Clinical trial NCT02762708
Conditions: Type 2 Diabetes Mellitus Obesity
Keywords: Bariatric Surgery Weight loss
Sponsor: Mayo Clinic
Start date: 2016-03-01; Completion date: 2018-01-06
Enrollment: 12 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
What are the benefits of undergoing Roux-en-Y gastric bypass surgery in improving type 2 diabetes outside of weight loss alone?

Detailed description:
Roux-en-y gastric bypass (RYGB) surgery leads to improvements in diabetes that occurs even before any significant weight loss is seen. What are the reasons for this? There is evidence in animals that changes in the composition of gut bacteria after RYGB may be responsible for improvements in how glucose is metabolized in the body. The study aims to correlate changes in the bacterial composition of the gut that occurs after surgery with improvements in glucose metabolism in people with type 2 diabetes. The investigators will do this by using validated methods to measure glucose metabolism and cutting edge technology to analyze the gut microbiome. A parallel group of patients with similar weight, height and age will undergo caloric restriction alone to mimic weight loss seen after RYGB. Participants will also undergo similar glucose metabolism studies and analysis of their gut bacterial composition. Thus, any difference seen between groups can be attributed to the surgery itself and may lead to identification of novel therapeutic options for diabetes.

Arms: arm 1: Subjects between the ages of 20-65 years of age seen for weight management in the Nutrition Clinic at Mayo Clinic, who have been approved for RYGB. arm 2: Subjects between the ages of 20-65 years of age, with diagnosis of Type 2 diabetes mellitus or impaired fasting glucose. Subjects will undergo caloric restriction alone to mimic weight loss seen after gastric bypass surgery. arm 3: Four subjects who have undergone RYGB surgery will be studied 4 weeks after surgery. Subjects will be randomized to receive an infusion of Exendin-9,39 or normal saline. arm 4: Four subjects who have undergone RYGB surgery will be studied 4 weeks after surgery. Subjects will be randomized to receive an infusion of Exendin-9,39 or normal saline.
Interventions: intervention 1: Roux-en-Y Gastric Bypass (RYGB) intervention 2: Caloric Restriction intervention 3: Exendin-9,39 intervention 4: Normal Saline
Intervention descriptions: intervention 1: Subjects will be studied prior to surgical intervention and 12 weeks post surgery. Subjects in surgical arm will start diet intervention after surgery. Follow up with dietary will be per standard of care following RYGB surgery. intervention 2: Subjects participating in caloric arm of this study will be asked to consume an identical diet that is controlled for caloric content and macronutrient composition. This is identical to that consumed by patient undergoing RYGB. Subjects in the caloric restriction arm will start the diet after the screening visit. Compliance will be monitored by alternating weekly meetings with a clinical psychologist and dietician. intervention 3: A subset of 4 subjects who have undergone RYGB surgery will be studied 4 weeks after surgery. Subjects will be randomized to receive an infusion of either Exendin-9,39 or normal saline. Exendin-9,39 blocks the action of specific hormones called "incretins" that are produced by the gut in health and in larger quantities after gastric bypass surgery. intervention 4: subset of 4 subjects who have undergone RYGB surgery will be studied 4 weeks after surgery. Subjects will be randomized to receive an infusion of either Exendin-9,39 or normal saline. Exendin-9,39 blocks the action of specific hormones called "incretins" that are produced by the gut in health and in larger quantities after gastric bypass surgery.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
The Effect of Bariatric Surgery on Metabolism, 
the Metabolome and the Microbiome in 
Patients With Type 2 Diabetes 
 
NCT02762708 
 
February 6, 2017 


Abbreviations: RYGB: Roux-en-Y Gastric Bypass; CR: Caloric Restriction; CRU: Clinical Research Unit; 
GLP-1: Glucagon-like peptide-1; DI: Disposition Index; φ: β-cell responsivity; Si: Insulin action; EGP: 
Endogenous Glucose Production; Meal Ra: Meal Appearance; Rd: Glucose Disappearance 
 
