A Dose-response Study Examining the Contribution of GLP-1 Receptor Signaling to Glucagon-stimulated Insulin Secretion
Phase: PHASE3
Conditions: Healthy
Sponsor: Adrian Vella

 
 
 
 
 
A dose-response study examining the contribution of GLP-1 receptor signaling to Glucagon-stimulated 
insulin secretion 
 
Adrian Vella  
26th March 2020 
IRB# 20-003995 
NCT04459338 
 
 

Abstract  
  
Glucagon is an important insulin secretagogue1,2 in the concentrations found within the islet3. α-cell dysfunction 
as occurs in T2DM4,5, in prediabetes6,7 or in response to FFA elevation23 results in increased glucagon 
concentrations. In these conditions glucagon signals through the GLP-1 receptor (GLP1R) present on β-cells8 
but its net contribution to insulin secretion is unknown. In this experiment we will determine the contribution of 
GLP-1R signaling to glucagon-induced insulin secretion in vivo. To do so, we will use a dose-response 
experiment for glucagon-stimulated insulin secretion. The experimental conditions will then examine how 
glucagon-stimulated insulin secretion is modified by blockade of GLP1R blockade using exendin-9,39. This 
approach will complement other experiments using a glucagon bolus known to stimulate β-cell function21,22 in 
the presence and absence of GLP1R blockade. 
 
I. Hypothesis and Specific Aims 
 
Glucagon within the islet can signal the β-cell through GLP1R, and acts as an insulin secretagogue3,8. This 
signaling is blocked by exendin-9,399,10. The relative importance of glucagon signaling through its cognate 
receptor or through GLP1R is unknown. Despite the lower affinity of GLP1R for glucagon11, intra-islet 
concentrations of glucagon are sufficiently high to stimulate GLP1R3. The other situation where this may occur 
is in response to pharmacologic doses of glucagon as used for β-cell function testing1,2 or raising peripheral 
glucagon concentrations above fasting values5,12,13. The experiments proposed will characterize the role of 
GLP1R in glucagon’s actions on the β-cell and the potential therapeutic role of dual (GLP-1R and 
glucagon receptor11) agonists for the treatment of T2DM and obesity. The experiments will also provide an 
opportunity to test glucagon’s actions on insulin secretion, independently of glucose concentrations. 
 
We will therefore address the following specific aim: - 
 
Specific Aim: Determine the contribution of GLP1R to glucagon-stimulated insulin secretion.  
 
1° Hypothesis: Glucagon-stimulated insulin secretion is decreased by GLP1R blockade with exendin-9,39  
 
2° Hypotheses: The contribution of GLP1R to glucagon-stimulated insulin secretion is more significant at 
higher glucagon concentrations 
 
II. Background and Significance 
 
Diabetes mellitus causes morbidity, mortality and costs in excess of ~$170 billion/year14. The discovery of gut-
derived proglucagon peptides, most specifically GLP-115, has fueled therapeutic innovations for T2DM16. 
Proglucagon is transcribed and translated from the glucagon gene (GCG) but its final fate depends on 
concomitant expression of prohormone-convertase (PC) enzymes17. PC-1/3 is expressed in intestinal L-cells 
resulting in production of GLP-1. On the other hand, PC-2 is expressed in α-cells so that glucagon is the main 
derivative of proglucagon produced by these cells. However, PC-1/3 expression is inducible, suggesting that 
extra-intestinal synthesis and secretion of GLP-1 is possible18. IL-619, hyperglycemia20 and FFA21 seem to 
increase PC-1/3 expression in islets. This raises the possibility that pancreatic production of GLP-1 
modulates islet function in response to glucolipotoxicity and other metabolic stressors9. 
 
In pharmacologic concentrations, GLP-1 is a powerful insulin secretagogue. It also inhibits glucagon 
secretion22, delays gastrointestinal transit and decreases appetite23. Endogenous concentrations of active 
GLP-1 are much lower and transient24, partly due to the ubiquity of DPP-4. Considerable GLP-1 degradation 
occurs close to its site of production25 resulting in a circulating T1/2 ~1min26. This may explain why inhibition of 
GLP-1 actions at GLP1R using a competitive antagonist (exendin-9,39) has little effect on postprandial glucose 
metabolism27,28. Indeed, only one study to date has shown a post-prandial effect of exendin-9,39 in healthy 
humans29 and this can be explained by increased gastric volume prior to meal ingestion30,31. On the other 
hand, after bariatric surgery when endogenous GLP-1 concentrations are raised several-fold, inhibition of GLP-
1 action impairs insulin secretion and raises glucose concentrations slightly, but significantly28.  
 
