Lixisenatide accelerates restoration of normoglycemia and improves human beta-cell function and survival in diabetic immunodeficient NOD–scid IL-2rg RIP-DTR mice engrafted with human islets

Abstract

Objective
Glucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents, increases proliferation and survival of pancreatic beta cells. To investigate the effects on human beta cells, we used immunodeficient mice transplanted with human islets. The goal was to determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human islet function and survival in vivo.

Methods
Five independent transplant studies were conducted with human islets from five individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with lixisenatide (50, 150, and 500 µg/kg) or vehicle. Islet function was determined by blood glucose, plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total beta- and alpha-cell number, percent proliferation, and levels of apoptosis.

Results
Diabetic mice transplanted with marginal human islet mass and treated with lixisenatide were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests, human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were significantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated mice. Nevertheless, in one experiment we found a significant twofold to threefold increase in human beta-cell numbers in lixisenatide-treated compared to vehicle-treated mice.

Conclusion
Diabetic human islet-engrafted immunodeficient mice treated with lixisenatide show improved restoration of normoglycemia, human plasma insulin, and glucose tolerance compared to vehicle-treated mice engrafted with the same donor islets. Because the proliferative capacity of human beta cells is limited, improved beta-cell survival coupled with enhanced beta-cell function following lixisenatide treatment may provide the greatest benefit for diabetic patients with reduced functional islet mass.

