Clinical trial NCT02170870
Conditions: Dyspepsia Diabetes Mellitus With Gastrointestinal Symptoms
Sponsor: Mayo Clinic
Start date: 2014-06-01; Completion date: 2017-11-17
Enrollment: 104 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to understand why people with indigestion have gastrointestinal symptoms and in particular to understand whether symptoms are related to increased sensitivity to nutrients in the small intestine and to a hormone (GLP1) which is normally released from the small intestine in response to nutrients. We propose to study the contribution of GLP1 to intestinal sensitivity with a drug (exendin 9-39) that blocks the effects of GLP1.

Detailed description:
Upper gastrointestinal symptoms (early satiety, pain, nausea, and vomiting) are not uncommon in diabetic (DM) enteropathy. While these symptoms are often attributed to accelerated or delayed gastric emptying, the precise contribution of abnormal gastric emptying to symptoms in patients with DM gastroparesis is often unclear.

The investigators recently observed that approximately 50% of patients with functional dyspepsia have increased sensation to duodenal nutrient (carbohydrate and lipid) perfusion. Another recent study suggests that patients with functional dyspepsia have low-grade mucosal inflammation, abnormalities of cell-to-cell adhesion proteins which predispose to increased epithelial permeability, and a leaky epithelial barrier. Type 1 DM is associated with increased small intestinal permeability even in subjects who do not have celiac disease.

Hence, the investigators proposed to evaluate the overall hypothesis that intestinal chemosensitivity related to increased epithelial permeability and GLP-1 explains symptom severity in patients with functional dyspepsia and in patients with DM and dyspepsia. Healthy subjects, Patients with DM and GI symptoms, and patients with functional dyspepsia underwent assessment of intestinal chemosensitivity during duodenal nutrient perfusion, gastric emptying (by scintigraphy), cardiovascular and GI vagal functions (plasma pancreatic polypeptide response to sham feeding and a comprehensive autonomic reflex screen), in vivo assessment of small intestinal permeability (urinary lactulose:mannitol ratio), and upper endoscopy with assessment of epithelial tight junction proteins and permeability on small bowel biopsies.

During the nutrient infusion, subjects in each group (i.e., healthy subjects, functional dyspepsia and DM) were randomized to lipid infusion and placebo or lipid infusion and exendin 9-39. Hormonal responses (i.e., GLP-1, cholecystokinin (CCK), gastric inhibitory polypeptide (GIP), glucagon, peptide tyrosine tyrosine (PYY), C-peptide, and insulin) and plasma glucose will also be evaluated during enteral nutrient infusion. GI symptoms during each perturbation (meal, nutrient infusion) will be evaluated by validated questionnaires. Blood will be collected for DNA-based genetic analyses, initially to assess the relationship of GI sensorimotor dysfunctions and symptoms with single nucleotide polymorphisms (SNPs) affecting CCK and GLP-1 receptors. The analysis will assess for disturbances in these parameters in functional and DM dyspepsia, investigate associations between symptoms during enteral infusion and hormonal-epithelial functions, and evaluate relationships between daily symptoms and results of testing.

Arms: arm 1: Exendin 9-39 was administered intravenously (1,200 pmol/kg bolus followed by infusion at 300 pmol/kg/min).

Lipid infusion 66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml). arm 2: Normal saline infusion was prepared to match the appearance of Exendin 9-39. Lipid infusion 66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml). arm 3: Exendin 9-39 was administered intravenously (1,200 pmol/kg bolus followed by infusion at 300 pmol/kg/min).

Lipid infusion 66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml). arm 4: Normal saline infusion was prepared to match the appearance of Exendin 9-39. Lipid infusion 66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml). arm 5: Exendin 9-39 was administered intravenously (1,200 pmol/kg bolus followed by infusion at 300 pmol/kg/min).

Lipid infusion 66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml). arm 6: Normal saline infusion was prepared to match the appearance of Exendin 9-39. Lipid infusion 66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml).
Interventions: intervention 1: Exendin 9-39 intervention 2: Microlipid intervention 3: Placebo
Intervention descriptions: intervention 1: Exendin 9-39 will be administered intravenously (1,200 pmol/kg bolus followed by infusion at 300 pmol/kg/min). intervention 2: Lipid infusion {66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml). intervention 3: Normal saline infusion will be prepared to match the appearance of Exendin 9-39

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
Gastrointestinal Sensorimotor 
Dysfunctions in Diabetes 
Mellitus 
NCT 02170870 
June 16, 2017 


1 
 
PROTOCOL 
 
Title of the study: 
Gastrointestinal Sensorimotor Dysfunctions in Diabetes Mellitus 
IRB Number 
14-002098 
Principal investigator: 
Adil E. Bharucha, MD 
Co-investigator(s): 
Ananda Basu, MD, Michael Camilleri, MD., Magnus Halland, MD, 
Yogish Kudva, MD, Alan Zinsmeister, PhD, Phillip Low MD, 
Wolfgang Singer MD, Lawrence Szarka MD, Tonette Gehrking, 
Jade Gehrking, Gopanandan Parthasarathy, Thomas Smyrk, 
Pratyusha Tirumanisetty 
Study Coordinator: 
Bridget Neja, Kelly Feuerhak 
Study Technicians: 
Duane Burton, Michael Ryks, Debbie Rhoten 
Version 
    6/16/17 
 
ABSTRACT 
Upper gastrointestinal symptoms (early satiety, pain, nausea, and vomiting) are not 
uncommon in diabetic (DM) enteropathy.  While these symptoms are often attributed to accelerated 
or delayed gastric emptying, the precise contribution of abnormal gastric emptying to symptoms in 
patients with DM gastroparesis is often unclear.   
We recently observed that approximately 50% of patients with functional dyspepsia have 
increased sensation to duodenal nutrient (carbohydrate and lipid) perfusion.  Another recent study 
suggests that patients with functional dyspepsia have low-grade mucosal inflammation, 
abnormalities of cell-to-cell adhesion proteins which predispose to increased epithelial permeability, 
and a leaky epithelial barrier.  Type 1 DM is associated with increased small intestinal permeability 
even in subjects who do not have celiac disease.   
Hence, we propose to evaluate the overall hypothesis that intestinal chemosensitivity related 
to increased epithelial permeability and GLP-1 explains symptom severity in patients with functional 
dyspepsia and in patients with DM and dyspepsia.  24 healthy subjects, 40 patients with DM and GI 
symptoms, and 40 patients with functional dyspepsia will undergo assessment of intestinal 
chemosensitivity during duodenal nutrient perfusion, gastric emptying (by scintigraphy), 
cardiovascular and GI vagal functions (plasma pancreatic polypeptide response to sham feeding and 
a comprehensive autonomic reflex screen), in vivo assessment of small intestinal permeability 
(urinary lactulose:mannitol ratio), and upper endoscopy with assessment of epithelial tight junction 
proteins and permeability on small bowel biopsies.  During the nutrient infusion, subjects in each 
group (ie, healthy subjects, functional dyspepsia and DM) will be randomized to lipid infusion and 
placebo or lipid infusion {66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml)} and exendin 
9-39 in the ratio of 1:1.  Hormonal responses (i.e., GLP-1, CCK, GIP, glucagon, PYY, C-peptide, 
and insulin) and plasma glucose will also be evaluated during enteral nutrient infusion.  GI 
symptoms during each perturbation (meal, nutrient infusion) will be evaluated by validated 
questionnaires.  Blood will be collected for DNA-based genetic analyses, initially to assess the 
relationship of GI sensorimotor dysfunctions and symptoms with SNPs affecting CCK and GLP-1 
receptors.  The analysis will assess for disturbances in these parameters in functional and DM 
dyspepsia, investigate associations between symptoms during enteral infusion and hormonal-
epithelial functions, and evaluate relationships between daily symptoms and results of testing.     
       