However, the implication that endogenous GLP-1 is of little relevance outside of bariatric surgery, is at odds 
with the conservation of the GLP-1 sequence across vertebrates15. GLP-1 may also contribute to the ‘incretin 
effect’ – the potentiation of post-prandial insulin secretion by gut (as opposed to I.V.) nutrient delivery. Salehi et 
al. reported that endogenous GLP-1 enhances postprandial insulin secretion32. However, the experiment 

Figure 1: Effect of GLP-1 receptor blockade using exendin-9,39. At time 0, 
subjects received an intravenous glucose challenge. Exendin-9,39 raised post-
challenge glucose concentrations (also fasting in T2DM). In people without 
diabetes, β-cell function (A) is impaired but this is not apparent (B) in people 
with T2DM. On the other hand, in non-diabetic subjects there is no effect on 
glucagon (C) whereas in T2DM, glucagon is increased during fasting and the 
first 50 minutes after the glucose challenge (D). * p < 0.05. Non diabetic data 
from Sathananthan, M., et al. (2013) Diabetes 62(8): 2752-2756 
utilized a hyperglycemic clamp as opposed to the transient meal (or meal-like) challenges in our experiments 
(Fig.1)28,33. More germane to this proposal, they also observed an effect of GLP1R blockade on insulin 
secretion in response to intravenous glucose alone32. 
 
Infusion 
of 
exendin-9,39 
to 
block 
GLP1R during an intravenous glucose 
challenge (that mimics the systemic 
appearance of a meal) impairs β-cell 
function in non-diabetic humans with 
little 
effect 
on 
glucagon 
secretion 
(Fig.1a,c)33. 
Similar 
effects 
are 
observed in cadaveric islets from people 
without T2DM (Fig.2). Conversely, in 
people with T2DM (Fig.1b,d) or in islets 
isolated from diabetic humans (Fig.5), 
there is no effect of exendin-9,39 on β-
cell function, perhaps due to the degree 
of 
β-cell 
dysfunction. 
However, 
in 
T2DM, 
exendin-9,39 
raises 
fasting 
glucagon and impairs its suppression by 
intravenous glucose in vivo (Fig.4). Our 
preliminary data also suggests that 
GLP-1 contributes to islet function 
outside the postprandial period; the 
effects differ in T2DM compared to 
non-diabetic subjects (Fig.1).  
 
Although the source of fasting GLP-1 
secretion is uncertain, complementary 
experiments in isolated human islets 
(and 
the 
literature9,19,34,35) 
strongly 
suggest an islet source for GLP-1 
(Fig.2). O’Malley et al. have suggested 
that islet GLP-1 expression increases with the development of diabetes36. It is also notable that exposure of 
islets to FFA and hyperglycemia also increases GLP-118,37,38. Our published33 and unpublished (Fig.1) 
preliminary data, as well as genetic data39 shows that GLP-1 signaling through its cognate receptor in the 
fasting state (or during an intravenous glucose challenge) plays a  role in islet function. Understanding the 
physiologic role of GLP-1 secretion independently of oral intake in humans will allow us to better 
modulate this pathway and prevent / treat islet dysfunction in prediabetes and T2DM.  
 
The other consideration is that glucagon within the islet can signal the β-cell through GLP1R, and acts as an 
insulin secretagogue3,8. This signaling is blocked by exendin-9,399,10. The relative importance of glucagon 
signaling through its cognate receptor or through GLP1R is unknown. Despite the lower affinity of GLP1R for 
glucagon11, intra-islet concentrations of glucagon are sufficiently high to stimulate GLP1R3. The other situation 
where this may occur is in response to pharmacologic doses of glucagon as used for β-cell function testing1,2 or 
raising peripheral glucagon concentrations above fasting values5,12,13. The experiments proposed will 
characterize the role of GLP1R in glucagon’s actions on the β-cell and the potential therapeutic role of 
dual (GLP-1R and glucagon receptor11) agonists for the treatment of T2DM and obesity.  
 
Glucagon dysregulation is likely to be an earlier contributor to the pathogenesis of T2DM than has been 
appreciated40. More significantly, although I.V. administration of glucagon is used as a test of β-cell function2, 
its role as an insulin secretagogue within the islet has been overlooked3. Glucagon can signal the β-cell 
through GLP1R in addition to the glucagon receptor8 (and its action blocked by exendin-9,39)10. What role 
does this play in physiology and in T2DM? The experiments proposed will quantify the effects of exendin-9,39 
on glucagon-stimulated insulin secretion. Complementary experiments will also explore whether glucagon-
stimulated insulin secretion is altered by rs3765467, by T2DM and by acute FFA elevation to mimic metabolic 
stress. 
 