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ORIGINAL RESEARCH
open access to scientific and medical research
Open Access Full Text Article
http://dx.doi.org/10.2147/DMSO.S87253
Lixisenatide accelerates restoration of
normoglycemia and improves human beta-cell
function and survival in diabetic immunodecient
NOD–scid IL-2rgnull RIP-DTR mice engrafted with
human islets
Chaoxing Yang1
Matthias Loehn2
Agata Jurczyk1
Natalia Przewozniak1
Linda Leehy1
Pedro L Herrera3
Leonard D Shultz4
Dale L Greiner1
David M Harlan5
Rita Bortell1
1Program in Molecular Medicine,
Diabetes Center of Excellence,
Uni versity of Massachusetts Med ica l
School, Worcester, MA, USA; 2Sanofi-
Aventis, Diabetes Division, Frankfurt,
Germany; 3University of Geneva,
Geneva, Switzerland; 4The J ack son
Laboratory, Bar Harbor, ME, USA;
5Department of Medicine, Diabetes
Center of Excellence, University
of Massachusetts Medical School,
Worcester, MA, USA
Correspondence: Rita Bortell
Program in Molecular Medicine, Diabetes
Center of Excellence, University of
Massachusetts Medical School,
368 Plantation Street, AS7-2055,
Worcester, MA 01605, USA
Tel +1 508 856 3788
Fax +1 508 856 4093
Email rita.bortell@umassmed.edu
Objective: Glucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents,
increases proliferation and survival of pancreatic beta cells. To investigate the effects on human
beta cells, we used immunodeficient mice transplanted with human islets. The goal was to
determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human
islet function and survival in vivo.
Methods: Five independent transplant studies were conducted with human islets from five
individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with
lixisenatide (50, 150, and 500 µg/kg) or vehicle. Islet function was determined by blood glucose,
plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total
beta- and alpha-cell number, percent proliferation, and levels of apoptosis.
Results: Diabetic mice transplanted with marginal human islet mass and treated with lixisenatide
were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests,
human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were signifi-
cantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic
beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated
mice. Nevertheless, in one experiment we found a significant twofold to threefold increase in
human beta-cell numbers in lixisenatide-treated compared to vehicle-treated mice.
Conclusion: Diabetic human islet-engrafted immunodeficient mice treated with lixisenatide
show improved restoration of normoglycemia, human plasma insulin, and glucose tolerance
compared to vehicle-treated mice engrafted with the same donor islets. Because the prolifera-
tive capacity of human beta cells is limited, improved beta-cell survival coupled with enhanced
beta-cell function following lixisenatide treatment may provide the greatest benefit for diabetic
patients with reduced functional islet mass.
Keywords: GLP-1 receptor agonist, lixisenatide, human islet transplant, beta cells, glucose
tolerance tests, plasma insulin
Introduction
Glucagon-like peptide-1 (GLP-1) and GLP-1 receptor agonists have been reported to
improve beta-cell function and viability.1–3 At the beta-cell level, GLP-1 and its receptor
agonists were found to induce beta-cell proliferation and decrease beta-cell apoptosis
in rodents and in vitro.3–6 In vivo, GLP-1 receptor agonists preserve beta-cell mass in
multiple animal models of diabetes,3,7–9 although an effect on beta-cell mass/number has
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not yet been demonstrated in humans. In clinical trials with
type 2 diabetes (T2D) patients, GLP-1 receptor agonists low-
ered both fasting and postprandial glucose concentrations;10,11
GLP-1 receptor agonists also potentiate glucose-dependent
insulin secretion, and thus have a low propensity to cause
hypoglycemia.12 However, in patients with long-standing type
1 diabetes, C-peptide secretion was not increased, although
insulin sensitivity improved.13,14
Lixisenatide is a recently developed GLP-1 receptor
agonist with potent binding affinity and extended biological
activity.15–18 In patients with T2D, once-daily administration
of lixisenatide improved glycemic control by decreasing
both postprandial and fasting glucose levels; moreover, these
effects were both immediate and sustained.19–22 In addition
