To Determine the Effect of Endogenous GLP-1 Secretion on Islet Function in People With and Without Type 2 Diabetes
Phase: PHASE3
Conditions: Healthy, Type 2 Diabetes
Sponsor: Adrian Vella

 
 
 
 
To determine the effect of endogenous GLP-1 secretion on islet function in people with and without 
Type 2 Diabetes 
 
 
Adrian Vella MD 
 
IRB #: 20-003993 
 
NCT: 04466618  
 
V3 1/10/2022 
 

Abstract 
  
Post-translational processing of proglucagon in enteroendocrine cells yields a highly conserved peptide 
hormone, Glucagon-Like Peptide-1 (GLP-1)1,2. Although GLP-1-based therapy is now used to treat type 2 
diabetes (T2DM) and obesity3, important questions about its physiological role remain4.  It has a t1/2 ~ 1 minute 
in the circulation due to rapid degradation by a ubiquitous peptidase – Di-Peptidyl Peptidase-4 (DPP-4)5 and 
indeed, a significant proportion of endogenous GLP-1 is degraded close to the site of production6. Competitive 
antagonism of the GLP-1 receptor (GLP1R) with exendin-9,39 has little effect on postprandial insulin secretion 
– unless post-prandial GLP-1 concentrations are markedly elevated7. These conditions are typically only fulfilled 
after Roux-en-Y Gastric Bypass (RYGB)8 and, to a lesser extent sleeve gastrectomy9. Nevertheless, data from 
our lab10,11 and others12 could suggest a physiologic role for GLP-1 secretion beyond the postprandial state.  
 
Accumulating evidence suggests that in rodents13,14 and humans15 GLP-1 is synthesized within islets and may 
act locally in a paracrine fashion. Indeed, mice with genetic loss of intra-islet GLP-1 exhibit decreased insulin 
secretion and impaired response to metabolic stressors14. ‘Pancreatic’ GLP-1 may contribute to the effects of 
DPP-4 inhibitors in rodents16,17 and humans. Antagonism of GLP1R with exendin-9,39 during fasting impairs the 
islet cell response to an I.V. glucose challenge11,12. Islet GLP-1 content is increased in T2DM and in islets from 
non-diabetic humans exposed to hyperglycemia and Free Fatty Acids (FFA). These observations imply that 
paracrine GLP-1 secretion supports islet function in the presence of glucolipotoxicity13. We will examine the role 
of endogenous GLP-1 secretion in people with and without T2DM and during β-cell stress induced by FFA 
elevation. 
  
I. Hypothesis and Specific Aims 
 
Defects in α-cell18 and β-cell function19 are central to the pathogenesis of T2DM and to the progression from 
prediabetes through to T2DM20. There is accumulating evidence that α-cells can produce GLP-1 which in 
pharmacologic doses has potent anti-diabetic effects4. It has been hypothesized that this ‘pancreatic’ GLP-1 
acts in a paracrine fashion to support islet function – at least early in the evolution of prediabetes to diabetes13. 
Indeed, the enzymatic machinery necessary for the production of GLP-1 from proglucagon is expressed in 
response to inflammation and caloric excess14,21. Islet GLP-1 content is increased by T2DM in humans. 
Moreover, our preliminary data, as well as recent genetic data22,23 suggest that even in the absence of an oral 
challenge, GLP-1 receptor signaling can alter islet function. This may identify a therapeutic pathway to prevent 
or reverse islet dysfunction in T2DM. 
 
Intra-islet expression of GLP-1 seems to be enhanced in T2DM13. Certainly, in rodent models and human islets 
(Fig.2,3) exposure to substrate excess or inflammation increases the presence of GLP-1 within the islet14,24. 
Perhaps as islet cells dedifferentiate or transdifferentiate25,26 in T2DM, GLP-1 production increases. Whether 
this is an adaptive or maladaptive response is uncertain. We will compare the effects of GLP1R blockade in 
people with and without T2DM (Fig.5). An acute rise in FFA decreases β-cell function (as quantified by 
Disposition Index) in humans27. In addition, fasting and nadir post-prandial glucagon concentrations are 
increased by FFA elevation18.  It is unknown if ‘pancreatic’ GLP-1 helps dampen these deleterious effects on 
islet function and if this applies equally to α-cell and β-cell function (Fig.4). By comparing the effect of FFA 
elevation on islet function in the presence and absence of GLP-1R blockade in people without T2DM, the 
experiments will help determine the role of GLP-1 in protecting islet function during metabolic stress (as 
recreated by FFA elevation). 
 