III. Preliminary Data 
   

We27,28 and others41-43 have shown that GLP-1 in the postprandial 
period has little effect on post-prandial insulin secretion outside of 
RYGB. Intriguingly, a variant in GLP1R that increases response to 
GLP-144 has been shown to decrease risk of T2DM and lower 
fasting glucose39,45. Also, DPP-4i which raise postprandial 
concentrations of active GLP-146 also lower fasting glucose 
concentrations, suggesting that fasting GLP-1 secretion alters islet 
function. The literature9,19,34,35 and our preliminary data (Fig.1,2), 
suggest a pancreatic source for GLP-1.  
 
T2DM, 
inflammation and 
metabolic 
stress 
increase 
islet 
expression of GLP-119, perhaps as an adaptive function to 
preserve α- and β-cell function. Blockade of GLP1R with exendin-
9,39 seems to have divergent effects in people with and without 
T2DM (Fig.4,7), perhaps because of the degree of β-cell 
dysfunction present (Fig.4,5).   In Specific Aim 2 (Fig.9) we will 
characterize differences (in people with and without T2DM) in islet 
function during fasting and hyperglycemic conditions attributable 
to endogenous GLP-1. We will also alter fasting FFA 
concentrations40 to examine the role of GLP-1 during metabolic 
stress in people without T2DM.  
 
Glucagon is a known insulin secretagogue1,2. Within islets, 
glucagon can act on the β-cell through GLP1R in addition to its 
cognate receptor3,10. The experiments will provide an opportunity 
to characterize the GLP1R contribution to glucagon-stimulated 
insulin secretion using a range of glucagon infusion rates.  
 
IV. Research Design and Methods 
 
Study Subjects: After approval 
from 
the 
Mayo 
Clinic 
Institutional Review Board, we 
will recruit 20 weight-stable, 
otherwise 
healthy 
subjects 
between the ages of 18-65,  
using 
local 
advertising 
and 
other 
methods 
employed 
previously40. Subjects will have 
no history of active disease or 
diabetes. Individuals who have 
expressed 
interest 
in 
participating in research will 
also be contacted and invited to 
participate 
in 
the 
study. 
Screening 
Visit: 
After 
an 
overnight fast of 
8 
hours, 
subjects will present to the 
Clinical Research Trials Unit 
(CRTU) at approximately 0700. 
Subjects will provide written, 
informed consent. To ensure 
they are otherwise healthy, 
subjects will undergo a history and limited physical examination; blood collection for complete blood count, 
fasting glucose, HbA1c, sodium, potassium, creatinine and urine collection to exclude pregnancy. An ECG will 
also be performed. The Minnesota Leisure-Time Physical Activity questionnaire will be used to assess habitual 
activity levels47. Body Composition: After informed written consent is obtained, body composition will be 
measured at the screening visit using dual-energy X-ray absorptiometry (iDXA scanner; GE, Wauwatosa, WI). 
All subjects will be instructed to follow a weight-maintenance diet (55% carbohydrate, 30% fat and 15% 
 
Figure 3: Experimental Design  
Variable glucose infusion labelled 
with 3-H3-glucose glucose to maintain 
glucose @ 160mg/dL
Fconstant(3-H3-glucose)
-180      -120       -60          0          60        120     180   
240        300
± Exendin-9,39 (300pmol/Kg/min)
Glucagon (ng/kg/min)
0.6
0.4
0.8
1.0
1.2
Figure 2: Acute GLP1R blockade with Ex-9,39 
in isolated human cadaveric islets (obtained 
from Prodo labs) from non-diabetic subjects 
(ND) decreased insulin secretion in response to 
hyperglycemia. This was not apparent in T2DM. 
Glucose-stimulated insulin secretion was 
assessed by static incubation of islets at low 
(4mM) and high (16mM) glucose ± 100nM Ex-
9,39. Insulin Stimulation Index represents fold 
change in insulin secretion at 16/4 mM glucose. 
*p<0.05 (n=3 technical repeats). 

protein) for at least 3 days prior to each study. Screening OGTT: At approximately 0900 subjects will ingest 
75g glucose. Arterialized venous blood will be sampled over 2 hrs48. Glucose values at 0 and 120 minutes will 
be used to categorize glucose tolerance status. Subjects who have had a DEXA or OGTT in the last 3 months 
as part of the Vella Lab research studies or another Mayo research group, and weight is stable (+) or (-) 5 
pounds, will not be retested and previously obtained values will be used.  
 