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Table of Contents 
ABSTRACT .......................................................................................................................................... 1 
1  
SPECIFIC AIMS .......................................................................................................................... 3 
2  
BACKGROUND AND SIGNIFICANCE .................................................................................... 5 
3  
RESEARCH DESIGN AND METHODS .................................................................................... 7 
3.1 
Study Design ......................................................................................................................... 7 
3.2 
Study Subjects ....................................................................................................................... 7 
3.3 
Details of Study Assessments ............................................................................................... 8 
3.4 
Gene and Epigenetic Studies .............................................................................................. 15 
3.5 
Data Analysis ...................................................................................................................... 16 
3.6 
Potential Pitfalls .................................................................................................................. 17 
4  
HUMAN SUBJECTS ................................................................................................................. 17 
5  
SAFETY AND ADVERSE EVENTS ........................................................................................ 19 
5.1 
Definitions........................................................................................................................... 19 
5.2 
Recording of Adverse Events ............................................................................................. 21 
5.3 
Reporting of Serious Adverse Events and Unanticipated Problems ................................... 22 
5.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ........................................... 22 
5.3.2 
Sponsor-Investigator reporting: Notifying the FDA ................................................... 22 
5.4 
Stopping Rules .................................................................................................................... 23 
5.5. 
Medical Monitoring ............................................................................................................ 23 
APPENDIX ......................................................................................................................................... 23 
REFERNCES ...................................................................................................................................... 26 
 


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1  
SPECIFIC AIMS 
Upper gastrointestinal symptoms (early satiety, pain, nausea, and vomiting) are not uncommon in 
diabetes mellitus (DM) 1.  These symptoms are often attributed to delayed gastric emptying or 
gastroparesis.  Over a 10-year time period, the risk of developing gastroparesis was 5% in type 1 DM 
(HR 33, 95% CI 4.0, 274 adjusted for age and gender versus controls), 1% in type 2 DM (HR 7.5 
(95% CI: 0.8, 68) adjusted for age and gender versus controls and 1% in controls 2.  However, 
similar to functional dyspepsia 3, the precise contribution of abnormal gastric emptying to symptoms 
in patients with DM gastroparesis is unclear.  Indeed, a majority of patients with DM and delayed 
gastric emptying are asymptomatic.  In our cohort of DM patients with GI symptoms who underwent 
scintigraphy, a substantial proportion, i.e., 55 of 129 patients (42%) had normal gastric emptying; the 
remainder had had delayed (36%) or rapid GE (22%) 4.  Moreover, symptoms did not predict 
abnormal (i.e., delayed or rapid) gastric emptying.  Other, small, studies reported impaired gastric 
accommodation and increased gastric sensation in DM 5,6 but did not describe the relationship of 
these abnormalities to upper GI symptoms.    
Nutrients and enteral hormones (e.g., CCK, GLP-1) released in response to nutrients also 
stimulate small intestinal mechano and chemoreceptors or tastant receptors, which are located on or 
activate vagal afferents.  Few studies have explored the contribution of duodenal sensitivity to 
symptoms in functional dyspepsia.  Duodenal acidification induces proximal gastric relaxation, 
increases sensitivity to gastric distension, and inhibits gastric accommodation to a meal 7.  Duodenal 
lipid infusion increased the sensitivity to gastric distention in functional dyspepsia 8-11.  This 
sensitizing effect was blocked by a lipase inhibitor or a CCK-A receptor antagonist 12, 13.  We 
recently observed increased sensitivity to duodenal nutrient (carbohydrate and lipid perfusion) in 
approximately 50% of patients with functional dyspepsia during lipid and separately carbohydrate 
infusion, without gastric distention (unpublished observations).  Moreover, increased sensitivity was 
associated with higher plasma concentrations of CCK during lipid infusion and more severe 
dyspeptic symptoms during daily life.  However, the contribution of intestinal chemosensitivity to 
symptoms in DM is unknown.  We recently observed that patients with functional dyspepsia 
reported more severe symptoms during enteral lipid and carbohydrate infusion.  Second, this 
increased sensitivity was associated with higher plasma concentrations of enteral hormones (e.g., 
GLP-1 during lipid and carbohydrate infusion), more severe dyspeptic symptoms, and a greater 
impact on quality of life.   
Vagal dysfunction has been implicated as a mechanism for GI sensorimotor dysfunctions in DM 
14.  The plasma pancreatic polypeptide (PP) response to sham feeding evaluates GI vagal function 15.  
In clinical practice, assessment of cardiovascular vagal functions is used as a surrogate measure of 
GI vagal dysfunction.  Only 1 study evaluated the relationship between cardiovascular and GI vagal 
dysfunction (i.e., gastric acid secretion, PP response to sham feeding) and observed a modest 
correlation 15.  The utility of the sham feeding response and cardiovascular measures to predict upper 
GI sensorimotor dysfunctions has not been evaluated. 
A recent study suggests that patients with functional dyspepsia have low-grade mucosal 
inflammation, abnormalities of cell-to-cell adhesion proteins which predispose to increased epithelial 
permeability, and a leaky epithelial barrier 16.  Type 1 DM is associated with increased small 
intestinal permeability even in subjects who do not have celiac disease 17, 18.  Moreover, a 
comparison of 339 type 1 DM patients and 89 first degree relatives observed higher serum zonulin 
levels in type 1 DM patients than controls or relatives; patients with increased zonulin had increased 
epithelial permeability 19.  The only study which evaluated small intestinal permeability in type 2 
DM used suboptimal techniques and reported normal permeability 20.   


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Our overall hypothesis is that intestinal chemosensitivity related to increased epithelial 
permeability and CCK explains symptom severity in patients with DM with upper GI symptoms.   
The specific aims of this study are to address the following hypotheses in 24 healthy subjects, 40 
patients with DM and GI symptoms, and 40 patients with functional dyspepsia: 
1) A) Patients with DM and GI symptoms have increased intestinal chemosensitivity 
compared to controls and B) Intestinal chemosensitivity predicts severity of ongoing upper 
gastrointestinal symptoms independent of gastric emptying.  Intestinal chemosensitivity will 
be recorded by evaluating symptoms during duodenal lipid infusion (222 kcal, 222 ml) and 
placebo or the GLp-1 receptor antagonist exendin 9-39 over 2 hours; plasma hormones (i.e., 
glucose, CCK, GLP-1, PYY, C-peptide,  will also be measured.  Delivering nutrients into the 
duodenum will ensure that hormonal responses are not affected by gastric emptying disturbances.  
Day-to-day symptoms will be evaluated by the Nepean dyspepsia severity index and the GCSI.  
The relationship between daily symptoms and intestinal chemosensitivity will be assessed in a 
model which also incorporates gastric emptying and accommodation.    
2) Intestinal chemosensitivity will be A) associated with higher plasma CCK and GLP-1 
concentrations B) reduced by the GLP-1 antagonist exendin 9-39 and C) inversely 
associated with impaired abdominal vagal function.  This hypothesis will be evaluated by 
comparing intestinal chemosensitivity (a) with plasma CCK and GLP-1 concentrations (b) with 
and without exendin 9-39 and (c) in patients with and without and GI vagal dysfunction as 
assessed by the plasma pancreatic polypeptide response to sham feeding. 
3)  A) Patients with DM and GI symptoms have abnormal epithelial tight junction proteins 
which predispose to increased epithelial permeability and increased small bowel 
permeability and B) These structural and functional markers of epithelial permeability are 
associated with intestinal chemosensitivity.  Intestinal permeability will be measured in vivo 
by two sugar urine excretion after oral ingestion.  Biopsies from the distal second or third portion 
of the duodenum will be obtained endoscopically, to measure various markers or epithelial 
structure and function. 


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2  
BACKGROUND AND SIGNIFICANCE 
 
Diabetic enteropathy 
The term diabetic enteropathy implies that diabetes mellitus (DM) can affect the entire 
gastrointestinal (GI) tract 1.  Diabetic enteropathy may be asymptomatic or manifest with upper (i.e., 
heartburn, dysphagia, dyspepsia, gastroparesis) or lower GI symptoms (diarrhea, constipation, and 
fecal incontinence).  Gastrointestinal dysmotility in DM is multifactorial: extrinsic and intrinsic (i.e., 
enteric) neural dysfunction, hyperglycemia, and hormonal disturbances have been implicated.   
Patients with DM may have accelerated or delayed gastric emptying (GE), increased and 
reduced gastric sensation, and impaired gastric accommodation.  Antral hypomotility and/or 
pylorospasm, which can result from a vagal neuropathy, can delay GE 21.  Much attention has 
focused on delayed gastric emptying or gastroparesis as an explanation for upper GI symptoms in 
DM.  Similar to idiopathic gastroparesis, the contribution of delayed gastric emptying to symptoms 
in patients with DM gastroparesis is unclear for several reasons.  A majority of patients report 
symptoms within 1 hour of eating.  However, even normally, only approximately 25% of solids have 
emptied from the stomach by then.  Hence, delayed gastric emptying is unlikely to explain early 
postprandial symptoms.  Second, a majority of patients with DM and delayed gastric emptying are 
asymptomatic.  Third, a substantial proportion of DM patients with GI symptoms who underwent 
scintigraphy, i.e., 55 of 129 patients (42%) in our cohort, had normal gastric emptying; the 
remainder had had delayed (36%) or rapid GE (22%) 4.  Symptoms did not predict abnormal (i.e., 
delayed or rapid) gastric emptying in patients with DM and GI symptoms 4.  Even in functional 
dyspepsia, gastric emptying and accommodation only explained one-third of the variance in 
postprandial symptoms 22, 23. 
Impaired gastric accommodation and increased gastric sensation have been implicated to 
cause early satiety in functional dyspepsia 24.  Our understanding of gastric accommodation and 
gastric sensation in DM is limited to 3 studies, which assessed a total of 37 patients.  While gastric 
sensation was increased in both studies, gastric compliance in DM was increased in 1 study 5 and 
reduced in 2 studies 6, 25.  Another study, with 10 healthy subjects and 10 DM patients, observed an 
association between a lack of symptoms in diabetic gastrointestinal motility disorders and visceral 
afferent neuropathy identified by esophageal electrical stimulation 26.  Thus, while sensory 
disturbances – increased or decreased – are recognized in diabetic peripheral neuropathy, there are 
minimal data on GI sensation.  Conceivably, GI sensory disturbances influence the expression of GI 
motor dysfunctions in DM.  For example, asymptomatic delayed gastric emptying may be explained 
by vagal neuropathy.  Conversely, in symptomatic patients, delayed gastric emptying is likely 
explained by enteric dysfunction rather than vagal neuropathy.     