We will therefore address the following specific aim: - 
 
Specific Aim: Determine the effect of endogenous GLP-1 secretion on islet function in people with T2DM.  
 
1° Hypothesis: GLP1R blockade with exendin-9,39 raises fasting glucagon secretion in people with T2DM  
 
2° Hypotheses: (i) GLP1R blockade with exendin-9,39 impairs α-cell suppression by hyperglycemia to a 
greater extent in people with T2DM (ii) GLP1R blockade with exendin-9,39 impairs β-cell 
responses to hyperglycemia to a lesser extent in people with T2DM (iii) FFA elevation 
exacerbates the effects of GLP1R blockade with exendin-9,39 on β-cell function 
 
   
 

II. Background and Significance 
   
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.1)11. Similar effects are observed in cadaveric islets from people without T2DM (Fig.3). Conversely, in 
people with T2DM (Fig.1) or in islets isolated from diabetic humans (Fig.2), 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.1). 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). In (slight) contrast to our findings Salehi et al. observed an 
effect of exendin-9,39 on β-cell function in T2DM. This may be due to the small sample size of our preliminary 
data or to differences in baseline β-cell function of each cohort12. 
 
Although the source of fasting GLP-1 
secretion is uncertain, complementary 
experiments in isolated human islets 
(and 
the 
literature14,16,17,24) 
strongly 
suggest an islet source for GLP-1. 
O’Malley et al. have suggested that islet 
GLP-1 expression increases with the 
development of diabetes13. This is borne 
out by our data in human islets 
documenting increased GLP-1 in T2DM. 
It is also notable that exposure of islets 
to 
FFA 
and 
hyperglycemia 
also 
increases GLP-1, in keeping with prior 
reports28-30. 
Our 
published11 
and 
unpublished preliminary data, as well as 
genetic 
data23 
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.  
 
Because the study of the GLP-1 system 
can be confounded by a lack of antibody 
specificity31, investigators have made use of genetic models or of GLP1R blockade with exendin-9,39. Chambers 
et al. used selective reintroduction of glucagon gene (Gcg) expression in the intestine versus the pancreas of 
Gcg-/- mice32. They reported that exendin-9, 39 infusion after selective reactivation of pancreatic Gcg expression 
impaired the response to intraperitoneal (i.e. not delivered via the intestine) glucose32. In contrast, exendin-9,39 
had no effect on the insulin response to glucose after selective reactivation of intestinal Gcg expression32. To 
ensure that these experimental results were not influenced by the α-cell hyperplasia present in Gcg-/- mice – 
conditions that favor GLP-1 production in dedifferentiated α-cells33 – two new lines of mice with the elimination 
of Gcg in the distal gut or throughout the small and large intestine were developed. These were used to re-
examine the importance of glucagon-derived peptides in the gut and in the pancreas. Using these models, 
without α-cell hyperplasia, the same investigators suggest that while gut GLP-1 is a contributor to glucose 
homeostasis in healthy mice without α-cell hyperplasia, detectable GLP-1 is present in mouse and human 
pancreas17. This led Hutch et al. to conclude that the response to DPP-4i is dependent on pancreatic production 
of GLP-1 and also (in the presence of an oral challenge) of intestinal Glucose-dependent Insulinotropic 
Polypeptide (GIP)16. 
 
