The Effect of the Interaction of Glucagon and Insulin on Endogenous Glucose Production in Humans
Phase: PHASE3
Conditions: Healthy
Sponsor: Adrian Vella

Title 
The Effect of the Interaction of Glucagon and Insulin on Endogenous 
Glucose Production in Humans 
 
 
 
Protocol Number: 20-001570 
Principal Investigator: Adrian Vella MD 
IND Sponsor: Adrian Vella MD, # 116569 (Somatostatin) 
Funded by: Intramural Funds  
Version Number:  2 
January 13, 2021 
NCT: 04461015 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Specific Aim: Determine the relative contribution of changes in insulin and glucagon 
concentration on endogenous glucose production in nondiabetic subjects.  
1° Hypothesis: Hyperinsulinemia restrains the effect of rising glucagon concentrations on 
endogenous glucose production 
2° Hypothesis: The % change in endogenous glucose production in response to rising 
glucagon concentrations is unchanged by hyperinsulinemia  
Rationale: The experimental design we propose will enable the dissection of the relative 
contribution of insulin and glucagon concentrations to endogenous glucose production. 
Acquisition of this data will allow us to develop a model of insulin and glucagon contributions to 
postprandial glucose metabolism (1; 2).  
A.  Significance   
Diabetes mellitus causes morbidity, mortality and costs in excess of ~$170 billion/year(3). 
Therefore preventing diabetes is important. T2DM is characterized by defects in insulin 
secretion & action(4), and impaired postprandial suppression of glucagon(1; 2). Recently, we 
have shown that diabetes-associated variation in TCF7L2 impairs postprandial suppression of 
glucagon secretion – an aspect of islet function that has been overlooked in prior genotype-
phenotype correlation studies and in the pathogenesis of prediabetes(5). This indicates that the 
abnormal regulation of postprandial glucagon secretion and glucose tolerance in the TT 
subjects cannot be explained by alterations in β-cell function, in keeping with recent reports 
where glucagon secretion is independent of β-cell function(6). Taken together, these data imply 
that to accurately quantify the ability of the pancreas to lower glucose concentrations in the 
post-prandial period, we have to account for both β-cell and α-cell function. The oral minimal 
model uses knowledge of C-peptide kinetics to measure insulin secretion and quantify β-cell 
function. In a prior series of experiments (IRB # 17-006493), we quantified the contribution of 
glucagon suppression to endogenous glucose production in the setting of changing insulin 
concentrations. This experiment will provide information of how changing glucagon 
concentrations alter endogenous glucose production when insulin concentrations are constant. 
 
B. Preliminary Data 
 
Figure 1: Prior experiment with manipulation of insulin (0.6, 0.8 and 1.0 of an insulin profile) and glucagon 

1. Glucagon alters postprandial glucose metabolism (2). 
2. We have expertise in the design and conduct of clamp experiments (7; 8). 
3. We have experience with the use of isotopes to measure glucose turnover (9). 
4. Previously we utilized data from similar in vivo experiments to develop novel models of 
insulin secretion (10), hepatic insulin extraction (11) and the effect of Glucagon-Like 
Peptide-1 (GLP-1) on insulin secretion (12). 
5. Data from IRB # 17-006493 demonstrate that with decreasing postprandial insulin 
concentrations, the effect of a lack of glucagon suppression on endogenous glucose 
production becomes more marked (Fig.1,2) 
 
C. Research Design and Methods 
 
Study Subjects: We will recruit 15 otherwise healthy subjects using intramural and local 
extramural advertising after approval from the Mayo Clinic Institutional Review Board. 
Individuals who express interest in participating will be invited for a screening visit. Individuals 
with a BMI < 19 or > 40 kg/m2 will be excluded from the study to avoid potential confounding 
effects that may result from extreme leanness or from obesity. Subjects will have no known 
systemic illness, taking any medication that could affect glucose metabolism and no history of 
upper gastrointestinal surgery.  
 
Exclusion Criteria: Subjects < 18 years of age or > 60 years of age will not be studied to 
minimize the potential confounding effects of age on glucagon and insulin action. The subjects 
will not be taking medications that affect glucose metabolism (to be determined by PI) and have 
no history of chronic illness or upper gastrointestinal surgery.  We are seeking to recruit subjects 
who do not have diabetes (fasting glucose <100mg/dL). 
 
Screening Visit: To ensure subjects are healthy, following written, informed consent, subjects 
will undergo a history and physical examination, vital signs, height, weight, blood collection 
(after an 8 hour fast) for complete blood count, creatinine, sodium, potassium, fasting glucose 
and urine collection for point of care pregnancy testing for females able to become pregnant. 
Subjects will be instructed to consume a weight maintenance diet (55% carbohydrate, 30% fat 
and 15% protein, caffeine free) for at least 3 days prior to each study visit. Body Composition: 
Body composition will be measured prior to participation using dual-energy X-ray absorptiometry 
(iDXA scanner; GE, Wauwatosa, WI).  
 