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Figure 1. Gastrointestinal symptoms during enteral 
nutrient infusion.  A higher proportion of patients with 
functional dyspepsia (n=25) than controls (n=33) reported 
symptoms of moderate severity or worse during enteral 
carbohydrate (p ≤ 0.01) and lipid (p ≤ 0.01) infusions. 
Carbohydrate Infusion
Lipid Infusion
Proportion (%) with symptoms
Proportion (%) with symptoms
Small intestinal 
dysmotility, more frequently 
characterized by reduced than 
by increased motility 27, may 
also contribute to gastric stasis 
in DM.  We recently observed 
increased sensitivity during 
duodenal carbohydrate and 
separately lipid perfusion in 
approximately 50% of patients 
with functional dyspepsia 
(Figure 1).  Moreover, increased 
sensitivity was associated with 
higher plasma concentrations of 
GLP-1 during lipid infusion and 
more severe dyspeptic 
symptoms during daily life 
(unpublished data).  Previous 
studies have evaluated the effects of duodenal nutrient infusion on glycemia, hormonal responses, 
glucose absorption, and gastroduodenal motility but not sensation in DM 28-31.  Moreover, no studies 
have integrated findings from assessments of gastric emptying, accommodation, and sensation.  This 
is a major limitation since the expression (symptoms) resulting from a disturbance may depend on 
other coexisting disturbances.  For example, it is conceivable that intestinal hypersensitivity may 
amplify the consequences of rapid gastric emptying.  Doing so is necessary to better understand the 
pathogenesis of symptoms and devise therapies targeted to the underlying disturbance in DM.      
Vagal neuropathy 
Vagal dysfunction has been implicated as a mechanism for GI sensorimotor dysfunctions in DM 
14.  The plasma pancreatic polypeptide (PP) response to sham feeding evaluates GI vagal function 15.  
In clinical practice, assessment of cardiovascular vagal functions is used as a surrogate measure of 
GI vagal dysfunction.  Only 1 study evaluated the relationship between cardiovascular and GI vagal 
dysfunction (i.e., gastric acid secretion, PP response to sham feeding) and observed a modest 
correlation 15.  The utility of the sham feeding response and cardiovascular measures to predict upper 
GI sensorimotor dysfunctions is unknown and will be evaluated in this study. 
 
Small intestinal epithelial permeability and mucosal inflammation 
A recent study suggests that patients with functional dyspepsia have low-grade mucosal 
inflammation, abnormalities of cell-to-cell adhesion proteins which predispose to increased epithelial 
permeability, and a leaky epithelial barrier 16.  Type 1 DM is associated with increased small 
intestinal permeability even in subjects who do not have celiac disease 17, 18.  Moreover, a 
comparison of 339 type 1 DM patients and 89 first degree relatives observed higher serum zonulin 
levels in type 1 DM patients than controls or relatives; patients with increased zonulin had increased 
epithelial permeability 19.  The only study which evaluated small intestinal permeability in type 2 
DM used suboptimal techniques and reported normal permeability 20.  However, a high fat diet 
induced glucose intolerance, decreased intestinal tight junction proteins, increased epithelial 
permeability, and plasma concentrations of circulating cytokines in OLETF mice, which is a 


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spontaneous CCK-1 receptor knockout mouse and an animal model of obesity 32.  Given the 
similarities between this mouse model and type 2-DM in humans, it is conceivable that epithelial 
permeability is also increased in type 2 DM.    
 
3  
RESEARCH DESIGN AND METHODS 
 
3.1 
Study Design 
In addition to questionnaires, the primary study procedures include assessment of: 
i) Screening visit – The participant will undergo a physical examination if one has not been 
completed within the last 3 months. 
ii) Study day 1 - GI vagal function by sham feeding, autonomic functions, small intestinal 
permeability test and enteral nutrient infusion of lipids, to be performed in that order, on another day 
(Charlton 7 CRU) 
iii) Study day 2 – Women of child bearing potential will have a urine pregnancy test within 48 hours 
before the GI Transit study, Gastric emptying of solids and liquids and small intestinal transit on 1 
day (Charlton 7 CRU) 
iv) Study day 3 - Upper gastrointestinal endoscopy with a small amount of fluid and  intestinal 
biopsies  
These tests will preferably be performed in the order listed above.  (ii) must always be performed 
before (iv).  Ancillary procedures include collection of blood for genetic and epigenetic studies and a 
stool sample for microbial assessment.  Vital signs and nurse assessment will be performed at each 
visit.    
 
3.2 
Study Subjects 
We plan to accrue up to 115 subjects to ensure that 104 subjects complete the study in its entirety. 40 
patients with DM and GI symptoms and 40 patients with functional dyspepsia will be recruited from 
the clinical practice; 24 healthy subjects will be recruited by public advertisement.  To reduce the 
possibility of demographic differences between controls and patients, we will "frequency match" on 
age and gender by recruiting 5 controls after each set of 5 patients is studied.  Doing so will provide 
similar age and gender distribution in each type.  We will also attempt to recruit an even number of 
subjects with type 1 and 2 DM.   
 
Inclusion criteria for controls 
1. 
Healthy male or non-pregnant, non-breastfeeding female volunteers; 
2. 
18-70 years old; 
3. 
Able to provide written informed consent before participating in the study; 
4. 
Able to communicate adequately with the investigator and to comply with the 
requirements for the entire study 
 
Additional inclusion criteria for patients 
1. 
Symptoms of dyspepsia (i.e., early satiety, postprandial discomfort, nausea, vomiting, 
regurgitation) 


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2. 
Patients in the DM group will also require Type 1 or 2 DM of ≥ 3 years duration; in 
patients with type 2 DM, the dyspepsia symptoms should have begun or worsened after 
DM was diagnosed  
 
Exclusion criteria – for patients and controls 
1. 
Major abdominal surgery (i.e., appendectomy, cholecystectomy, tubal ligation,  
hysterectomy, and limited colonic resection are permissible) 
2. 
Clinical evidence (including physical exam and EKG) of significant cardiovascular, 
respiratory, renal, hepatic, gastrointestinal, hematological, neurological, psychiatric or 
other disease that may interfere with the objectives of the study and/or pose safety 
concerns   
3. 
Opiates, alpha adrenergic agonists, metoclopramide, and high doses of anticholinergic 
agents (eg, amitriptyline greater than 50 mg daily).  If medically safe, these drugs may be 
discontinued for four half lives prior to study assessments   
4. 
Treatment with GLP-1 agonists and amlyin which cause vagal blockade and may affect 
central processing of pain 
5. 
Use of tobacco products within the past 1 week or NSAIDs or aspirin within the past 
week (since they all may affect intestinal permeability)  
6. 
Bleeding or clotting disorders or medications that increase risk of bleeding from mucosal 
biopsies 
7. 
Positive tissue transglutaminase antibodies (TTG),  
8. 
For two days prior to studies, subjects will be instructed to avoid ingestion of artificial 
sweeteners such as SplendaTM (sucralose), NutrasweetTM (aspartame), foods containing 
lactulose or mannitol 
9. 
Pregnant or breast-feeding females 
10. 
Known intolerance or allergy to eggs 
11. 
Poor peripheral venous access, if central venous access is not available 
12. 
Any other condition or prior therapy that, in the opinion of the investigator, would make 
the patient unsuitable for the study 
 
Exclusion criteria for controls only 
1. 
Current symptoms of a functional gastrointestinal disorder assessed by questionnaire 
 
Exclusion criteria for patients only 
1. 
Severe vomiting that would preclude tube placement or participation in the study 
2. 
Structural cause for symptoms by endoscopy within the past 48 months 
3. 
Patients with gastric pacemakers 
 
3.3 
Details of Study Assessments 
In patients with DM, fingerstick bedside reflectance meter blood glucose concentrations will be 
assessed during enteral infusion and gastric emptying studies at time 0, every 30 minutes for 2h, then 
at 120 and 180 minutes.  For patients with type 1 and type 2 DM who are treated with insulin, insulin 
will be given according to the patient’s sliding scale.  For type 2 DM patients who are not on insulin, 
no insulin will be given.   
 