The effect of Bariatric Surgery on Metabolism, the Metabolome and the Microbiome in patients with 
Type 2 Diabetes 
 
Version 3: 2/6/2017 
 
Bariatric surgery is associated with resolution of 
type 2 diabetes, and remission rates of ~84% have been 
observed after Roux-en-Y Gastric Bypass (RYGB), 
influenced by factors such as diabetes duration and 
treatment [1]. Improvements in glycemic control are seen 
well before any significant weight loss is produced and 
patients are often dismissed from the hospital with a 
marked decrease in their diabetes medications. The 
necessary calorie restriction that occurs as a result of post-
surgical anatomical changes also results in better glycemic 
control [2]; however this does not explain the known 
superiority of RYGB over equivalent caloric restriction in 
improving metabolic parameters, particularly glucose 
metabolism (Figure 2). Although it is clear that over the 
longer-term RYGB may be superior in terms of sustainable effects on fat loss and β-cell function, it remains 
uncertain to what extent CR contributes to the effect of bariatric surgery on glucose metabolism. 
Recently, we have demonstrated that CR and RYGB are both associated with a decrease in fasting 
endogenous glucose production (EGP) in people with type 2 diabetes (Figure 2). It is unclear if the mechanism 
by which this is achieved is shared by the two interventions.  
One newly established methodology to enable untargeted elucidation of the metabolic pathways that 
are altered by a given intervention is large-scale metabolomics. Metabolomics is used to profile the 
metabolome – defined as the range of endogenous small-molecule chemicals produced in an organism. 
Metabolomic profiling via mass spectroscopy in our comprehensive metabolomics center will enable 
quantitative assessment of the metabolomic changes that occur with CR and RYGB and elucidate changes 
that are shared or otherwise by these two interventions. Metabolomic profiling via mass spectroscopy is 
already enhancing the understanding of complex metabolic mechanisms and pathways involved in, for 
example, cardiovascular disease where it has aided with risk stratification and event prediction thus allowing 
care to be individualized [3]. The metabolomic changes produced by calorie restriction in overweight healthy 
volunteers have been shown to be associated with an improvement in insulin action [4]. RYGB also produces 
differences in the circulating metabolome, in particular decreases in branched chain amino acid and increases 
in certain metabolites which are of gut origin [5, 6]. In this application we provide preliminary data to show 
differences in the Amino Acid metabolome between RYGB and CR. 
The microbiome, or gut host environment, consists of approximately 104 cells and has been the subject 
of much interest as it relates to energy homeostasis [7]. It changes in response to food intake and surgical 
intervention. There is compelling evidence to suggest that changes in the gut microbiome after RYGB are 
associated with improved insulin sensitivity [8, 9]. Using 16S rRNA-encoding gene sequences we will 
characterize the structure and dynamics of complex microbial communities, specifically how they change with 
intervention and then subsequently how the microbiome influences the circulating metabolome.  
Whether the changes seen in the metabolome and microbiome are cause or consequence of the 
weight loss and resultant improvements in glucose homeostasis following Roux-en-Y gastric bypass has not 
been fully investigated and may reveal novel mechanisms in the study of glucose metabolism following weight 
loss surgery. The bioinformatics aspects of the Microbiome Program supported by the Mayo Clinic Center for 
Individualized Medicine (CIM) and the Metabolomic Core will be integral parts of this project. 
 
Results of this proposal will be used to support an application for extramural funding at the end of this 
funding period. 
Approach (Figure 1): Volunteers with type 2 diabetes and obesity who are scheduled to undergo RYGB as 
part of their clinical care will be studied immediately prior to surgery and at 12 weeks post-operatively using a 
labeled mixed meal to enable simultaneous measurement of glucose metabolism and insulin secretion and 


action [10]. Blood samples for targeted metabolic profiling will also be drawn. A fecal sample to characterize 
the microbiome will be collected at both visits.  A matched control group of subjects with type 2 diabetes and 
obesity who are not scheduled to undergo bariatric surgery will also be recruited and studied at baseline and 
12 weeks following caloric restriction similar to that required post-operatively after RYGB. We will utilize weekly 
meetings with a dietician and a psychologist to ensure compliance with the prescribed diet. 
The overall objective of this proposal is to form the basis of a line of investigation that will underpin my 
research career and future extramural funding. We will address the following specific aims: - 
 
