar. J Clin

Gastroenterol, 43, 214-20.

DROSSMAN, D. A. 2005. What does the future hold for irritable bowel syndrome and the

functional gastrointestinal disorders? J Clin Gastroenterol, 39, S251-6.

EISSELE, R., GÖKE, R., WILLEMER, S., HARTHUS, H. P., VERMEER, H., ARNOLD,

R. & GÖKE, B. 1992. Glucagon-like peptide-1 cells in the gastrointestinal tract and

pancreas of rat, pig and man. Eur J Clin Invest, 22, 283-91.

ELLINGSGAARD, H., HAUSELMANN, I., SCHULER, B., HABIB, A. M., BAGGIO, L.

L., MEIER, D. T., EPPLER, E., BOUZAKRI, K., WUEEST, S., MULLER, Y. D.,

HANSEN, A. M., REINECKE, M., KONRAD, D., GASSMANN, M., REIMANN,

F., HALBAN, P. A., GROMADA, J., DRUCKER, D. J., GRIBBLE, F. M.,

EHSES, J. A. & DONATH, M. Y. 2011. Interleukin-6 enhances insulin secretion

33

by increasing glucagon-like peptide-1 secretion from L cells and alpha cells. Nat

Med, 17, 1481-9.

FITZGERALD, L. Z., KEHOE, P. & SINHA, K. 2009. Hypothalamic--pituitary-- adrenal

axis dysregulation in women with irritable bowel syndrome in response to acute

physical stress. West J Nurs Res, 31, 818-36.

FUKUDO, S., NOMURA, T. & HONGO, M. 1998. Impact of corticotropin-releasing

hormone on gastrointestinal motility and adrenocorticotropic hormone in normal

controls and patients with irritable bowel syndrome. Gut, 42, 845-9.

GILLESPIE, C. F. & NEMEROFF, C. B. 2005. Hypercortisolemia and depression.

Psychosom Med, 67 Suppl 1, S26-8.

GONSALKORALE, W. M., MILLER, V., AFZAL, A. & WHORWELL, P. J. 2003a.

Long term benefits of hypnotherapy for irritable bowel syndrome. Gut, 52, 1623-9.

GONSALKORALE, W. M., PERREY, C., PRAVICA, V., WHORWELL, P. J. &

HUTCHINSON, I. V. 2003b. Interleukin 10 genotypes in irritable bowel syndrome:

evidence for an inflammatory component? Gut, 52, 91-3.

GUTNIAK, M. K., LINDE, B., HOLST, J. J. & EFENDIĆ, S. 1994. Subcutaneous

injection of the incretin hormone glucagon-like peptide 1 abolishes postprandial

glycemia in NIDDM. Diabetes Care, 17, 1039-44.

GUYTON, A. C. & HALL, J. E. 2001. Textbook of medical physiology, Philadelphia,

Saunders.

HELLSTRÖM, P. M. 2009. GLP-1: broadening the incretin concept to involve gut

motility. Regul Pept, 156, 9-12.

HELLSTRÖM, P. M. 2011. GLP-1 playing the role of a gut regulatory compound. Acta

Physiol (Oxf), 201, 151-6.

HELLSTRÖM, P. M., HEIN, J., BYTZER, P., BJÖRNSSÖN, E., KRISTENSEN, J. &

SCHAMBYE, H. 2009. Clinical trial: the glucagon-like peptide-1 analogue ROSE-

010 for management of acute pain in patients with irritable bowel syndrome: a

randomized, placebo-controlled, double-blind study. Aliment Pharmacol Ther, 29,

198-206.

HELLSTRÖM, P. M., NÄSLUND, E., EDHOLM, T., SCHMIDT, P. T., KRISTENSEN,

J., THEODORSSON, E., HOLST, J. J. & EFENDIC, S. 2008. GLP-1 suppresses

gastrointestinal motility and inhibits the migrating motor complex in healthy

subjects and patients with irritable bowel syndrome. Neurogastroenterol Motil, 20,

649-59.

KELLER, C., STEENSBERG, A., PILEGAARD, H., OSADA, T., SALTIN, B.,

PEDERSEN, B. K. & NEUFER, P. D. 2001. Transcriptional activation of the IL-6

gene in human contracting skeletal muscle: influence of muscle glycogen content.

FASEB J, 15, 2748-50.

34

KIANK, C., TACHÉ, Y. & LARAUCHE, M. 2010. Stress-related modulation of

inflammation in experimental models of bowel disease and post-infectious irritable

bowel syndrome: role of corticotropin-releasing factor receptors. Brain Behav

Immun, 24, 41-8.

MARTINI, F., NATH, J. L. & BARTHOLOMEW, E. F. 2012. Fundamentals of anatomy

& physiology, San Francisco, Benjamin Cummings.