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i) Screening items include questionnaires (for overall functional GI symptoms [Rome III 
criteria], severity of dyspepsia [Nepean severity index], gastroparesis cardinal symptom index 
[GCSI], anxiety and depression [HAD], and autonomic symptoms), blood tests (CBC, glycosylated 
hemoglobin, fasting glucose, sodium, potassium, bicarbonate, calcium, chloride, phosphorous, AST, 
total bilirubin, alkaline phosphatase, creatinine, high sensitive C-reactive protein, tissue 
transglutaminase antibodies, and lipase), and EKG (to be reviewed by study physician).  Some 
patients with diabetes mellitus have low serum lipase.  Patients with serum lipase < lower limit of 
normal (i.e., < 10 IU/ml) will not be eligible to participate in the study. 
 
Study procedures will be conducted over 3 days.  Items (ii), (iii), (iv), and (v) below will be 
performed on the same day preferably before other study procedures.  A tentative schedule is as 
follows: (ii) between 8 and 8.45 am, (iii) between 8.45 am and 11 am, (iv) can start while (iii) is in 
progress and (v) between 12.30 pm and 3 pm.    
 
ii) Plasma pancreatic polypeptide (PP) response to sham feeding. Pancreatic polypeptide (PP) 
is secreted by the pancreas in response to hypoglycemia, ingestion of food or sham feeding secondary to 
vagal nerve stimulation.  Vagal nerve injury blocks secretion.  This assessment will be performed in the 
Charlton 7 CRU.  Subjects will be fasting with NPO for 8 hours prior to this test. They must abstain 
from coffee or  for 12 hours and stop all cholinergic agents for 48 hours before the test.  During the 
sham feeding test, food is chewed and spit out (not swallowed)   .  After placement of an IV in the 
hand or forearm vein for blood draws, this hand will be placed in a hotbox heated to 55ºC.  Blood 
will be drawn in a lavender-top (EDTA) tube(s) at -5, 0, 5, 10, 15, 20, 25, and 30 minutes for PP.  Spin 
down, keep specimen cold, and send 1.0 mL (0.5 mL minimum) of EDTA plasma frozen in plastic vial.  
 
iii) Small intestinal permeability in vivo.  As in prior studies 33, 34, lactulose, 1000 mg, and 
mannitol, 200 mg (L7877 and M8429 from Sigma-Aldrich, St. Louis, MO 63103), will used to 
determine the urine sugar excretions at different times as markers of small bowel mucosal 
permeability after oral ingestion of the sugars in aqueous solution.  500 mL of tap water will be 
given 30 minutes after the start of the small bowel permeability test.  Because colonic permeability 
will not be assessed, urine will be collected every 30 minutes for the first 2 hours (when the 
participant was able to provide a specimen, and cumulated for the entire 2 hours).  The total volume 
of each collection is measured, and an aliquot from each collection will be obtained to estimate the 
total content of each sugar for the different time intervals.  The urine aliquot will be stored at -20° 
Celsius until it was thawed for analysis.   Cumulative and ratio excretions of the two sugars at 0-2 
hours will be used to estimate small bowel and colonic mucosal permeability, respectively, based on 
recent validation studies.  Urinary saccharide concentrations will be measured by high performance 
liquid chromatography-tandem mass spectrometry.  Details of this method were previously described 
elsewhere; the assay was adapted from the method of Lostia et al. 35.  
 
The cumulative excretion = [concentration of sugar (µg/mL)]* total urine volume (mL). 
 
The lactulose:mannitol ratio (L:M ratio) was calculated by:  
 
L:M ratio = 0.2 x (cumulative excretion lactulose) / (cumulative excretion mannitol) 
 
iv) Autonomic reflex screen.  Patients will complete a standardized autonomic symptom 
questionnaire developed by Dr. Low and undergo a standardized evaluation of autonomic functions 


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in Dr. Low’s laboratory (Charlton CRU).  This assessment is routinely used in clinical practice and 
comprises: 1) the Quantitative Sudomotor Axon Reflex Test (QSART) at proximal and distal 
standard sites to assess sympathetic postganglionic sudomotor axon function 36; 2) heart rate 
responses to deep breathing and the Valsalva maneuver to assess cardiovagal function 37; and 3) 
beat-to-beat blood pressure responses to the Valsalva maneuver and head-up tilt (HUT) to assess 
sympathetic adrenergic function 38.  Subjects will be studied under standardized conditions.   
 
v) Hormonal responses and symptoms during duodenal infusions.  A small bore nasogastric 
tube will be inserted through the nostril into the stomach over a guidewire and advanced under 
fluoroscopic guidance until the infusion ports are in the 2nd part of the duodenum.   
 
Thereafter, subjects in each group (ie, functional dyspepsia and DM) will be randomized to 
lipid infusion {66.7 mL Microlipid (0.5 gm/mL diluted in water to 222 ml)} together with placebo or 
exendin 9-39 in the ratio of 1:1.  Lipids will be delivered into the duodenum at an infusion rate 
which matches the rate at which glucose enters the systemic circulation after oral glucose.  After 
placement of an IV in the hand or forearm vein for blood draws, this hand will be placed in a hotbox 
heated to 55ºC.  We have considerable preliminary data for symptoms and hormonal responses with 
this paradigm.  Plasma glucose, insulin, C-peptide, GLP-1, CCK, ghrelin, and PYY will be collected 
at 5 minute intervals for 30 minutes, 10 minute intervals from 30 – 60 minutes, and at 15 minute 
intervals from 60 – 180 minutes (18 times including 0 minutes).    
 
 
vi) GLP-1 antagonist exendin 9-39 will be sourced from C.S. Bio, reconstituted in the Mayo 
Pharmacy by an established process used in previous studies, and undergo endotoxin testing, sterility 
testing and bacterial testing, and held in quarantine before it will be used in humans.  Research 
compounded sterile preparations (CSP) are prepared and assessed for end preparation testing 
according to USP <797> in the hospital pharmacy services production lab.  Each batch is tested for 
sterility  according to USP <71> utilizing membrane filtration. The sterility testing is validated by a 
formulation Bacteriostasis Fungistasis test.  The amount of  each batch tested is determined by USP 
<71>.  Each batch is batch is tested for endotoxin according to USP <85>.  High risk CSP are 
quarantined for 14 days and when all end preparation testing is completed.  
Exendin 9-39 will be administered intravenously at the same dose (1,200 pmol/kg bolus 
followed by infusion at 300 pmol/kg/min) as previously described at Mayo Clinic (Dr. A Vella) 39, 40.  
This dose has previously been shown to block the effects of GLP-1 infused at supraphysiologic 
doses and the effects of endogenous GLP-1 on gastrointestinal motility and insulin secretion 41-43.  
The effects of this dose are qualitatively similar to studies using 2.5- to 3.0-fold higher infusion rates 
44-46.  It is possible that endogenous GLP-1 still contributes to insulin secretion in the presence of 
exendin-9,39; however the residual effect is likely to be small 45.  For example, the 25% increase in 
integrated glucose concentrations with an infusion of 900 pmol/kg/min 46 is comparable to the 
change in glucose concentrations in other studies 40.  The half life of exendin 9-39 is 33 minutes.  
Exendin 9-39 is very safe and well tolerated and the increase in blood glucose concentration with 
exendin 9-39 infusion is modest.  For example, in 1 study blood glucose concentrations after a 
duodenal meal were on average only 6 mg/dl higher in healthy subjects and 19 mg/dl higher in 
T2DM for exendin 9-39 versus saline 47.  Even in type 1DM, the effects of exendin 9-39 on blood 
glucose are modest 48.  [Blood glucose data was provided as AUC rather than mg/dl].  We will 
monitor blood glucose by fingerstick and administer insulin as necessary (Section 3.3).        