to increased insulin secretion in response to meal-related or
glucose stimulation, a reduction in endogenous glucose pro-
duction and slowing of gastric emptying also contribute to the
reduction in postprandial blood glucose levels observed with
lixisenatide and other GLP-1 receptor agonists.22–25 In vitro
studies with an INS-1 rat pancreatic beta-cell line indicates
that GLP-1 and its receptor agonists, including lixisenatide,
protect against lipid- and cytokine-induced apoptosis.26 Even
so, it is not possible to directly assess beta-cell apoptosis in
patients due to the inaccessibility of pancreatic islets, and
clinical studies are difficult to control due to the variability
between patients in regards to age, sex, diet, and other life-
style factors.
A useful method to interrogate human islet function
in vivo is to transplant human islets into diabetic immu-
nodeficient nonobese diabetic–severe combined immu-
nodeficiency (NOD–scid) IL-2 receptor common gamma
chain (IL-2rgnull) (NSG) mice.27 Many diabetes-inducing
chemicals, such as streptozotocin, may cause damage to
other organs. Also, the chemical-induced destruction of
endogenous beta cells is not always complete, rendering
problematic the interpretation of long-term transplantation
studies with exogenous islets. To circumvent these issues,
we utilized a strain of transgenic NSG mice that uses the
rat insulin promoter (RIP) to drive human diphtheria toxin
receptor (DTR) expression in the animal’s beta cells. When
treated with low doses of diphtheria toxin, the NSG RIP-
DTR mouse model allows complete and specific ablation of
mouse pancreatic beta cells and thereby avoids broadly toxic
agents such as streptozotocin. The goal of this study was to
investigate the efficacy of lixisenatide to promote human
beta-cell function, proliferation, and survival using diabetic
NSG RIP-DTR mice engrafted with marginal amounts of
human pancreatic islets.
Methods
Mice and diabetes induction
NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(Ins2-HBEGF)6832
Ugfm/Sz mice, referred to as NSG RIP-DTR mice, were
developed at the Jackson Laboratory, Bar Harbor, ME, USA,
by backcrossing the RIP-DTR transgene from a B6;CBA-RIP-
DTR stock kindly provided by Pedro Herrera. The original
B6;CBA Tg(Ins2-HBEGF)6832 Ugfm/Sz mice were made
by injecting the construct into B6;CBA eggs. The transgene
was backcrossed using a marker-assisted speed congenic
method to the NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (abbreviated
as NOD–scid IL-2rgnull or NSG) strain background. These
NSG RIP-DTR mice express the human DTR driven by a
RIP. The RIP-DTR transgene was then fixed to homozygosity.
All mice were housed in a specific pathogen-free facility
and maintained in accordance with the Institutional Animal
Care and Use Committee of the University of Massachusetts
Medical School.28
To induce diabetes, male NSG RIP-DTR mice
(8–12 weeks old) were injected intraperitoneally (ip) with
20 ng diphtheria toxin (List Biological Laboratories, Camp-
bell, CA, USA) diluted in sterile phosphate-buffered saline
(PBS). Blood glucose was monitored with an Accu-chek
Aviva Plus glucometer (Hoffman-La Roche Ltd, Basel,
Switzerland) to confirm diabetes (blood glucose .300
mg/dL on 2 consecutive days). Diabetic mice that were not
transplanted with human islets within 1 week were given
insulin implants (LinShin Canada Inc., Scarborough, ON,
Canada) to prevent metabolic decompensation until human
islets were available.
Pharmacokinetic analyses
An initial pharmacokinetic study with unengrafted,
euglycemic NSG mice was performed to determine the
plasma levels of lixisenatide over a 24-hour period fol-
lowing treatment. All mice were injected subcutaneously
(sc) with vehicle alone or with 50 µg/kg, 150 µg/kg, or
500 µg/kg lixisenatide (provided by Sanofi-Aventis,
Frankfurt, Germany). Blood was collected in potassium
ethylenediaminetetraacetic acid (K-EDTA) tubes at 0, 5,
15, 30, 60, and 120 minutes and 4, 8, and 24 hours from
three mice/group at each time point; plasma was stored at
−80°C. High-performance liquid chromatography analy-
sis of lixisenatide levels in blood plasma was performed
by Sanofi-Aventis. Based on these time-course data
( Figure S1), the mice in the transplant studies were treated
twice daily with the same concentrations of lixisenatide as
in the pharmacokinetic study.