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 

In animal models where GLP-1 signaling is impeded, GIP secretion 
in response to an oral (not I.V.) challenge increases to 
compensate16,32,34. Understanding the physiologic role of GIP 
(unlike GLP-1) has been hampered by the lack of a competitive 
antagonist useful in humans. Gasbjerg et al. have recently 
described such a compound but as yet it has not been used by 
other investigators35. However, the insulinotropic effect of GIP 
seems to be lost in T2DM36. In addition, although it is possible that 
intestinal GIP compensates for GLP1R blockade, a negative effect 
of exendin-9,39 is still observed in non-diabetic human islets and 
humans (± T2DM) exposed to glucose – given intravenously in the 
case of the in vivo experiments11,12. 
 
Intra-islet expression of GLP-1 seems to be enhanced in 
T2DM13. Certainly, in rodent models and human islets exposure to 
substrate excess or inflammation increases the presence of GLP-
1 within the islet14,24. Perhaps as islet cells dedifferentiate or 
transdifferentiate25,26 in T2DM, GLP-1 production increases. 
Whether this is an adaptive or maladaptive response is uncertain. 
We will compare the effects of GLP1R blockade in people with and 
without T2DM. An acute rise in FFA decreases β-cell function (as 
quantified by Disposition Index) in humans27. In addition, fasting 
and nadir post-prandial glucagon concentrations are increased by 
FFA elevation18.  It is unknown if ‘pancreatic’ GLP-1 helps dampen 
these deleterious effects on islet function and if this applies equally 
to α-cell and β-cell function. By comparing the effect of FFA 
elevation on islet function in the presence and absence of GLP-1R blockade in people without T2DM, the 
experiments will help determine the role of GLP-1 in protecting islet function during metabolic stress (as recreated 
by FFA elevation).  
 
 III. Preliminary Data 
 
The rationale and rigor of our hypotheses are supported by our Data (In addition to Fig.1-3): -  
 
1. Manipulation of FFA concentrations; measurement of β-cell function and of glucose metabolism: We 
have experience in manipulating FFA27,37 to alter β-cell function. We have extensive experience with tracer 
methodology38-41 and with deconvolution of insulin 
secretion from peripheral C-peptide concentrations42-
44. We also have extensive experience7,45-48 with the 
application of the minimal model49 to experimental 
data.  
 
2. Use of exendin-9,39 and GLP-1: We have used 
these peptides extensively as attested by our 
preliminary data and publication record7,11,39-41,50-52.  
 
IV. Research Design and Methods 
 
Subjects: After approval from the Mayo Clinic 
Institutional Review Board, we will recruit 20 weight-
stable, subjects with T2DM managed with diet or with 
metformin alone using local advertising, EHR search 
tools and other methods employed previously27. 
Subjects will have no history of microvascular or 
macrovascular complications of T2DM. Individuals 
who have expressed interest in participating in 
research will also be contacted and invited to 
participate in the study. In addition, we will recruit 20 
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). 
Figure 3: Human islets exhibit GLP-1 immunoreactivity. (Left 
panel). Images show examples of sections from cadaveric 
pancreata from 2 patients with T2DM stained with a GLP1 
specific antibody and counterstained with insulin. (Right panel) 
In addition, total islet GLP1 content (measured by GLP-1-
specific ELISA) is increased in human islets with T2DM 
compared to non-diabetic (ND), (n=4 repeats). Islet GLP-1 
content is also increased after 72 hours incubation in 
hyperglycemic (7 and 11 mM glucose) conditions + FFA (0.5 
mM palmitate). *p<0.05 for all conditions  vs. ND islets. 
Insulin
GLP-1

age- weight- and sex-matched subjects without diabetes using similar methods. Subjects <18 years of age or 
>65 years of age will not be studied. 
 
Inclusion criteria – non-diabetic subjects. 
We will recruit up to 20 weight-stable, non-diabetic subjects using the methods described above.  
 