We will study 20 subjects a total of 2 times at least a week apart in random order. All subjects will be admitted 
to the Mayo Clinic CRTU at 1700 hours the evening before the study. Women who could become pregnant will 
have a urine collection for point of care pregnancy testing.  Following ingestion of a standard 10 kcal/kg 
caffeine-free meal, subjects will fast overnight. The following morning at approximately 0530 (-210 min), a 
dorsal hand vein will be cannulated and placed in a heated Plexiglas box maintained at 55°C to allow sampling 
of arterialized venous blood. The contralateral forearm vein will be cannulated for glucose, and hormone 
infusions. 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). 
Subsequently, the infusion will be varied so as to mimic the anticipated pattern of fall of EGP to minimize 
changes in Specific Activity49. At that time another glucose infusion, also labeled with [3-3H] glucose will 
commence and infusion rate varied so as to produce peripheral glucose concentrations of ~160mg/dL. A 
glucagon infusion at 0.4ng/Kg/min will start at 0900 (0 min), increasing by 0.2ng/kg/min every hour (Fig.3). 
Blood will be sampled till the end of the study (1400 – 300 min) when I.V. cannulas will be removed; 
participants will consume a late lunch and leave the CRTU. This will be referred to as the saline (Sal) day 
because 0.9% Saline will be infused between 0 and 300 min. On another study day, exendin-9,39 (Exe) will be 
infused at 300pmol/kg/min between 0 and 300 min. 
 
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 
collected in protease inhibitor-containing tubes (BD800, BD Franklin Lakes, NJ) and 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).  Total and Intact GLP-1 will be collected in protease inhibitor-containing tubes 
(BD800, BD Franklin Lakes, NJ) and measured using an ELISA (ALPCO Diagnostics, Salem, NH). [3-3H] 
glucose specific activity will be measured by liquid scintillation counting after deproteinization and passage 
over anion- and cation-exchange columns50. 
 
Calculations: In addition to fasting, peak and nadir hormone concentrations, we will calculate area above 
basal (AAB) or area under the curve (AUC) during the experiment using the trapezoidal rule as previously 
described50. Si, Insulin Secretion Rate (ISR) and β-Cell responsivity (Φ) indices will be calculated51 from the 
glucose, insulin and C-peptide concentrations observed during the experiments using the minimal model52, 
incorporating age-associated changes in C-peptide kinetics53. Φ is derived from the sum of φ1 (1st phase insulin 
secretion) and φ2 (2nd phase insulin secretion) as before44. Disposition index (DI) will express β-cell function as 
a function of the prevailing insulin action51. Glucose disappearance (Rd) will be calculated using steady state 
Steele equations54,55 after Specific activity is smoothed as previously described56. Endogenous glucose 
production (EGP) will be calculated as the difference between the tracer determined rate of glucose 
appearance and the glucose infusion rate. All infusion rates will be expressed as Kg per lean body mass.  
 
Statistical Analysis: A paired 2-tailed student t-test 
(parametric) or a Wilcoxon rank-sign test (non-
parametric) will be used to examine differences 
between Sal and Exe days for the 1° hypothesis 
(Table 3).  
 
3) Power Calculation: Please refer to Table 3.  
 
3) Interpretation: If glucagon stimulates insulin secretion by acting, at least partially, through GLP1R we would 
expect that exendin-9,39 will decrease the response to glucagon by blocking GLP1R. To test our 1° 
hypothesis, we will compare Φ during each glucagon infusion rate in the presence and absence of exendin-
9,39. Assuming that glucagon concentrations as encountered in the islet are necessary to stimulate insulin 
secretion via GLP1R, we expect differences to be more marked at the higher glucagon infusion rates (2° 
hypothesis). The experiments will be conducted in relatively lean, healthy and insulin sensitive humans. Under 
these experimental conditions, we expect EGP to be suppressed and insulin secretion stimulated by 
hyperglycemia and glucagon infusion. The rising infusion rates of glucagon will enable us to examine the 
Power Calculation  
Mean ± SD 
Detectable 
difference  
1° hypothesis (n = 20 - paired) 
Φ (10-9min-1) - hyperglycemia 
149 ± 45 
30 (20%) 
Table 1: Means ± SD used to estimate power  

effects of the latter on endogenous glucose production (EGP) and endogenous insulin secretion in the 
presence and absence of GLP1R blockade. 

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