MARTÍNEZ, V., RYTTINGER, M., KJERLING, M. & ASTIN-NIELSEN, M. 2007.

Characterisation of colonic accommodation in Wistar Kyoto rats with impaired

gastric accommodation. Naunyn Schmiedebergs Arch Pharmacol, 376, 205-16.

MCKENDRICK, M. W. 1996. Post Salmonella irritable bowel syndrome--5 year review. J

Infect, 32, 170-1.

MCKENDRICK, M. W. & READ, N. W. 1994. Irritable bowel syndrome--post salmonella

infection. J Infect, 29, 1-3.

MCKERNAN, D. P., GASZNER, G., QUIGLEY, E. M., CRYAN, J. F. & DINAN, T. G.

2011. Altered peripheral toll-like receptor responses in the irritable bowel

syndrome. Aliment Pharmacol Ther, 33, 1045-52.

MURPHY, D. L., LERNER, A., RUDNICK, G. & LESCH, K. P. 2004. Serotonin

transporter: gene, genetic disorders, and pharmacogenetics. Mol Interv, 4, 109-23.

NAKADE, Y., TSUKAMOTO, K., IWA, M., PAPPAS, T. N. & TAKAHASHI, T. 2007.

Glucagon like peptide-1 accelerates colonic transit via central CRF and peripheral

vagal pathways in conscious rats. Auton Neurosci, 131, 50-6.

O' MAHONY, S. M., COELHO, A. M., FITZGERALD, P., LEE, K., WINCHESTER, W.,

DINAN, T. G. & CRYAN, J. F. 2011. The effects of gabapentin in two animal

models of co-morbid anxiety and visceral hypersensitivity. Eur J Pharmacol, 667,

169-74.

O'MAHONY, S., CHUA, A. S., QUIGLEY, E. M., CLARKE, G., SHANAHAN, F.,

KEELING, P. W. & DINAN, T. G. 2008. Evidence of an enhanced central 5HT

response in irritable bowel syndrome and in the rat maternal separation model.

Neurogastroenterol Motil, 20, 680-8.

O'MAHONY, S. M., BULMER, D. C., COELHO, A. M., FITZGERALD, P.,

BONGIOVANNI, C., LEE, K., WINCHESTER, W., DINAN, T. G. & CRYAN, J.

F. 2010. 5-HT(2B) receptors modulate visceral hypersensitivity in a stress-sensitive

animal model of brain-gut axis dysfunction. Neurogastroenterol Motil, 22, 573-8,

e124.

O'MAHONY, S. M., MARCHESI, J. R., SCULLY, P., CODLING, C., CEOLHO, A. M.,

QUIGLEY, E. M., CRYAN, J. F. & DINAN, T. G. 2009. Early life stress alters

behavior, immunity, and microbiota in rats: implications for irritable bowel

syndrome and psychiatric illnesses. Biol Psychiatry, 65, 263-7.

35

O'MALLEY, D., DINAN, T. G. & CRYAN, J. F. 2011a. Altered expression and secretion

of colonic interleukin-6 in a stress-sensitive animal model of brain-gut axis

dysfunction. J Neuroimmunol, 235, 48-55.

O'MALLEY, D., DINAN, T. G. & CRYAN, J. F. 2012. Interleukin-6 modulates colonic

transepithelial ion transport in the stress-sensitive wistar kyoto rat. Front

Pharmacol, 3, 190.

O'MALLEY, D., JULIO-PIEPER, M., GIBNEY, S. M., DINAN, T. G. & CRYAN, J. F.

2010. Distinct alterations in colonic morphology and physiology in two rat models

of enhanced stress-induced anxiety and depression-like behaviour. Stress, 13, 114-

22.

O'MALLEY, D., LISTON, M., HYLAND, N. P., DINAN, T. G. & CRYAN, J. F. 2011b.

Colonic soluble mediators from the maternal separation model of irritable bowel

syndrome activate submucosal neurons via an interleukin-6-dependent mechanism.

Am J Physiol Gastrointest Liver Physiol, 300, G241-52.

OLDEN, K. W. 2003. Approach to the Patient with Severe, Refractory Irritable Bowel

Syndrome. Curr Treat Options Gastroenterol, 6, 311-317.

ORTIZ-LUCAS, M., SAZ-PEIRÓ, P. & SEBASTIÁN-DOMINGO, J. J. 2010. Irritable

bowel syndrome immune hypothesis. Part two: the role of cytokines. Rev Esp

Enferm Dig, 102, 711-7.

REINDERS, C. I., HERULF, M., LJUNG, T., HOLLENBERG, J., WEITZBERG, E.,

LUNDBERG, J. O. & HELLSTRÖM, P. M. 2005. Rectal mucosal nitric oxide in

differentiation of inflammatory bowel disease and irritable bowel syndrome. Clin

Gastroenterol Hepatol, 3, 777-83.