 
Figure 2: The glucose concentrations resulting from the interaction between a lack of glucagon suppression and 
progressive decreases in insulin secretion 

 
Figure 3: experimental design  
Fasting
Variable glucose infusion labelled with 3-H3-
glucose to maintain glucose @ ~95mg/dL
Fconstant(3-H3-glucose)
-180      
0 
90                    180                   270  
Somatostatin (60ng/Kg/min)
Glucagon
0.70ng/Kg/min
0.35ng/Kg/min
0
Insulin @ 0.4 or 0.8 mU/Kg/min
Experimental Design: Volunteers will participate in a total of 2 protocols. The overnight studies 
will be performed at least a week apart in random order.  
 
Study A (Insulin 
0.4mU/Kg/min): Subjects will 
be admitted to the CRTU at 
approximately 1700 on the day 
prior to study. They will then 
consume a standard 10kcal/kg 
meal (55% carbohydrate, 30% 
fat, 15% protein, caffeine free) 
and fast overnight. Blood will 
then be sampled for baseline 
enrichment and 2H20 (1.67g/kg 
of body water) will be given in 3 
divided doses at 2200, 2400 and 
0200 (13). The following 
morning (approximately 0530), a 
forearm vein will be cannulated 
to allow infusions to be 
performed. In addition, a 
cannula will be inserted retrogradely into a vein of the contra-lateral dorsum of the hand. This 
will be placed in a heated Plexiglas box maintained at around 120oF to allow sampling of 
arterialized venous blood. At approximately 0600 (-180 min), a primed, (10µCi prime, 
0.1µCi/min continuous) infusion containing trace amounts of glucose labeled with [3-3H] glucose 
will be started and continued till 0900 (0 min).  
At 0900 (0 min), the infusion will be varied so as to mimic the anticipated pattern of fall of 
EGP.  In addition, glucose also labeled with [3-3H] glucose will be infused so as to maintain 
glucose concentrations at ~ 95mg/dL. Peripheral venous glucose concentrations will be 
measured every 10 minutes to allow the infusion rate of glucose to be adjusted as necessary.  
Simultaneously, an infusion of somatostatin (60ng/kg/min) will be started  at time 0 to 
inhibit endogenous islet secretion and therefore ensure identical portal insulin concentrations on 
the two study days(8). Insulin will be infused at a constant rate known to produce ~ 50% 
suppression of EGP (0.4mU/Kg/min).  
From 0900 (0 min) to 1030 (90 min) no glucagon will be infused. Subsequently, a 
glucagon infusion will commence (91 – 180 min) at 0.35 ng/Kg/min and then increase to 0.70 
ng/Kg/min (181- 270 min), a rate which will be maintained till the end of the study (1330).  
 
Study B (0.8mU/Kg/min): Approximately 1-2 weeks after the first study visit, subjects will be 
asked to return to the CRTU. This study visit will be similar to Study A. However, insulin will be 
infused at 0.8mU/Kg/min 0 to 270 minutes. 
 
.Analytical Techniques: All blood will be immediately placed on ice, centrifuged at 4oC, 
separated and stored at -80oC until assay. Glucose will be measured using a Yellow Springs 
glucose analyzer. Glucagon will be measured using an ELISA (Mercodia, Winston-Salem, NC). 
C-peptide will be measured using EMD Millipore (Billerica, MA) reagents.  Insulin will be 
measured using a chemiluminescence assay with reagents obtained from Beckman (Access 
Assay, Beckman, Chaska, MN). [3-3H] glucose specific activity will be measured by liquid 
scintillation counting following deproteinization(14).  Endogenous glucose production (EGP) and 
glucose disposal (Rd) will be measured as before(9; 15). 

 
Mean ± SD 
Detectable 
difference (0.4 vs. 
0.8 insulin) 
Detectable 
difference within 
each sub-group  
Nadir EGP (µmol/kg/min) 
3.9 ± 1.1 
0.4 (10%) 
0.7 (18%) 
AUC EGP (µmol/kg per 5hr) 
2816 ± 182 
67 (2%) 
120 (4%) 
Table 1: Pooled Means ± SD used to estimate power.  (Data from Ref. 1) 
 
Calculations: Glucose appearance and disappearance will be calculated using the steady-state 
equations of Steele et al., in which the actual tracer infusion rate for each interval is used in the 
calculation. The volume of distribution of glucose will be assumed to equal 200 ml/kg, and the 
pool correction factor assumed to be 0.65(16). Endogenous glucose production will be 
determined by subtracting the glucose infusion rate from the tracer-determined rate of glucose 
appearance(9). All rates of infusion and turnover will expressed per Kg/lean body mass. 
Statistical 
Analysis: A 
paired, two 
way student t-
test 
(parametric) 
or a Wilcoxon 
matched-pairs 
signed rank test (non-parametric) will be used to examine changes of EGP at the various time 
points of each study day. Differences between the 3 glucagon infusion rates will be assessed 
using a repeated-measures ANOVA which assumes the absence of a Gaussian distribution 
(Friedman test). Subsequently, in the presence of significant between group differences (p < 
0.05), a post-hoc Student’s t-test will used to examine differences between the 3 infusion rates 
of glucagon. If values are not normally distributed, a Dunn’s test will be used instead. 
 