Specific Aim 1: Characterize the changes in the circulating metabolome that occur after Roux-en-Y Gastric 
Bypass in contrast to caloric restriction alone. 
Hypothesis: Metabolomic changes after Roux-en-Y gastric bypass surgery are associated with a decrease in 
endogenous glucose production in subjects with type 2 diabetes. 
Specific Aim 2: Characterize the changes in gut microbiome in subjects with type 2 diabetes after Roux-en-Y 
gastric bypass, in contrast to caloric restriction alone. 
Hypothesis: Gut microbiome changes unique to Roux-en-Y gastric bypass influence remission of type 2 
diabetes. 
 
Preliminary Studies 
A.Mathematical modelling of glucose turnover. 
The meal minimal model can measure insulin 
secretion and action [11, 12] [13] and has been 
validated using hyperglycemic and euglycemic 
clamps as well as tracer infusions to directly 
measure glucose turnover. We have shown that 
insulin sensitivity (Si ) and β-cell responsiveness (φ) 
can be estimated from measured glucose and C-
peptide concentrations [12, 14]. The 
appropriateness of insulin secretion for the 
prevailing degree of insulin action is assessed by 
calculating the total disposition index (DItotal), which 
equals the product of insulin secretion and insulin 
sensitivity (φtotal x Si). 
 
B. Triple-tracer methodology for reliable 
measurement of postprandial glucose fluxes. The dual-isotope method 
pioneered by Steele et al. enabled simultaneous measurement of both the 
systemic rate of appearance of ingested glucose (Ra meal) and postprandial 
endogenous glucose production [15]. We developed and validated a novel 
triple-tracer approach to minimize changes in both meal and endogenous 
plasma tracer-to-tracee ratios thereby allowing simultaneous measurement 
of Ra meal and endogenous glucose production [10, 16]. We will use the 
validated triple-tracer method to measure glucose disappearance, 
endogenous glucose production and meal appearance in study subjects. 
 
C. Differences in the metabolomic profile in subjects after RYGB vs. CR. 
Targeted mass spectroscopy of the metabolomic profile in subjects after 
RYGB and CR showed clear differences in amino acid profiles between 
groups, despite comparable weight loss (Table 1). The changes shared by 
the two interventions were an increase in glycine and a decrease in alanine 
concentrations. Alanine is extracted by the liver and utilized in hepatic 
gluconeogenesis [17]; hence decreased concentrations may contribute to 
improvements in endogenous glucose production seen in both groups 
(Figure 2). Another interesting finding is the decrease in alpha aminoadipic 
acid concentration unique to the RYGB group. An elevated aminoadipic acid 
concentration is a robust biomarker for increased risk of developing diabetes 
[18].  
 
Research and Methods 


Study Subjects: Individuals aged 20-65 years seen for weight management in the Nutrition Clinic at the Mayo 
Clinic, will be recruited to the study prior to undergoing Roux-en-Y Gastric Bypass (RYGB). We will recruit 15 
subjects who have a fasting glucose concentration of ≥ 126 mg/dl on two or more occasions or who have a 
history of type 2 diabetes or impaired fasting glucose treated with one or two oral agents other than 
thiazolidinediones. Potential participants will attend the Mayo Clinic Clinical Research Trial Unit (CRTU) for a 
screening visit. In addition, we will also recruit 15 subjects with type 2 diabetes or impaired fasting glucose who 
are not interested in pursuing surgical intervention but are willing to undergo supervised caloric restriction as 
detailed below. The 2 groups will be matched for age, gender and BMI and duration and severity (HbA1c and 
no. of oral medications needed to achieve glycemic control) of diabetes.  
 