P a g e  | 11 
 
vii) Upper GI Endoscopy and Biopsies.  Upper GI endoscopy will be performed in the 
endoscopy lab of Clinical Research Unit, Charlton 7.  Patients will receive conscious sedation as 
required for the procedure to be conducted with comfort.  A full endoscopy report will be placed in 
the medical record.  There are 3 changes in this revision (6/15/15). Because electron microscopy 
studies suggest that duodenal epithelial integrity is impaired in diabetes, we propose to (i) assess 
mucosal impedance during endoscopy and (ii) assess routine histology with mucosal biopsies. Also, 
as explained below, we propose to evaluate the mucosal microbiome with biopsies 
 During endoscopy, mucosal impedance will be measured with a new endoscopically placed 
probe that measures epithelial impedance over a 2 cm area 49, and is or has being used to measure 
transepthelial resistance in the esophagus and duodenum in other IRB-approved research protocols at 
Mayo Clinic 50.  By measuring current generated by ion flow, this device provides a measure of 
transepithelial resistance and permeability.  The soft mucosal impedance (MI) catheter will be 
advanced through the working channel of an upper endoscope.  Measurements will be obtained until 
approximately 4 optimal recordings are obtained from the mucosa in the second part of the 
duodenum.  The electrodes are connected to an impedance voltage transducer at the bedside via thin 
wires, which ran the length of the catheter.  The voltage generated by the transducer was limited to 
produce at most 2.5 microamps RMS of current.  The frequency for the measuring circuit will be set 
at 2 kHz.  Mucosal impedance will be expressed in ohms as the ratio of voltage to the current, 
according to Ohm’ law (V=IR).  Data will be acquired with a stationary impedance data acquisition 
system (InSight; Sandhill Scientific, Inc) and were viewed and analyzed on BioView Analysis 
software (Sandhill Scientific, Inc).  A small amount of fluid and 15 biopsies will be collected from 
the second part of the duodenum for the following assessments:   
a) ICS diameter using transmission electron microscopy, performed at the Optical Imaging Core (5-
10 micron sections, 2 bites, separate medium (Trump solution) or even formalin)  
b) Transepithelial resistance (TER) and fluorescein flux across the squamous epithelium, using mini-
Ussing chambers, in collaboration with Dr.Farrugia’s laboratory  
TER: Four biopsies will be mounted in 4 ml Ussing chambers (Physiological Instruments, San 
Diego, CA) exposing 0.03 cm2 area.  Using a pair of Ag/AgCl electrodes with agar-salt bridges 
current-giving platinum electrodes, TER will be measured.  These assessments need to be performed 
within 30 minutes of collecting samples (3 bites, Kreb’s solution). 
Paracellular flux: Macromolecular flux across biopsies will be studied using FITC Dextran (4 kDa, 
Molecular Probes, NY) added to the mucosal compartment. At 30 min intervals for a total of 3 hrs, 
fluorescence in the basal compartment will be analyzed using a Synergy Multi-Mode Microplate 
Reader (BioTek, VT, USA).  The same specimen used for TER can be used for this.  
c) Expression of tight junction gene expression with quantitative real-time RT-PCR will be assessed 
using Taqman gene expression assay.  The following mRNA levels will be measured: ZO-1, ZO-2, 
ZO-3, occludin, and Claudins-1-4.  The expression of each gene will be normalized to housekeeping 
genes beta-actin, beta-2-microglobulin, and GAPDH.  Protein concentrations will be determined by 
Pierce bicinchoninic acid assay as previously described.  Primary immunoglobulin (Ig)G antibodies 
directed against ZO-1, ZO-2, ZO-3, occludin and claudins 1-4 will be used.  Corresponding 
secondary antibodies will be used and densitometric comparison will be carried out on the 
immunoblot.  In non-post-infectious IBS, occludin and claudin-1 were found to be “internalized” 


P a g e  | 12 
 
into the cytosolic space. Similar changes have been seen with occludin expression in the jejunal 
tissue from non-post-infectious IBS making distribution of TJ proteins an important component of 
studying expression.  Immunohistochemistry will be used to determine expression and distribution 
of ZO-1, occludin, phosphorylated occludin and claudin-1.  This requires 2 tissue biopsies (fresh 
frozen in liquid nitrogen at -80). 
d) Small bowel inflammation: will be assessed with RT-PCR for global markers of macrophages 
(CD68), pro-inflammatory macrophage (M1) markers: TNFα, IL1β IL6 and anti-inflammatory (M2) 
macrophage markers (CD206, CD36) using previously described methods.  This requires 2 tissue 
biopsies (fresh frozen in liquid nitrogen at -80). 
e) Assessment of microbiome (2 specimens snap frozen in liquid nitrogen at -80C) – Currently, we 
are collecting stool but not mucosal biopsy specimens for microbial analysis. Our recent studies 
(IRB 12-6091) indicate that the mucosal microbiome discriminated between health and constipation 
with 94% accuracy independent of colonic transit. In contrast, the fecal microbiome predicted 
colonic transit and breath hydrogen production. Hence, and given documented associations between 
the gut microbiota and diabetes mellitus cited below, we propose to collect 2 mucosal biopsies for 
this purpose.     
f) Three specimens for mRNA expression to be collected in RNA later [No change]. 
g) Routine histology – Because preliminary analysis of electron microscopy analysis demonstrates 
widening of intercellular spaces and because there is evidence for low grade intestinal mast cell and 
eosinophil infiltration in functional disorders 51, we propose to evaluate routine histology in 2 
mucosal biopsies collected in formalin; Dr. Thomas Smyrk will examine the same. 
viii) Gastric emptying and small intestinal transit of a mixed solid-liquid meal. Using 
established techniques, gastric emptying of solids and liquids and small bowel transit will be 
simultaneously assessed by scintigraphy. 52  99mTc-sulfur colloid (1 mCi) will be added to 2 raw eggs 
during the scrambling and cooking process. The eggs will be served on 1 slice of bread and with 240 
mL of 1% milk (296 kcal, 32% protein, 35% fat, 33% carbohydrate) labeled with 111In-
diethylenetriaminepentaacetate (0.1 mCi).  During the gastric emptying study, patients will complete 
VAS scales for severity of 6 symptoms (nausea, fullness, bloating, abdominal pain, belching and 
burning), at 15 minute intervals, on a Likert scale with the descriptors (absent, light, moderate, 
severe, and intolerable).  During the study, patients will eat  a standardized meal, a lunch of chicken 
breast, during this study visit  this meal  is not radioactive. Data will be analyzed as in previous 
studies.   
 
ix) Stool Collection. Two recent metagenome-wide association studies have highlighted 
associations between specific gut microbiota and type 2 diabetes mellitus 53, 54.  To build on these 
observations and extend our ongoing studies evaluating the relationship between gastrointestinal 
transit and gut microbiome (IRB 12-6091) we will collect a stool sample in each subject.  Using a 
stool kit and standardized instructions, patients will collect stool a sample according to the procedure 
in the Appendix.  The stool sample will be frozen and stored in a -20°C freezer.  The stool specimen 
will be collected without a laxative.   


P a g e  | 13 
 
x) Symptom diary.  Participants will complete a validated daily diary (i.e., Gastroparesis 
Cardinal Symptom Index-Daily Diary (GCSI-DD) for 2 weeks at home 55, 56.  This will probably 
require 5 minutes daily. 
xi) Immunochemical Analysis.  Arterialized venous plasma samples will be placed in ice, 
centrifuged at 4oC, separated and stored at -20oC until assay.  Glucose will be measured by the 
Hitachi 912 (Roche Diagnostics, Indianapolis, IN), hexokinase catalyzes the phosphorylation of 
glucose by ATP. G-6-P is oxidized to 6-phosphogluconate in the presence of NADP by the enzyme 
glucose-6-phosphate dehydrogenase. No other carbohydrate is oxidized. The amount of NADH 
formed during the reaction is equivalent to the amount of D-glucose in the specimen and can be 
measured photometrically by the increase in absorbance at 340nm. 
GLP-1 has been developed to measure biologically active GLP-1(7–36, 7-37) amide level will be 
measured by enzyme linked immunosorbent assay (Linco Research), with the lowest levels of 
detection 3 pM with no cross reactivity to GLP-1-(9–36) amide, GLP-2 and glucagon also have no 
cross reactivity. 
CCK  – concentrations will be measured by an immunoassay (Alpco Diagnostics) which utilizes 
rabbit antiserum to a synthyetic cholecystokinin 26-33 sulphate (CCK 8 sulphate) and binds to most 
biological active forms with nearly equimolar potency. 57  It has essentially no cross-reactivity with 
gastrin and plasma CCK immunoreactivity did not increase during gastrin-17 infusion into healthy 
subjects.  With this RIA, plasma CCK concentrations averaged ~ 1 pmol/L under basal conditions 
and increased to ~ 5 pmol/L after a meal. 
PYY will be measured by radioimmunoassay (Linco Research, Inc.). PYY exists in at least 2 
molecular forms, 1-36 and 3-36, both of which are physiologically active. There is no measureable 
crossreactivity to glucagon, ghrelin, insulin, GLP-1.   
C-peptide levels will be measured by a 2-site immunometric (sandwich) assay using 
electrochemiluminescence detection (Cobas e411, Roche Diagnostics Indianapolis, IN). Patient 
specimen, biotinylated monoclonal C-peptide specific antibody, and monoclonal C-peptide-specific 
antibody labeled with ruthenium react to form a complex. Streptavidin-coated micro particles act as 
the solid phase to which the complex becomes bound. Voltage is applied to the electrode inducing a 
chemiluminescent emission from the ruthenium. (red top, needs 0.5 ml serum, 3 ml tube) 
 
Plasma insulin concentrations will be measured using a chemiluminescence assay (Access Assay; 
Beckman Coulter, Chaska, MN).  Established approaches will be used to estimate  cell 
responsiveness and insulin sensitivity from plasma glucose, insulin, and C-peptide concentrations.   
Total ghrelin will be measured by a radioimmunoassay technique (Linco Research, Inc.). The assay 
uses 125I-labeled ghrelin and a ghrelin antiserum to determine the level of total ghrelin in plasma. 
There is no measurable crossreactivity to glucagon, GLP-1(7-36), insulin. (0.8 microL) 