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Lixisenatide improves human islet grafts
Human islet transplantation and
lixisenatide treatment
Human islets were obtained from the Integrated Islet Dis-
tribution Program under protocols approved by the Insti-
tutional Review Board of the University of Massachusetts
Medical School. Islets were transplanted into the subrenal
capsular space as previously described;29 insulin implants
were removed upon transplant. Five independent transplant
studies were performed, each with human islets from a single
donor. One day post-transplant, the mice were randomized
into four groups, with five to seven mice in each group. The
mice were injected sc twice daily with lixisenatide (50, 150,
or 500 µg/kg/injection) or vehicle until graft removal at
∼4 weeks post-transplant.
Glucose tolerance test, plasma insulin/C-
peptide, and glucose stimulation index
For the glucose tolerance test (GTT), mice were fasted for
5–6 hours, and blood glucose was measured following ip
injection of glucose (2.0 g/kg body weight). To measure
plasma levels of human insulin and C-peptide, heparinized
blood from transplanted mice was collected with protease
inhibitor (aprotinin; Sigma-Aldrich Co, St Louis, MO,
USA). Non-fasting blood samples were collected just
prior to drug/vehicle treatment. On alternate weeks, the
mice were fasted for 5–6 hours prior to glucose injection
(2 g/kg, ip); in the Donor 1 study, arginine (1 g/kg, ip)
was given in addition to glucose. Blood was collected at
0 (fasted) and 15 minutes (stimulated) post-injection; the
glucose stimulation index was determined as the ratio of
plasma insulin at 15 and 0 minutes. All plasma was stored
at −80°C until analyzed by human-specific enzyme-linked
immunosorbent assay (ELISA) (ALPCO Diagnostics,
Salem, NH, USA).
Bromodeoxyuridine treatment,
immunouorescence staining,
and TUNEL assay
Human islet-engrafted mice were provided drinking water
containing 0.8 mg/mL of bromodeoxyuridine (BrdU) ad
libitum for 7 days prior to nephrectomy of the graft-bearing
kidney. Euglycemic mice at the time of nephrectomy were
followed for reversion to hyperglycemia for confirmation
of human islet graft function. Islet graft-bearing kidneys
were fixed in 10% neutral-buffered formalin. Paraffin-
embedded sections were stained with guinea pig anti- insulin
(Dako, Carpinteria, CA, USA), mouse anti-glucagon
(Abcam, Cambridge, England), and rat anti-BrdU (Accu-
rate Chemical, Westbury, NJ, USA); secondary Alexa Fluor
antibodies (Alexa Fluor 647, 594, 488) were from Life
Technologies (Carlsbad, CA, USA), and 4′,6-diamidino-2-
phenylindole (DAPI) was from Sigma-Aldrich Co. Insulin+,
insulin+BrdU+, glucagon+, and glucagon+BrdU+ cells were
visualized by fluorescence microscopy (Nikon Eclipse Ti
series; Nikon Corporation, Tokyo, Japan). Terminal deoxy-
nucleotidyl transferase-mediated dUTP nick end labeling
(TUNEL) assay was performed as per manufacturer’s instruc-
tions (Hoffman-La Roche Ltd). All counts were performed
with Nikon NIS Elements software.
Total beta- and alpha-cell counts in human
islet grafts
To determine total beta- and alpha-cell numbers in the islet
grafts, 5 µm serial sections were cut through the entire graft
and immunostained for insulin and glucagon. Beginning at
the outer edge of the graft, images of the entire section were
taken and stitched with a Nikon Eclipse Ti series microscope
with motorized x–y stage. Subsequent serial sections were
counted at 20 µm intervals to avoid duplicate counting of
the same cells; all counts were performed with Nikon NIS
Elements software.
Statistical analyses
Time-course data were analyzed by two-way analysis of
variance (ANOVA) with Tukey’s or Holm–Sidak’s multiple
comparisons test; insulin levels, cell counts, proliferation,
and TUNEL data were analyzed by one-way ANOVA
with Bonferroni’s or Tukey’s multiple comparisons test
when comparing the four groups. Percent diabetes survival
(Kaplan–Meier) was analyzed by Mantel–Cox log rank test.
All statistical analyses were performed with GraphPad Prism
(San Diego, CA, USA); P-values ,0.05 were considered
significant.
Results
Lixisenatide treatment accelerates
restoration of normoglycemia in diabetic
mice engrafted with human islets
Diabetic NSG RIP-DTR mice were engrafted with human
islets from a single donor, randomized into four groups, and
treated with vehicle (control) and lixisenatide at 50 µg/kg
(low dose), 150 µg/kg (medium dose), and 500 µg/kg (high
dose). A total of five independent studies were done with
human islets from five donors; none of the donors were
diagnosed as diabetic. The demographic characteristics of