Exclusion Criteria: Subjects <20 years of age will not be studied to minimize the possibility of type 1 diabetes. 
Subjects > 65 years of age will not be studied to minimize the potential confounding effects of age on glucose 
tolerance. Subjects on chronic antibiotic therapy will be excluded due to their influence on gut bacteria. 
Subjects will refrain from using antibiotics for 2 weeks prior to the screening visit until completion of the study, 
except for routine peri-operative use and as clinically indicated. Healthy status will indicate that the participant 
has no known active systemic illness and no active microvascular or macrovascular complications of their 
diabetes.  
 
Screening Visit: Subjects will provide written informed consent and undergo a history and physical 
examination along with blood collection for complete blood count, fasting glucose, HbA1c and chemistry group; 
urine collection to exclude infection, proteinuria or pregnancy and ECG. Subjects will ensure that they have not 
taken any antibiotics for 2 weeks prior to the screening visit. The Minnesota Leisure-Time Physical Activity 
questionnaire will be administered to assess habitual activity levels [19]. They will be given instructions on 
managing their diabetes medications. Subjects will also complete the validated bowel disease questionnaire to 
ensure that there are no functional or organic bowel symptoms [20]. A receptacle to collect a baseline fecal 
sample prior to Study 1 will be provided to subjects. 
Body Composition: Body composition will be measured at the screening visit using dual-energy X-ray 
absorptiometry (iDXA scanner; GE, Wauwatosa, WI) and abdominal CT Scan with cuts at L2/3 and T11/12 to 
determine percent body fat and visceral fat [21].  
 
Experimental Design: Subjects will be studied on two occasions, the second study will occur approximately 
12 weeks after Roux-en-Y gastric bypass or equivalent caloric restriction. 
 
Study 1 (baseline): All subjects will be admitted to the Mayo Clinic CRTU at 17.00 hours the evening before 
the study. A fecal sample, produced within 48 hours of the start of the study, will be collected. Following 
ingestion of a standardized low calorie meal (197 Kcal: 40% carbohydrate, 30% fat, and 30% protein) subjects 
will fast overnight. The meal will occupy a volume that is tolerable to patients who have undergone restrictive 
upper gastrointestinal procedures. Approximately at  05:30 a cannula will be inserted retrogradely into a vein of 
the dorsum of the hand. This will be placed in a heated Plexiglas box maintained at 55oC to allow sampling of 
arterialized venous blood. A cannula will also be placed in the vein of the contralateral hand to allow infusion of 
tracers. Approximately at 06:30 hrs (-180 minutes) subjects will receive a primed continuous infusion of [6,6-
2H2] glucose. At the start of the study, approximately 09:30 hrs (0 minutes) subjects will consume a mixed meal 
consisting of one scrambled egg, 55g of ham), and Jell-O containing 35g of glucose enriched with [1-13C] 
glucose (to~4%) as previously described. An infusion of [6-3H] glucose will be started at the same time at a rate 
that mimics the anticipated glucose appearance of the [1-13C] glucose contained within the meal.  At the same 
time, the rate of infusion of the [6,6-2H2] glucose will be altered so as to approximate the anticipated pattern of 
fall in endogenous glucose production as previously described in detail [21-23].  Blood will be collected to allow 
measurement of glucose, tracer and hormone concentrations. At the end of the study (15:30, 360 minutes), the 
cannulae will be removed; participants will consume a late lunch and leave the CRTU.  
 
 
Intervention: Subjects participating in both arms of this study will be asked to consume an identical diet that is 
controlled for caloric content and macronutrient composition (Table 2). This is identical to that consumed by 
patients undergoing Roux-en-Y gastric bypass at the Mayo Clinic. Subjects in the surgery arm will start the diet 
after surgery, while subjects in the caloric restriction arm will do so after the screening visit. 