The following are Exclusion Criteria: -   
 
(a) Age < 25 or > 65 years (to avoid studying subjects who could have latent type 1 diabetes, or the effects 
of age extremes in subjects with normal or impaired fasting glucose).  
(b) HbA1c ≥ 6.5% 
(c) Use of glucose-lowering agents.  
(d) For female subjects: positive pregnancy test at the time of enrollment or study  
(e) History of prior upper abdominal surgery such as adjustable gastric banding, pyloroplasty and vagotomy.  
(f) Active systemic illness or malignancy. 
(g) Symptomatic macrovascular or microvascular disease. 
 
Subsequently, subjects will then undergo an Oral Glucose Tolerance Test to ensure that they have a fasting 
glucose ≤ 125mg/dl and a two-hour glucose ≤ 200mg/dl. 
 
Inclusion criteria – diabetic subjects. 
We will recruit up to 20 weight-stable, diabetic subjects treated with diet and lifestyle alone or with metformin 
monotherapy using the methods described above.   
 
The following are Exclusion Criteria: -   
 
(a) Age < 25 or > 65 years (to avoid studying subjects who could have latent type 1 diabetes, or the effects 
of age extremes in subjects with normal or impaired fasting glucose).  
(b) Use of any glucose-lowering agent other than metformin.  
(c) 2 or more fasting glucose values > 250mg/dl on medication or after medication withdrawal. 
(d) Unwillingness or inability to withdraw medication for three weeks prior to, and for the duration of the 
study. 
(e) For female subjects: positive pregnancy test at the time of enrollment or study  
(f) History of prior upper abdominal surgery such as adjustable gastric banding, pyloroplasty and vagotomy.  
(g) Active systemic illness or malignancy. 
(h) Symptomatic macrovascular or microvascular disease. 
 
The two groups will be matched for age, sex and weight. 
 
 

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 levels53. 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% protein) 
for at least 3 days prior to each study. Screening 
OGTT:  At approximately 0900 subjects without T2DM will ingest 75g glucose. Subjects with T2DM will not 
undergo OGTT. Arterialized venous blood will be sampled over 2 hrs54. 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 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 
 
Medication withdrawal: Participants (with T2DM) who are taking metformin will be asked to discontinue 
medication for three weeks before and then continue off medication for the duration of the study. While off 
medication, they will be asked to self-monitor their fasting glucose at least twice daily. Values consistently > 
250mg/dL will result in discontinuation of their participation in the study. 
 
Experimental Design (Fig.4): A total of 40 subjects (20 with T2DM and 20 age- weight- and sex-matched 
subjects without diabetes) will undergo 2 experiments similar to those described in Specific Aim 1 in random 
order. The two study days will be similar to the Sal and Exe days outlined in Specific Aim 1. The nondiabetic 
subjects will undergo two additional study days where at approximately 0600 (-180 min) an additional infusion of 
Intralipid (20%, 0.011ml/kg/min; Baxter, Healthcare, Deerfield, IL) and heparin (200 units prime, 0.2 unit/kg/min 
continuous) will commence as previously described55. The infusion will continue till the end of study (1230 – 210 
min). In previous experiments, these infusions raised FFA concentrations three-fold (~1200pmol/l) impairing 
insulin secretion and action as well as raising fasting and post-prandial glucagon concentrations27. The two study 
days will be referred to as the Sal+FFA and the Exe+FFA study days. 
 
Subjects with T2DM will undergo point of care glucose testing at 2000, 0000, and 0400. If the glucose value is 
found to be greater than 180 mg/dL, a mild correction scale as outlined in the existing “Subcutaneous Insulin 
Order Set” will be used to administer aspart insulin subcutaneously. The mild correction scale reads as follows: 
glucose 180-219 mg/dL, give 2 units, 220-259 mg/dL give 3 units, and so on up to 7 units of rapid acting insulin 
above 380 mg/dL. This order set also includes orders for nurses to check glucose at anytime if there is concern 
for hypoglycemia. If hypoglycemia is detected at any point, nurses will administer 15 grams of oral carbohydrate 
and recheck point of care glucose 15 minutes later to monitor for improvement in glucose and symptoms. The 
goal is to prevent morning glucose values above 180 mg/dL which have occurred after the standardized meal in 
some subjects with T2DM who control their diabetes with strict carbohydrate restriction. Administering 
subcutaneous insulin at 0400 would give sufficient time for exogenous insulin to be metabolized before blood 
draws are performed to measure endogenous insulin levels occur at 0830.  
 