SAITO, Y. A., MITRA, N. & MAYER, E. A. 2010. Genetic approaches to functional

gastrointestinal disorders. Gastroenterology, 138, 1276-85.

SAULNIER, D. M., RINGEL, Y., HEYMAN, M. B., FOSTER, J. A., BERCIK, P.,

SHULMAN, R. J., VERSALOVIC, J., VERDU, E. F., DINAN, T. G., HECHT, G.

& GUARNER, F. 2013. The intestinal microbiome, probiotics and prebiotics in

neurogastroenterology. Gut Microbes, 4, 17-27.

SCHELLER, J., CHALARIS, A., SCHMIDT-ARRAS, D. & ROSE-JOHN, S. 2011. The

pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim

Biophys Acta, 1813, 878-88.

SCHIRRA, J., WANK, U., ARNOLD, R., GÖKE, B. & KATSCHINSKI, M. 2002. Effects

of glucagon-like peptide-1(7-36)amide on motility and sensation of the proximal

stomach in humans. Gut, 50, 341-8.

SCHJOLDAGER, B. T., MORTENSEN, P. E., CHRISTIANSEN, J., ORSKOV, C. &

HOLST, J. J. 1989. GLP-1 (glucagon-like peptide 1) and truncated GLP-1,

fragments of human proglucagon, inhibit gastric acid secretion in humans. Dig Dis

Sci, 34, 703-8.

36

SEELEY, R. R., STEPHENS, T. D. & TATE, P. 2003. Anatomy & physiology, Boston,

McGraw-Hill.

SHAH, V., LYFORD, G., GORES, G. & FARRUGIA, G. 2004. Nitric oxide in

gastrointestinal health and disease. Gastroenterology, 126, 903-13.

THARAKAN, A., NORTON, I. T., FRYER, P. J. & BAKALIS, S. 2010. Mass transfer

and nutrient absorption in a simulated model of small intestine. J Food Sci, 75,

E339-46.

TJONG, Y. W., IP, S. P., LAO, L., WU, J., FONG, H. H., SUNG, J. J., BERMAN, B. &

CHE, C. T. 2011. Role of neuronal nitric oxide synthase in colonic distension-

induced hyperalgesia in distal colon of neonatal maternal separated male rats.

Neurogastroenterol Motil, 23, 666-e278.

TOLESSA, T., GUTNIAK, M., HOLST, J. J., EFENDIC, S. & HELLSTRÖM, P. M.

1998a. Glucagon-like peptide-1 retards gastric emptying and small bowel transit in

the rat: effect mediated through central or enteric nervous mechanisms. Dig Dis Sci,

43, 2284-90.

TOLESSA, T., GUTNIAK, M., HOLST, J. J., EFENDIC, S. & HELLSTRÖM, P. M.

1998b. Inhibitory effect of glucagon-like peptide-1 on small bowel motility. Fasting

but not fed motility inhibited via nitric oxide independently of insulin and

somatostatin. J Clin Invest, 102, 764-74.

TURNBULL, A. V. & RIVIER, C. L. 1999. Regulation of the hypothalamic-pituitary-

adrenal axis by cytokines: actions and mechanisms of action. Physiol Rev, 79, 1-71.

VAN DER VEEK, P. P., VAN DEN BERG, M., DE KROON, Y. E., VERSPAGET, H.

W. & MASCLEE, A. A. 2005. Role of tumor necrosis factor-alpha and interleukin-

10 gene polymorphisms in irritable bowel syndrome. Am J Gastroenterol, 100,

2510-6.

XIA, Y., HU, H. Z., LIU, S., REN, J., ZAFIROV, D. H. & WOOD, J. D. 1999. IL-1beta

and IL-6 excite neurons and suppress nicotinic and noradrenergic

neurotransmission in guinea pig enteric nervous system. J Clin Invest, 103, 1309-

16.

YALE, S. H., MUSANA, A. K., KIEKE, A., HAYES, J., GLURICH, I. & CHYOU, P. H.

2008. Applying case definition criteria to irritable bowel syndrome. Clin Med Res,

6, 9-16.

YAZAR, A., BÜYÜKAFPAR, K., POLAT, G., PATA, C., KANÝK, A., TIFTIK, E. N. &

BAĞDATOĞLU, O. 2005. The urinary 5-hydroxyindole acetic acid and plasma

nitric oxide levels in irritable bowel syndrome: a preliminary study. Scott Med J,

50, 27-9.

YUAN, P. Q., WU, S. V., WANG, L. & TACHÉ, Y. 2010. Corticotropin releasing factor

in the rat colon: expression, localization and upregulation by endotoxin. Peptides,

31, 322-31.

37