The diet consists of three small pureed meals (740kcal daily: 30% protein, 15% fat, 55% carbohydrate) per day 
for the first 4 weeks after surgery. In the group not undergoing surgery, compliance will be monitored in all 
participants by weekly weigh-ins and weekly meetings in the Division of Endocrinology or CRTU. A meeting 
with the dietician, to review the daily dietary record, will alternate with a meeting with the clinical psychologist to 
reinforce compliance techniques. Subjects will then progress to a soft diet consisting of 3 small meals of 
tender, easily-chewed pieces (each meal is approximately 1 cup of food), for an approximate daily intake of 
875 kcal. We have previously been able to accomplish this in subjects with type 2 diabetes (see Figure 2).  
 
 
 
 
 
 
 
 
Table 2: Post-RYGB surgery diet, weeks 0-12. 
 
 
Post-op (Wk)  
 
Time 
Liquid Intake 
Food Intake 
0-4 
7:30 to 8 am 
 
Scrambled egg (75kcal) 
¼ cup cooked cereal (40kcal) 
8:30 am to noon 
8 fl oz. skim milk (80kcal) + 
sugar-free instant breakfast mix 
(100kcal) 
8 fl oz. water 
 
12:30 to 1 pm 
 
¼ cup pureed lean meat (77kcal) 
¼ cup pureed vegetables (15kcal) 
1:30 to 5 pm 
8 fl oz. skim milk (80kcal) 
8 fl oz. water 
 
5:30 to 6pm 
 
¼ cup pureed lean meat (77kcal) 
¼ cup pureed vegetables (15kcal) 
6:30 to 9:30pm 
8 fl oz. skim milk (80kcal) + 
sugar-free instant breakfast mix 
(100kcal) 
8 fl oz. water 
 
Approximately 740kcal daily – 30% protein, 15% fat, 55% carbohydrate 
5-12 
7:30 to 8 am 
 
Scrambled egg (75kcal) 
½ cup cooked or dry cereal 
unsweetened (80kcal) 
¼ cup  unsweetened  canned  fruit 
(30kcal) 
8:30 am to noon 
8 fl oz. skim milk (80kcal) + 
sugar-free instant breakfast mix 
(100kcal) 
8 fl oz. water  
 
12:30 to 1 pm 
 
½ cup cottage cheese low fat  (110 
kcal)   
¼ cup cooked  vegetables (15kcal) 
¼ cup  unsweetened canned  fruit 
(30kcal) 
 
1:30 to 5 pm 
8 fl oz. skim milk (80kcal) 
8 fl oz. water  
 
5:30 to 6pm 
 
2 ounces of  lean meat (well 
chewed) (110 kcal) 
¼ cup mashed potatoes (40kcal) 
¼ cup cooked  vegetables (15kcal) 
¼ cup sugar free “lite”  yogurt 
(20kcal) 
6:30 to 9:30pm 
8 fl oz. skim milk (80kcal) 
8 fl oz. water  
 
Approximately 875kcal daily – 30% protein, 15% fat, 55% carbohydrate 


Study 2: This study will occur in the 12th postoperative (or equivalent) week to ensure that the participants 
have recovered, have adequate hemoglobin and are tolerating oral intake. The study will be identical to Study 
1. The same meal will be consumed in both Study 1 and 2.  Subjects will not resume antidiabetic medication in 
the 12 weeks after surgery. Body composition will be measured the week prior to study. Height and weight will 
be measured in the CRTU at the time of admission. Analytic Techniques: All analytic techniques for blood 
samples are either established in the applicant’s mentor’s laboratory or are routinely performed in the Mayo 
GCRC Mass Spectrometry, Immunochemical Core laboratories or GI imaging and Endoscopy core of the Mayo 
CTSA.  
 