Figure 4: Experimental Design  
Fasting
Variable glucose infusion labelled 
with 3-H3-glucose to maintain 
glucose @ ~160mg/dL
1mg Glucagon
Sal study day: 0.9% Saline
Exe study day: Exendin-9,39 @ 300pmol/kg/min
Fconstant (3-H3-glucose)
-180      -120       -60           0           60        120       180   210
Sal + FFA study day: 0.9% Saline + Intralipid
and heparin
Exe + FFA study day: Exendin-9,39 @ 
300pmol/kg/min + Intralipid and heparin
Intralipid
+
heparin

 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 
columns48. 
FFA 
concentrations will be measured using LC/MS as 
previously56. 
 
 
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 
described48. Si, Insulin Secretion Rate (ISR) and β-
Cell responsivity (Φ) indices will be calculated49 from 
the glucose, insulin and C-peptide concentrations observed during the experiments using the minimal model57, 
incorporating age-associated changes in C-peptide kinetics58. Φ is derived from the sum of φ1 (1st phase insulin 
secretion) and φ2 (2nd phase insulin secretion) as before50. Disposition index (DI) will express β-cell function as 
a function of the prevailing insulin action49. Glucose disappearance (Rd) will be calculated using steady state 
Steele equations59,60 after Specific activity is smoothed as previously described61. 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. The GLP-1 
model42,62, will be used to quantify the potentiation of insulin secretion by GLP-151. The model assumes that 
insulin secretion is linearly modulated by GLP-1 through an index (π), which quantifies the increase of insulin 
secretion due to (changes in) circulating GLP-1concentrations50. 
 
Statistical Analysis: The primary endpoints for the hypotheses being tested represent differences in a given 
parameter e.g. glucagon for each subject between the Sal and Exe days. These will be used in an analysis of 
covariance (ANCOVA), which will include the baseline value (Sal) as a covariate together with age, BMI and 
sex, to test for differences attributable to T2DM. Within-group differences attributable to study conditions will be 
assessed using a Student’s paired t-test (parametric) or Wilcoxon matched-pairs signed rank test (non-
parametric). Other response variables (e.g. hormone area under the curve) will also be examined.  
 
Power Calculation: Please refer to Table 1 for the relevant effect size that is detectable for fasting glucose, 
glucagon and EGP – since the primary analysis will use ANCOVA to detect between group differences in 
response to exendin-9,39 we anticipate the ability to detect even smaller changes in these parameters. As 
regards the ability to detect an effect on Φ – prior studies using a meal challenge show values of 50±1745 vs. 
29±1910 10-9min-1 – non-diabetic vs. T2DM respectively – a ~40% difference. Assuming similar between-group 
differences and similar variation in Φ, we will be amply powered to detect biologically significant variation in β-
cell responses. 
 
Interpretation: To test our 1° hypothesis, we will compare differences (T2DM vs. non-diabetic controls) in the 
change between Sal and Exe days for fasting glucagon. Our preliminary data (Fig.1) has suggested a greater 
effect of GLP-1 receptor blockade on α-cell function in people with T2DM compared to non-diabetic subjects. If 
the increase in fasting glucagon concentrations is higher in people with T2DM we will accept our 1° hypothesis. 
If this is the case, we will then determine if this is accompanied by a corresponding change in fasting glucose. 
Glucose concentrations could remain unchanged because of a compensatory rise in fasting insulin secretion, 
preventing an increase in EGP. An alternative scenario would be that the glucagon increase raises EGP, 
unrestrained by insulin secretion but this is counteracted by a reciprocal and equal increase in glucose 
disappearance. The use of tracer-based measurement will enable us to measure glucose uptake and glucose 
release separately. It is also possible, but unlikely2, that GLP1R blockade changes hepatic insulin sensitivity. 
This will be quantified by comparing EGP with prevailing insulin concentrations on each study day.    
 