Power calculation: Assuming similar variation to that previously observed(1), 60 subjects 
would provide approximately 80% power (at a 2-sided 0.05 α level), to detect small, biologically 
significant, differences in peak glucose, glucose area above basal and EGP in the presence and 
absence of glucagon suppression   (Table 1, Col 3) as would 20 per subgroup  (Table 1, Col 
4).   
 
D. Interpretation 
 
We will accept our 1° hypothesis if endogenous glucose production (EGP) is higher during the 
0.4 vs. the 0.8 study day. This would imply that despite rising glucagon concentrations higher 
insulin concentrations can still restrain EGP. It also will allow us to quantify the fold changes in 
EGP at low or high insulin concentrations. 
 
To test our 2° hypothesis we will express EGP at 80, 180 and 270 minutes as a percentage of 
fasting EGP (0 min).  The relative changes with increments in glucagon will demonstrate 
whether the relationship is linear or asymptotic. Comparison of the two study days will enable us 
to determine the effect of insulin on this relationship. We will accept our 2° hypothesis if % 
suppression at the 80, 180 and 270 minute time points does not differ between study days.  
 
We will calculate changes in nadir and integrated rates of EGP, as previously described(17-20), 
to compare suppression between the two study days. We will also undertake exploratory 
analysis of the rates of glucose disappearance (Rd) to determine if differences in glucagon 
concentrations affect Rd as well as EGP.  
 
Limitations / Precautions/ Additional or Alternative Analyses:  
i. Inhibition of glucagon secretion by somatostatin lowers portal concentrations. In the case of 
glucagon, an infusion that maintains peripheral concentrations of glucagon constant likely will 
result in a ~10% decrease in portal glucagon concentrations(21-23). However, in the current 
experimental design, the portal glucagon concentrations on the two study days will not differ. 
 
E. Human Subjects 

Detailed Description:  Suitable volunteers will be asked to visit the CRTU on a total of 3 
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. After an 
overnight fast, a cannula will be placed to allow IV infusion. Infusions will be started on the study 
days. In addition a retrograde hand vein for blood draws will be placed. This hand will be placed 
in a Perspex hot-box heated to about 120 oF.  Blood will be sampled several times (via the 
retrograde hand vein) to obtain arterialized venous samples.   
 
Population: Subjects will be recruited using intramural and local extramural advertising after 
approval from the Mayo Clinic Institutional Review Board. 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.  
 
Recruitment of Subjects: Subjects will be recruited using IRB-approved advertising.  Eligible 
subjects who express interest in participating in the study will be invited to the CRTU for a 
screening visit.  
 
The following are exclusion criteria:  
 
• 
Age <18 or >40 
• 
BMI < 19 or > 28 kg/m2 
• 
Medication that can affect glucose metabolism (determined by PI) 
• 
Any chronic illness 
• 
Upper GI surgery 
• 
History of diabetes 
• 
FBG >100 mg/dL 
• 
For female subjects a positive pregnancy test 
 
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 a 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. 
  
Radiation. Subjects will be exposed to radiation in this study. Radioactive tracers will be used in 
the proposed studies. Lean body mass and percent body fat will be measured at the time of 
screening using DEXA (dual energy x-ray absorptiometry). 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. The Mayo CRTU body composition core has a QDR4500 with fan 
scan technology which allows body composition to be performed in only a few minutes. 
 
Deuterated Water. Deuterated water will be given in 3 divided doses at 2200, 2400 and 0200. 
Risk Monitoring / Risk Reduction: Deuterated water is sometimes associated with dizziness and 
vertigo which is reduced by giving the tracer in divided doses. The fact that it is given late at 
night while the patient is in bed will reduce the risk of falls. Prior experiments using deuterated 
water have suggested that the occurrence of this uncommon. 
 
Infusion of Tracers, Hormones. All 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 (under the supervision of a research pharmacist) in a laminar 
flow cabinet using aseptic technique. Infusions take place on the CRTU where facilities are 
available should untoward reactions occur. Somatostatin infusion will be undertaken under the 
auspices of an IND obtained from the FDA. Somatostatin use can be associated with nausea 
and hypotension. In prior experience these effects are minor and disappear within minutes of 
cessation of somatostatin infusion. Insulin can lower the levels of glucose in the blood and 
cause symptoms of hypoglycemia. Plasma glucose will be monitored at regular intervals during 
study visits. Vital signs will be monitored hourly as per our standard practice during infusions in 
the CRTU.    
 
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) 
Weekly 
Adverse event/safety rates (injuries) 
Weekly 
Study data 
Weekly 
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 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, as detailed it will 
advance our knowledge of how to treat diabetes and help make clinicians make rational 
therapeutic choices in the future.  

 
F. 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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