Exendin-(9,39) Amide Study: 
Of the fifteen subjects undergoing Roux-en-Y Gastric Bypass (RYGB), we propose to study four of these 
subjects  in the 4th postoperative week, to ensure that the participants have recovered, have adequate 
hemoglobin (as determined by PI) and are tolerating oral intake. Subjects will not resume antidiabetic 
medication in the 4 weeks after surgery. The study will be identical to Study 1 and 2. The same meal meal will 
be consumed. Body composition will be measured using dual-energy X-ray absorptiometry (iDXA scanner; GE, 
Wauwatosa, WI), the week prior to study. Height and weight will be measured in the CRTU at the time of 
admission.  At  approximately 0930 (0 minutes) an infusion of exendin-(9,39) amide, an inhibitor of active GLP-
1 in humans, will be infused at 300pmol/kg/min in half of the study subjects ([24, 25]. The infusion will be 
continued till the end of the study, at 360 minutes. The remainder of the subjects will receive a saline infusion. 
Assignment of exendin-(9,39) amide or saline infusion wil be in a randomized fashion (1:1).   
Metabolomic analysis: A tandem mass spectrometer with stable isotope dilution for quantification will be used 
to define the metabolomic profile in both cohorts.  
Microbiome Laboratory.  The microbiome lab in the Medical Sciences Building at Mayo Clinic houses 2 
computers containing 32-core AMD processors and 64 GB of RAM on which the calculations and modeling 
proposed will be carried out. The DNA extraction and library preparation facility includes a FastPrep 24 
beadbeater, 2 PCR machines, a qrtPCR machine, a biosafety cabinet, automated and manual gel 
eletrophoresis, and all other equipment and reagents necessary for DNA extraction and 16S rDNA 
amplification. The Medical Genomics Facility houses 6 Illumina Hi-Seq, 2 Illumina Mi-Seq, Sanger, and Pac-
Bio sequencers. 
  
Calculations:  Insulin sensitivity (Si), β-cell responsivity indices and total disposition index (DItotal) will be 
calculated from plasma glucose and insulin concentrations using the “oral” glucose minimal model [26]. [6-3H] 
glucose will be used to trace the appearance of the ingested [1-13C] glucose and [6,6-2H2] glucose will be used 
to trace endogenous glucose production. As previously described, meal appearance will be calculated by 
multiplying the systemic rate of appearance of [1-13C] glucose by the [1-13C] glucose in the meal [10]. In 
addition to measurement of peak hormone concentrations, we will calculate area above basal for the first 2 
hours and for all 6 hours after meal ingestion using the trapezoidal rule as previously described [27]. 
Differences in the metabolomic profile between groups will be quantified. Differences in the microbiome profile 
between groups will be quantified. 
We will subsequently explore correlations between changes in glucose metabolism and multiple metabolites. 
The metabolome and microbiome analysis will be undertaken in close collaboration with the bioinformatics 
core (Dr. Surendra Dasari, Ph.D.) to examine qualitative and qualitative differences as well as undertake 
pathway analysis to potentially identify specific pathways affected uniquely by CR or RYGB.  
 
Statistical Analysis. We will examine between-group (RYGB vs. CR) differences from the baseline study to 
the post-intervention study (Study 2- Study 1). To do so we will utilize an analysis of covariance adjusting for 
relevant covariates (e.g. age, gender and change in BMI or lean body mass after surgical intervention) as 
previously described. We will primarily examine between group differences in:   
 
1. Endogenous Glucose Production: Based on data from previous studies [27-29], 15 subjects per group 
would provide approximately 80% power to detect a 25% difference in nadir EGP values  and 87% power 
to detect a 25% difference in integrated EGP values. 
2. Disposition Index: 15 subjects per group would provide approximately 80% power to detect an 
association of intervention type corresponding to a 35% difference in mean DI values relative to the overall 
mean [28, 30]. 
3. Alpha-Aminoadipic Acid: 15 subjects per group would provide approximately 90% power to detect a 15% 
difference in mean values. 
 


Interpretation. In the RYGB group, if the improvements in glucose metabolism are associated with a 
metabolomic signature that is unique, then we will accept our primary hypothesis that metabolomic changes 
after surgery are associated with a decrease in endogenous glucose production in subjects with type 2 
diabetes. We will be able to differentiate between changes in insulin secretion (φtotal), insulin action (Si) and DI 
to determine the mechanistic basis for the observed improvements. If the improvements in glucose metabolism 
are associated with a unique change in the gut microbiome, then we will accept our secondary hypothesis that 
unique changes in the gut microbiome influence remission of type 2 diabetes. We will specifically be looking for 
metabolomic markers of the gut microbiome, which have been shown to influence insulin sensitivity [8, 9] and 
energy harvest, such as short-chain fatty acids [31]. 
 