P a g e  | 14 
 
Summary of Blood Collections. The total blood collected for this study is 403 ml.   
Item 
Test ID 
Lab 
Tube (amount required)  
Complete blood count # 
9109 
Central 
Clinical Lab 
3 ml EDTA  
Tissue transglutaminase 
antibody # 
83671 
Immunology 
Lab 
5 ml red top (0.5 ml serum)* 
High sensitive CRP # 
82044 
ICL  
1 ml serum  
Fasting glucose # 
7702 
ICL 
 2 ml Sodium fluoride (0.5 ml) 
Lipase # 
8328 
Central 
Clinical Lab 
5 ml SST (1 ml serum) 
Glycosylated hemoglobin # 
82080 
Central 
Clinical Lab 
3 ml EDTA (2 ml whole blood) 
Sodium 
81692 
Central 
Clinical Lab 
5 ml SST **  
Potassium 
81390 
Central 
Clinical Lab 
5 ml SST ** 
Bicarbonate 
876 
Central 
Clinical Lab 
5 ml SST ** 
Calcium 
8432 
Central 
Clinical Lab 
5 ml SST ** 
Chloride 
8460 
Central 
Clinical Lab 
5 ml SST ** 
Phosphorus 
8029 
Central 
Clinical Lab 
5 ml SST ** 
AST 
8360 
Central 
Clinical Lab 
5 ml SST ** 
Total bilirubin 
81785 
Central 
Clinical Lab 
5 ml SST ** 
Alkaline phosphatase 
8340 
Central 
Clinical Lab 
5 ml SST ** 
Glucose 1  
7702 
ICL 
2 ml Sodium fluoride (0.5 ml) 


P a g e  | 15 
 
GLP-1 1 
80075 
ICL 
3 ml EDTA (1 ml plasma) 
CCK 1 
90162 
ICL 
4 ml EDTA (2 ml plasma) 
PYY 1 
7670 
ICL 
3 ml EDTA (1 ml plasma) 
Total ghrelin 1 
83334 
ICL 
1.6 ml EDTA (0.8 ml plasma) 
C-peptide 1 
7653 
ICL 
5 ml red top (0.5 ml serum)* 
Insulin 1 
7661 
ICL 
5 ml red top (0.8 ml serum) * 
Plasma PP 2 
 
ICL 
3 ml EDTA (1 ml plasma) 
Epigenetic analysis 
(RRBS) 3 
31669 
BAP Lab 
500 ng DNA (EDTA tube buffy coat - 
snap-frozen in liquid nitrogen)  
DNA for SNPs 3 
31669 
BAP Lab 
Use buffy coat from EDTA tubes 
RNA analysis 4 
31669 
BAP Lab 
3 PAX gene tubes (2.5 ml each, total = 7.5 
ml) 
* Same tube for these specimens; ** Same tube for these specimens;  # Safety and screening 
assessments, one time only. 
1 Collected 18 timepoints during each study; 2 Sham feeding test – 8 collections; 
3 Requires DNA. Will be obtained from EDTA tubes for ghrelin and a separate 10 ml tube;  
3 PAX gene tube 
3.4 
Gene and Epigenetic Studies 
i) Towards our long-term objective of uncovering associations between SNPs and dyspepsia and 
gastroparesis, we propose to extract DNA from blood, to be drawn from study participants.  
Genome-wide analysis will be conducted using Illumina 610 QUAD microarray or comparable 
approaches.  Genotype-phenotype correlations will be examined using these patients and other 
patients in ongoing studies.   
 
ii) Epigenetic studies.  The association of differentially methylated CpG or genomic regions with 
gastroparetic symptoms, abnormal (delayed or rapid) gastric emptying, intestinal chemosensitivity, 
presence or absence of autonomic neuropathy, nephropathy, and retinopathy, will be evaluated; age, 
gender, glycosylated hemoglobin, and duration of DM are potential covariates. 
Reduced representation bisulfite sequencing (RRBS) and analysis will be performed.  RRBS 
is base resolution methylation sequencing to assess genome-wide DNA methylation enriched in CpG 
rich regions in the genome, particularly in the coding and its neighboring regions.  We will use Next 
Gene Sequencing to determine methylation status as outlined by Gu et al 58 and Illumina.  Briefly, 
DNA is digested with Msp1 and the digested DNA is purified.  Purified DNA is end repaired and 
adenylated.  DNA is ligated to Illumina adapters using T4 DNA ligase.  Ligated DNA is purified and 


P a g e  | 16 
 
size selected using Ampure beads.  Purified size selected DNA undergoes bisulfite modification and 
clean-up.  Modified DNA is PCR amplified and purified using Ampure beads.  This RRBS library 
DNA will be run on the Illumina HiSeq2000, indexing 4 samples per lane, using a standard 
operating procedure based on Illumina’s protocol.   
An additional 7.5 ml of blood will be collected for mRNA-sequencing and miRNA 
sequencing in 3 Paxgene tubes (2.5 ml each).   
 
3.5 
Data Analysis 
Chemosensitivity will be measured using a 100mmVAS anchored at each end (left end:No 
symptoms, right end: Severe symptoms).  The distribution of this measure will be checked for its 
approximation to a gaussian distribution.  An analysis to provide preliminary data for a new grant 
submission will be examined after approximately 15 subjects (5 DM, 10 controls) have completed 
the studies, but there is no intention of stopping the study based on the preliminary results.  We 
anticipate conducting this analysis in July 2014 or thereafter.  
 
Question 
Summary 
Parameter 
Statistical analysis 
Sensation (symptoms) 
during enteral infusion 
(Aim 1) 
VAS  score  
i) compare VAS scores in response to 
lipid infusion + exendin vs. lipid infusion 
+ placebo separately for each group using 
a 2-sample t-test or Wilcoxon rank sum 
test ii)compare the delta VAS scores 
(exendin – placebo) to assess the 
association with subject status (FD,DM,  
and controls)  using an ANOVA model  
(adjusting for covariates, eg, type of DM) 
or Kruskal-Wallis test.    
Relationship between 
intestinal chemosensitivity 
and severity of ongoing 
upper GI symptoms  
Mean symptom score 
and QoL defined by 
the  Nepean 
dyspepsia index and 
GCSI diary 
Multiple linear regression with the 
dependent variable = mean symptom 
score, and separately the QoL score  
(possibly after rank transformation). 
Potential predictor variables = VAS score  
during enteral lipid + placebo infusion, 
gastric emptying, age, gender, BMI  
Relationship between 
sensation and plasma 
hormones during lipid 
infusion (Aim 2) 
Using the VAS score 
from the enteral 
infusions and for 
hormones, the AUC  
values  
Spearman correlations between the VAS 
score and the AUC values for the various 
hormones,  overall and separately by 
group (FD,DM, controls)  
Small intestinal tight 
junction proteins, 
Protein expression, 
lactulose:mannitol 
Assess the associations between 
permeability and protein expression, vs.  
the VAS score, based on Spearman 


P a g e  | 17 
 
permeability, intestinal 
chemosensitivity  
ratio, VAS  score  
correlations.  
The sample size estimate is based on our previous study which evaluated intestinal 
chemosensitivity in functional dyspepsia in which the proportions with severe symptoms were 
assessed.  [In the current proposal, symptoms during lipid infusion will be evaluated on a continuous 
VAS scale, likely providing the ability to detect smaller effect sizes].  The effect of lipid plus 
placebo vs. lipid plus exendin in response to enteral infusion can be based on a comparison of the 
proportions with severe symptoms using a two sample t-test.  The association of group status with 
response to lipid plus exendin can also be assessed using a two-sample t-test for proportions.  There 
is ~80% power (alpha =0.05, 2-sided) to detect the specified differences listed in the table below.  
 
3.6 
Potential Pitfalls 
To reduce the likelihood of patient intolerance, a smaller diameter tube will be used for enteral 
nutrient infusion.  Only lipid and not carbohydrate infusions will be given; hence the duration of this 
study is shorter and hyperglycemia during nutrient infusion is less likely.   
 
4  
HUMAN SUBJECTS 
Description:  Studies will be conducted over 3 days.  Subjects will be remunerated $ 600.  A 
screening assessment will be performed to ensure subjects fulfill entry criteria.  Subjects will fast 
overnight prior to study days.  Study procedures will be performed using established techniques by 
experienced technicians in our programs.   
Research Materials:  Blood samples (hormones, glucose, extract DNA and RNA) and stool 
samples will be obtained for this study.  
Population and Recruitment of Subjects: Healthy subjects will be recruited from the community 
and patients with DM and dyspepsia will be recruited from the clinical practice and to patients who 
have provided research authorization.  The Institutional Review Board will approve all recruitment 
materials.  After discussing the study procedure and its risks, informed consent will be obtained, 
before study procedures.  No children or prisoners will be recruited.  
 