To test our 2° hypothesis (i) we will compare (T2DM vs. non-diabetic controls) the change in nadir glucagon 
concentrations, between Sal and Exe days, during the hyperglycemic clamp (120 – 180 min).  Previously, we 
Power Calculation  
Mean ± SD 
Detectable 
difference  
(n = 20 / 20) 
Φ (10-9min-1) - hyperglycemia 
149 ± 45 
41 (28%) 
Secondary Endpoints  
Mean ± SD 
Detectable 
difference  
φb (10-9) - fasting 
8 ± 2 
1.8 (22%) 
Fasting glucagon (ng/l) 
71 ± 17 
15 (21%) 
Fasting glucose (mmol/l) 
5.0 ± 0.4 
0.4 (8%) 
Fasting EGP (μmol/kg/min) 
14.4 ± 2.2 
1.5 (10%) 
Φ (10-9min-1) – GLP-1 
316 ± 124 
113 (36%) 
Table 1: Means ± SD used to estimate power  

had shown heterogeneity in glucagon suppression to hyperglycemia in subjects across the spectrum of glucose 
tolerance – with some of the heterogeneity explained by impaired insulin action52. In our preliminary experiments 
in people with T2DM, inhibition of endogenous GLP-1 secretion by exendin-9,39 raised both fasting and nadir 
glucagon in response to an intravenous glucose profile that mimics the systemic appearance of a 50g OGTT40 
(Fig.1). If this is the case, then we will accept our 2° hypothesis (i). It is possible that the nadir observed is a 
reflection of fasting glucagon concentrations and therefore we will also compare the % suppression from fasting 
values to determine whether effects on α-cell suppression by hyperglycemia are due to differences in 
dysregulation of baseline glucagon secretion. If we reject our 2° hypothesis (i), we will design studies to 
determine if α-cell suppression in response to protein and fat (greater stimuli of post-prandial glucagon secretion) 
is also impaired by GLP1R blockade with exendin-9,39. This is relevant to people with T2DM who have 
paradoxical rises in glucagon after mixed meals which cause postprandial hyperglycemia63,64.   
 
The relative disparity in response (between people with and without T2DM) to exendin-9,39 in the fasting state 
observed previously may reflect the underlying degree of β-cell dysfunction. People with T2DM have impaired 
β-cell function perhaps to the degree that they are much less responsive to any change in endogenous GLP-1 
secretion and action – reflecting a general decreased response to secretagogues65,66. Accordingly, we will test 
our 2° hypothesis (ii) by comparing the change in Φ (T2DM vs. non-diabetic controls) between Sal and Exe days 
during the hyperglycemic clamp. If we accept our hypothesis, in the non-diabetic subjects, we will correlate the 
change in Φ with β-cell function as measured by the Disposition Index49 (calculated from the screening OGTT67) 
to determine if the effect of exendin-9,39 on insulin secretion is a function of baseline DI. We would expect the 
effect of GLP1R blockade on Φ to be less marked as DI decreases. Examination of the effect on the different 
components of Φ i.e. φ1 and φ2 will enable us to determine whether these effects occur via increased storage of 
preformed insulin granules (φ1 – 1st phase), increased synthesis during a challenge (φ2– 2nd phase), or a 
combination.  
 
People with T2DM have elevated FFA concentrations and acute elevation of FFA impairs β-cell function in non-
diabetic individuals18,27. There are minimal effects of raising FFA in people with already elevated FFA 
concentrations68 hence our experimental design where we will only raise FFA in people without T2DM to test 2° 
hypothesis (iii). Intra-islet GLP-1 seems to increase after β-cell exposure to FFA (Fig.3). We will compare the α-
cell and β-cell responses to exendin-9,39 infusion in the presence and absence of FFA elevation. If we accept 
our 2° hypothesis (iii) this would imply that endogenous secretion of GLP-1 (in the fasting state) serves as an 
adaptive response to dampen the negative effects of acute FFA elevation on islet function18.  
 
 
 

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