Limitations. Changes in the gut microbiome and circulating metabolome are influenced by dietary intake. Our 
study design is such that both groups will have similar, but not identical macronutrient composition of their 
meals. We will minimize differences by utilizing a registered dietician as well as a clinical psychologist to help 
ensure compliance with the prescribed diet and discourage food consumption outside of what is provided by 
the study.  
 
Future Directions. Identification of a unique metabolomic profile after RYGB will permit manipulation of 
concentrations of these substances in overweight subjects with type 2 diabetes to determine their effect on 
weight loss and glucose metabolism. I hope to use the results of this study to secure extramural funding at the 
end of this award and be able to continue working on the role of bariatric surgery in the amelioration of type 2 
diabetes. 
 
 Human Subjects 
Detailed Description:  Suitable volunteers will be asked to visit the CRTU on a total of 14 occasions, including 
screening. During the first or screening visit they will meet with a member of the study team and undergo a 
history and physical examination to ensure that they fulfill entry criteria. If eligible, they will be asked to undergo 
determination of body composition. Subjects will be admitted on the eve of the study day. After an overnight 
fast, a cannula will be placed to allow IV infusion. Infusions will be started. In addition a retrograde hand vein 
for blood draws will be placed. This hand will be placed in a Perspex hot-box heated to 55degC. Subsequently 
they will ingest a mixed meal and blood will be sampled several times (via the retrograde hand vein) to obtain 
arterialized venous samples. The study will be repeated once at twelve week intervals.  
Population: Subjects will be recruited from the Mayo Nutrition Clinic and from Olmsted County residents and 
surrounding counties, who have previously indicated a willingness to participate in research – and who meet 
entry criteria. The racial composition of the county is outlined in the table below (%) using data from the 2000 
population census. No children or prisoners will be recruited.  
 
 
American 
Indian or 
Alaskan 
Native 
Asian or 
Pacific 
Islander 
Black, not 
of Hispanic 
Origin 
Hispanic 
White, not 
of Hispanic 
Origin 
Other 
Total 
Female 
0.15 
2.8 
1.8 
1.5 
43.8 
0 
51.5 
Male 
0.15 
2.8 
1.8 
1.5 
43.7 
0 
48.5 
Total 
0.3 
5.6 
3.6 
3.0 
87.5 
0 
100.0 
 
Research Materials:  We will be obtaining blood samples to measure hormone and glucose concentrations in 
response to meal challenge.  
Recruitment of Subjects: Subjects will be recruited by means of advertisements placed within our institution 
and from the nutrition clinic. Potentially eligible patients in the Mayo Nutrition Clinic will be identified by the 
physicians caring for the patient, and if interested in participating will contact the research team. If insufficient 
subjects are recruited by this approach, advertisements will be taken out in local newspapers until recruitment 


is complete.  Eligible subjects will be invited to participate in the study and to come to the CRTU for a 
screening visit.  
 
The following are exclusion criteria;  
(a) Age < 20 (to avoid studying subjects who could have type 1 diabetes)  
(b) > 65 years of age will not be studied to minimize the potential confounding effects of age on 
glucose tolerance.. 
(c) For female subjects: positive pregnancy test at the time of enrollment or study visits 
(d) Previous treatment with thiazolidinediones. 
(e) chronic antibiotic therapy 
(f) active systemic illness  
(g)  active microvascular or macrovascular complications of their diabetes.  
 