Group   
                                   Proportions (%) with severe symptoms   
exendin+lipid 
vs. placebo+lipid  
Controls vs. FD 
(exendin+lipid)  
Controls vs. DM 
(exendin+lipid)   
FD vs. DM 
(exendin+lipid) 
Controls(n=12 vs n=12)   10% vs. 68%  
10% vs. 60%  
10% vs. 60%  
30% vs. 73%  
functional dyspepsia 
(n=20 vs. 20) 
 10% vs. 50%  
DM (n=20 vs. 20)  
 10% vs. 50%  


P a g e  | 18 
 
Potential Risks: Blood sampling. Blood samples are collected by venipuncture for this study.  
Assays for measuring blood glucose and plasma hormone will require 19ml blood at each of 18 time 
points during the enteral infusion study.  The total blood drawn is 408 ml.  Bruising can occur with 
venipuncture, as can fainting, etc.  Risk Monitoring/Risk Reduction: The samples are collected using 
aseptic technique in the venipuncture area of the Clinical Research Unit where facilities are available 
should untoward reactions (fainting, etc.) occur. Given the aseptic nature of the sample collection 
and the small risk of bruising, the monitoring plan is focused on advising volunteers to call the co-
investigators (physician) should they have unusual pain or discomfort from the venipuncture site. 
Enteral tube placement This will require exposure to radiation and may be associated with 
discomfort.  The risk of perforation with a soft-tipped feeding tube is very low. Risk Monitoring / 
Risk Reduction: The radiation safety committee will review all projected exposure to radiation prior 
to approval of the protocol by the IRB.  The small bore soft-tipped feeding tube will be positioned 
by trained personnel under fluoroscopic guidance in the CRU.  Subjects will be closely monitored 
for tolerance.  Enteral nutrition. The may be associated with nausea and abdominal cramping 
symptoms. Risk Monitoring/Risk Reduction: Subjects will fast overnight prior to the lipid infusion.  
To minimize the effect on blood glucose, lipid instead of glucose infusion is being administered.  
They will be closely monitored by trained personnel.  Exendin 9-39 infusion.  The effects of 
exendin infusion on postprandial blood glucose concentrations is modest.  For example, in 1 study 
blood glucose concentrations after a duodenal meal were on average only 6 mg/dl higher in healthy 
subjects and 19 mg/dl higher in T2DM for exendin 9-39 versus saline 47.  Risk Monitoring / Risk 
Reduction: Blood glucose concentrations will be monitored regularly and insulin will be 
administered if necessary.  Gastric emptying. Gastric emptying and small intestinal transit will be 
measured by scintigraphy. This will require exposure to radiation. Risk Monitoring / Risk 
Reduction: The radiation safety committee will review all projected exposure to radiation prior to 
approval of the protocol by the IRB.  Pregnancy tests will be checked within 48 hours prior to each 
study day.  Vital signs will be recorded before the study.  
 
Dosimetry and Organ Exposure in mrad (see attached under APPENDIX) 
 
In view of the radiation exposure, all females of childbearing age will be required to have a negative 
urine pregnancy test within 1 week of this study. 
Protection: The key personnel in this application have completed the required education on the 
protection of human research participants. The institution has established a formal program entitled 
the Mayo Investigator Training Program or MITP. The MITP is a web based educational course 
designed to provide all personnel involved in human subject research with training about human 
subject protection. All Mayo personnel engaged in human subject research are required to complete 
the course.  
Data Safety and Monitoring Plan. The ultimate goal of this application is to further our 
understanding of GI symptoms in patients with dyspepsia and diabetes. The DSMP utilized will 
adhere to the protocol approved by the Mayo Clinic IRB. We propose the following plan: - 
Data quality and management: The principal investigator will review all data collection forms on a 
three-monthly basis for completeness and accuracy of the data as well as protocol compliance.  
Adverse events grading: The common grading scale listed below will be used to grade AEs: 
 
0 
No adverse event or within normal limits or not clinical significant 


P a g e  | 19 
 
1 
Mild AE, did not require treatment 
2 
Moderate AE, resolved with treatment 
3 
Severe AE, resulted in inability to carry on normal activities and required 
professional medical attention 
4 
Life threatening or disabling AE 
5 
Fatal AE 
 
Attribution scale: An adverse event includes both, an expected side effect that is of a serious nature, 
or an unexpected side effect/ event regardless of severity. All events will be graded as to their 
attribution (unrelated to protocol, or possibly, probably, or definitely related to protocol). Any event 
that is reported to either the principal investigator or his designated research associates by the subject 
or medical staff caring for the subject and which meets the criteria will be documented as such. 
Data Monitoring. The majority of data generated from these protocols will be from analyses 
performed in our laboratory or the immunochemical core laboratory. Standard quality control 
procedures are in place for each assay.  The frequency of data review for this study differs according 
to the type of data and can be summarized in the following table: 
 
Data type 
Frequency of 
review 
Subject accrual (adherence to protocol regarding 
demographics, inclusion/exclusion) 
Weekly 
Adverse event/safety rates (injuries) 
Weekly 
Annual report 
Yearly for IRB  
 
Informed Consent. Written informed consent will be obtained from all individuals who participate 
in the study. The principal investigator or his co-investigators meet with each participant, review the 
consent form in detail and confirm the subjects understanding of the study. They answer all 
questions posed by the participants and when convinced that the subject verbally demonstrates 
understanding of the protocol obtains a signed consent. Only designated staff are authorized to 
obtain informed consent. 
Benefits: This study exposes subjects to risks detailed above. However, it will advance our 
knowledge of the mechanisms of GI sensorimotor dysfunctions in patients with DM.  
 
5  
SAFETY AND ADVERSE EVENTS 
 
5.1 
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria:  
 Serious: Serious problems or events that results in significant harm, (which may be physical, 
psychological, financial, social, economic, or legal) or increased risk for the subject or others 
(including individuals who are not research subjects). These include: (1) death; (2) life 
threatening adverse experience; (3) hospitalization - inpatient, new, or prolonged; (4) 
disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) breach of 


P a g e  | 20 
 
confidentiality and (7) other problems, events, or new information (i.e. publications, DSMB 
reports, interim findings, product labeling change) that in the opinion of the local investigator 
may adversely affect the rights, safety, or welfare of the subjects or others, or substantially 
compromise the research data, AND 
 Unanticipated: (i.e. unexpected) problems or events are those that are not already described as 
potential risks in the protocol, consent document, not listed in the Investigator’s Brochure, or 
not part of an underlying disease. A problem or event is "unanticipated" when it was 
unforeseeable at the time of its occurrence. A problem or event is "unanticipated" when it 
occurs at an increased frequency or at an increased severity than expected, AND 
 Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, 
device, biologic) in a patient or research subject. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  Serious problems/events can be well defined 
and include; 
 death 
 life threatening adverse experience 
 hospitalization 
 inpatient, new, or prolonged; disability/incapacity 
 persistent or significant birth defect/anomaly 
 
and/or per protocol may be problems/events that in the opinion of the sponsor-investigator may 
have adversely affected the rights, safety, or welfare of the subjects or others, or substantially 
compromised the research data. 
 
Other important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance.  They may jeopardize the subject, and may require intervention 
to prevent one of the other serious outcomes noted above.  For example, drug overdose or abuse, a 
seizure that did not result in in-patient hospitalization or intensive treatment of bronchospasm in an 
emergency department would typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious, should be regarded as non-serious 
adverse events.  
 
Withdrawal criteria 
 Inability to place nasoduodenal feeding tube 
 Inability to obtain intravenous access 
 Severe abdominal discomfort, nausea or vomiting during enteral nutrient infusion 
 Severe hyperglycemia (> 400 mg/dl) unresponsive to insulin therapy 
 
Adverse Event Reporting Period 


P a g e  | 21 
 
 
Example 
For this study, the study treatment follow-up period is defined as 1 days following the last 
administration of study treatment. The study period during which adverse events must be reported is 
normally defined as the period from the initiation of any study procedures to the end of the study 
treatment follow-up.   
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition should 
be recorded as an adverse event if the frequency, intensity, or the character of the condition worsens 
during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting condition.  
At the end of the study, any new clinically significant findings/abnormalities that meet the definition 
of an adverse event must also be recorded and documented as an adverse event.  
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the sponsor-investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At the last 
scheduled visit, the sponsor-investigator should instruct each subject to report, to the sponsor-
investigator, any subsequent event(s) that the subject, or the subject’s personal physician, believes 
might reasonably be related to participation in this study.  
 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be documented 
and reported as a serious adverse event unless specifically instructed otherwise in this protocol.  Any 
condition responsible for surgery should be documented as an adverse event if the condition meets 
the criteria for an adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an 
adverse event in the following circumstances:  
 Hospitalization or diagnostic or elective surgical procedures for a preexisting condition.  
Surgery should not be reported as an outcome of an adverse event if the purpose of the 
surgery was elective or diagnostic and the outcome was uneventful. 
 Hospitalization or prolonged hospitalization for therapy of the target disease of the study (e.g., 
diabetes mellitus), unless it is a worsening or increase in frequency of hospital admissions as 
judged by the clinical investigator. 
5.2 
Recording of Adverse Events 
At each contact with the subject, the study team must seek information on adverse events by specific 
questioning and, as appropriate, by examination.  Information on all adverse events should be 
recorded immediately in the source document, and also in the appropriate adverse event section of 
the case report form (CRF).  All clearly related signs, symptoms, and abnormal diagnostic, 
laboratory or procedure results should recorded in the source document. 
 