 
Potential Risks: Blood sampling. Blood samples are collected by venipuncture for this study. Bruising can 
occur with venipuncture, as can fainting, etc. Risk Monitoring / Risk Reduction: The samples are collected 
using aseptic technique in designated venipuncture areas of the CRTU 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 investigators should they have 
unusual pain or discomfort from the venipuncture site. Blood drawn within an 12 week period will not exceed 
550mL (one pint).  
Vascular catheter placement.  Catheter insertion, intravenous infusion and blood withdrawal are associated 
with a small risk of phlebitis.  Risk Monitoring / Risk Reduction: This will be minimized by careful attention to 
sterile technique.  If phlebitis occurs, it will be treated conservatively with heat and when appropriate, with 
antibiotics.  The catheters will be cared for by experienced CRTU nurses in order to minimize the risk of these 
complications.  In all protocols, “arterialized – venous” blood will be obtained by placing a hand in which a 
catheter has been inserted in a heated box during the study.  The temperature inside the box is maintained at 
~55°C.  With prolonged exposure to continuous heat, there is a potential risk of local skin irritation or a minor 
burn.  If this occurs, it will be treated appropriately.  Catheter risks will be discussed with the volunteers prior to 
obtaining consent for the study. 
Tracer and Hormone Infusion. Tracers infusions carry a risk of allergic reactions, bruising, discomfort at the 
site of infusion, and infection. Risk Monitoring / Risk Reduction: All infusates are prepared by trained personnel 
in a laminar flow cabinet using aseptic technique. Infusions take place on the CRTU where facilities are 
available should untoward reactions occur. Exendin-(9,39)  (CS Bio, Menlo Park CA) will be prepared in a 
similar manner. 
Radiation. Subjects will be exposed to radiation in this study. Radioactive tracers will be used in the proposed 
studies. Lean body mass, percent body fat, and visceral adiposity will be measured at the time of screening 
using DEXA (dual energy x-ray absorptiometry) and CT-scan. Risk Monitoring / Risk Reduction: The lowest 
dose of radioactive tracers that can be reliably counted in plasma will be used. In all instances, the amount of 
radiation that a volunteer will receive will be well below levels that result in significant risk of harmful effects.  
Proposed radiation exposure will be reviewed by the Mayo Clinic Radiation Safety Board prior to initiation of 
any study. Women who could become pregnant will be required to have a negative pregnancy test prior to 
participation in each study utilizing radioactive tracers.  
Confidentiality. All studies expose participants to the psychosocial risks arising from any breach in 
confidentiality. The risks include anxiety, confusion, damage to family relationships or a compromised ability to 
obtain insurance or employment. These risks may not be confined to the individual but may extend to other 
family members. Risk Monitoring/Risk Reduction: The nature of the information obtained will be explained in 
detail to each participant   All information will be stored anonymously in the database and only the PI or one of 


his designates will have access to the data.  
 
Data Safety and Monitoring Plan. The ultimate goal of this application is to further our understanding the role 
of different therapies in the management of 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 an 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 
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. Genotyping results are examined for consistency between repeated genotypes of a given sample 
chosen at random. 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) 
three-monthly basis 
Adverse event/safety rates (injuries) 
Weekly 
Study data 
three-monthly basis 
Annual report 
Yearly for CRTU, 
IRB  
 
Informed Consent. Written informed consent will be obtained from all individuals who participate in the study. 
The principal investigator or member of study team,  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, as detailed it will advance our 
knowledge of how to treat diabetes and help make clinicians make rational therapeutic choices in the future.  


 
VI. Gender/Minority Mix 
The majority of residents in Rochester, Minnesota and surrounding counties are White, recent population 
estimates from over the past decade have indicated that minorities make up a significantly larger segment of 
our communities. According to census area data such cultural groups as the Somalis, Hispanic/ Latinos, and 
South East Asians have increased the percentage of minorities living in Rochester and the surrounding 
counties from 3% in 1990 to nearly 10% in 2000. Despite the increases in the number of minorities within rural 
Rochester, Minnesota, it is likely that recruitment will fall short in the area of minority participation. However, 
we are actively working with the minority outreach specialist in the Center for Patient Oriented Research and 
Tribal Elders from the local minority populations, to develop complementary community-based strategies for 
recruitment of minorities at Mayo Clinic Rochester. 
 
 
 
 
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