P a g e  | 22 
 
All adverse events occurring during the study period must be recorded.  The clinical course of each 
event should be followed until resolution, stabilization, or until it has been ultimately determined 
that the study treatment or participation is not the probable cause.  Serious adverse events that are 
still ongoing at the end of the study period must be followed up, to determine the final outcome.  
Any serious adverse event that occurs after the study period and is considered to be at least possibly 
related to the study treatment or study participation should be recorded and reported immediately. 
 
5.3 
Reporting of Serious Adverse Events and Unanticipated Problems 
When an adverse event has been identified, the study team will take appropriated action necessary to 
protect the study participant and then complete the Study Adverse Event Worksheet and log.  The 
sponsor-investigator will evaluate the event and determine the necessary follow-up and reporting 
required. 
5.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
This section is written with the intent that a specifically designed adverse event worksheet will be 
completed for any SAE.  The information on that worksheet will be reported to the IRB in a de-
identified manner.. 
 
The IRB requirements reflect the guidance documents released by the Office of Human Research 
Protections (OHRP), and the Food and Drug Administration (FDA) in early 2007 and are 
respectively entitled “Guidance on Reviewing and Reporting Unanticipated Problems Involving 
Risks to Subjects or Others and Adverse Events” and “Guidance for Clinical Investigators, Sponsors, 
and IRBs: Adverse Event Reporting – Improving Human Subject Protection.” 
 
(Refer to the IRB Policy and Procedure on Reporting Unanticipated Problems Involving Risks to 
Subjects or Others to the IRB, on the IRB web site.  Describe how this will be carried out by the 
study team and investigator.  Also address notification of other investigators if necessary. 
http://mayocontent.mayo.edu/irb/DOCMAN-0000047812  
 
The sponsor-investigator will report to the Mayo IRB any UPIRTSOs and Non-UPIRTSOs 
according to the Mayo IRB Policy and Procedures.  According to Mayo IRB Policy any serious 
adverse event (SAE) which the Principal Investigator has determined to be a UPIRTSO must be 
reported to the Mayo IRB as soon as possible but no later than 5 working days after the investigator 
first learns of the problem/event. 
 
The sponsor-investigator will review all adverse event reports to determine if specific reports need to 
be made to the IRB and FDA.  The sponsor-investigator will sign and date the adverse event report 
when it is reviewed.  For this protocol, only directly related SAEs/UPIRTSOs will be reported to the 
IRB. 
5.3.2 Sponsor-Investigator reporting: Notifying the FDA 
The sponsor-investigator will report to the FDA all unexpected, serious suspected adverse reactions 
according to the required IND Safety Reporting timelines, formats and requirements. 
 


P a g e  | 23 
 
Unexpected fatal or life threatening suspected adverse reactions where there is evidence to suggest a 
causal relationship between the study drug/placebo and the adverse event, will be reported as a 
serious suspected adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no later 
than 7 calendar days after the sponsor-investigator’s initial receipt of the information about the 
event. 
 
Other unexpected serious suspected adverse reactions where there is evidence to suggest a causal 
relationship between the study drug/placebo and the adverse event, will be reported as a serious 
suspected adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no later than 15 
calendar days after the sponsor-investigator’s initial receipt of the information about the event. 
 
Any clinically important increase in the rate of serious suspected adverse reactions over those listed 
in the protocol or product insert will be reported as a serious suspected adverse reaction.  This will 
be reported to the FDA on FDA Form 3500A no later than 15 calendar days after the sponsor-
investigator’s initial receipt of the information about the event. 
 
Findings from other studies in human or animals that suggest a significant risk in humans exposed to 
the drug will be reported.  This will be reported to the FDA on FDA Form 3500A, no later than 15 
calendar days after the sponsor-investigators initial receipt of the information about the event. 
 
5.5 
Stopping Rules  
 
All study procedures (blood draws, upper endoscopy, enteral nutrient infusion, exendin 9-39) are 
very safe.  If any subject develops an intestinal perforation or clinically significant bleeding during 
endoscopy or intestinal lipid infusion, the study will be placed on hold until the cause of the 
complication is determined.   
 
5.6. 
Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at his/her site.  
This safety monitoring will include careful assessment and appropriate reporting of adverse events 
as noted above, as well as the construction and implementation of a site data and safety-monitoring 
plan (see section 10  “Study Monitoring, Auditing, and Inspecting”).  Medical monitoring will 
include a regular assessment of the number and type of serious adverse events. 
 
 
APPENDIX 
 
Stool Specimen Collection Instructions 
 
Attaching the collection container 
1. Open the kit 
 


P a g e  | 24 
 
2. Lay the collection container on a flat surface with the labeled side facing 
up.  Fold up the two cardboard sides (Figure 1). 
 
 
 
3. Remove the backing from the tape on each of the cardboard sides. 
 
4. Insert the collection container into the toilet bowl and attach the tape to 
the top of the toilet seat toward the back half of the bowl (Figure 2).  The 
cardboard sides should be up against the bottom of the toilet seat. 
 
  
5. Shape the paper dish (middle part of the collection container) into a bowl 
by gently pushing down the center. 
 
Collection a sample 
1. Do not urinate into the collection container.  (You may wish to urinate 
before attaching the collection container to the toilet seat.) 
 
2. Have a bowel movement into the paper dish 
 
3. Take out the collection tube and unscrew the cap.  Use the spoon 
attached to the cap to scoop a marble-sized sample.  Insert filled spoon 
back into the tube and tightly screw the camp onto the collection tube 
(Figure 3). 
 
 
 


P a g e  | 25 
 
Disposal 
1. Remove the paper dish holding the stool by gently lifting up the four 
attachment sites.  Flush the paper dish and stool. 
 
2. Remove the cardboard frame from the toilet seat and discard it in the 
wastebasket. 
 
3. Wash your hands thoroughly with soap and water. 
 
 
Returning the sample to Mayo Clinic 
1. Be sure the collection tube cap is tightly fastened. 
 
2. Place the collection tube containing your sample into the small white bag. 
 
 
 
 
If you are at Mayo Clinic: 
At your earliest convenience, return the white bag containing your specimen 
to: Station S/Specimen Collection Cart 
Monday through Friday, 7 a.m. to 5 p.m. 
 
If you are mailing your stool specimen to Mayo Clinic: 
At your earliest convenience, mail your specimen to Mayo Clinic using the 
prepaid mailer provided. 
 
 
 
 
 


P a g e  | 26 
 
Dosimetry and Organ Exposure in mrad 
Gastric and Small Bowel Transit 
 
99m Tc-sulfur colloid 
 
 
Effective Dose Equivalent (He) 
organ doses in mrad 
  
Organ/Wt 
Administered 
Body  
Gonads 
Breast 
RBM 
Lung 
Thyroid 
Bone 
O#1: 
ULI 
O#2: 
LLI 
O#3: SI 
O#4: 
Stomach 
O#5: 
Uterus 
RAM  
Activity (mCi) 
  
0.25 
0.15 
0.12 
0.12 
0.03 
0.03 
0.06 
0.06 
0.06 
0.06 
0.06 
Tc-99m      
Non-
Absorbable 
Markers 
(solids) 
1 
21 
96 
2 
19 
4 
0 
17 
444 
307 
226 
218 
59 
Effective Dose 
  
24 
0 
2 
0 
0 
1 
27 
18 
14 
13 
4 
He 
(mrem): 
103 
 
 
 
 
111In-DTPA  
 
 
Effective Dose Equivalent (He) 
organ doses in mrad 
  
Organ/Wt 
Administered 
Body  
Gonads 
Breast 
RBM 
Lung 
Thyroid 
Bone 
O#1: 
ULI 
O#2: 
LLI 
O#3: SI 
O#4: 
Stomach 
O#5: 
Bladder 
RAM  
Activity (mCi) 
  
0.25 
0.15 
0.12 
0.12 
0.03 
0.03 
0.06 
0.06 
0.06 
0.06 
0.06 
In-111      Non-
Absorbable 
Markers 
(solids) 
0.1 
16 
155 
2 
36 
3 
0 
11 
407 
740 
185 
93 
41 
Effective Dose 
  
39 
0 
4 
0 
0 
0 
24 
44 
11 
6 
2 
He 
(mrem): 
132 
 
 
 
 
 
 
 
 
 
 
 


P a g e  | 27 
 
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