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Yang et al
the islet donors and the numbers of islet equivalents (IEQs)
transplanted into each mouse for each study are shown in
Table 1.
Blood glucose and body weight measurements of the
mice were taken prior to islet transplant and then twice
weekly following transplantation (Figure 1A and B). In
four of the five transplant studies, mice treated with lixisen-
atide exhibited significant improvement in blood glucose
control more rapidly than mice receiving vehicle alone
(Figure 1A). In the Donor 2 study, only the high-dose lix-
isenatide mice showed significant improvement in blood
glucose (at 9 and 13 days post-transplant). The mice in all
other groups remained hyperglycemic; consequently, all
mice engrafted with Donor 2 islets were euthanized without
further analysis.
A combined survival curve analysis for transplant studies
with islets from Donors 1, 3, 4, and 5 showed that lixisen-
atide treatment improved recovery from diabetes with high
statistical power (Figure 2). The median time for the mice
to become diabetes free was 12 days for the vehicle control
group, and 5, 3, and 4 days for low-, medium-, and high-dose
lixisenatide-treated groups, respectively. On surgical exci-
sion of the graft-bearing kidney, all mice became acutely
hyperglycemic (blood glucose .500 mg/dL, Figure 1A),
thus verifying that the human islet graft was responsible for
maintaining blood glucose levels.
Lixisenatide treatment is associated
with improved body weight maintenance
Mouse body weights were measured during each transplant
study. In the Donor 1 study, all mice lost significant weight
between the time of transplant and nephrectomy, except
the medium-dose group (Figure 1B), which also showed
the best blood glucose control. In transplant studies with
islets from Donors 2 and 5, mice in the control group lost
a small, but significant, amount of weight. This weight
loss was not unexpected because these control mice were
hyperglycemic throughout the trial. However, it is interest-
ing to note that none of the lixisenatide-treated mice in the
Donor 2 and 5 studies showed significant weight loss, even
though some of the mice in the lower dose groups were
also hyperglycemic. In transplant studies with islets from
Donors 3 and 4, in which normoglycemia was eventually
restored in all mice, there was no significant change in body
weight of the mice in either the control or lixisenatide-treated
groups.
Glucose-stimulated human insulin
secretion is increased in islet-engrafted
mice treated with lixisenatide
At 2 weeks post-transplant, the fasting levels of human insu-
lin showed no significant differences between control and any
of the lixisenatide-treatment groups (Figure 3A). However,
glucose-stimulated levels of human insulin were signifi-
cantly increased with medium-dose lixisenatide treatment
in Donors 1 and 4 transplant studies (Figure 3B). Of note,
the stimulation index (ratio of stimulated to fasting human
insulin) was significantly increased compared to controls in
the medium-dose group for Donor 1, and both medium- and
high-dose groups for Donors 3 and 4 transplants (Figure 3C).
Mice in the Donor 2 and 5 transplant studies were excluded
because the controls (as well as some of the treated mice)
were hyperglycemic.
Lixisenatide treatment improves GTTs
in human islet-engrafted mice
Consistent with the increased human insulin levels in
lixisenatide-treated mice in response to glucose, lixisenatide
treatment also significantly improved responses in GTTs in
both the Donor 3 and 4 studies at 4 weeks post-transplant
(Figure 4). Although mice in the Donor 4 study received
uninterrupted drug treatment until the GTT, mice in the
Donor 3 study continued to exhibit significantly improved
glucose tolerance, even though lixisenatide treatment
had been discontinued for 4 days prior to the GTT. In the
Donor 3 transplant study, the mice treated with lixisenatide
showed significant differences from controls in the medium-
dose (P,0.01) and high-dose (P,0.05) groups, whereas
in the Donor 4 transplant study, the low-dose lixisenatide
group showed significant differences from the control group
(P,0.05).
Table 1 Demographic characteristics of human islet donors
Donor 1 Donor 2 Donor 3 Donor 4 Donor 5
Age, years 30 53 43 51 61
Sex (M/F) M F nr M F
Ethnicity Hispanic/
Latino
Hispanic/
Latino
nr Hispanic/
Latino
nr
Body
weight, kg
95 105 nr 86 nr
BMI, kg/m230.9 42.7 34.7 28.9 22.2
Time
in culture*
1 day,
0 hour
3 days,
12 hours
1 day,
3 hours
2 days,
10 hours
nr
Transplanted
IEQs/mouse
2,500 2,500 3,000 3,000 3,000
Note: *Refers to the amount of time for which the human islets were cultured
following isolation until shipment to our laboratory.
Abbreviations: BMI, body mass index; F, female; IEQs, islet equivalents; M, male;
nr, not recorded.

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Lixisenatide improves human islet grafts
ADonor 1
600
Control **
**
***
*
***
****
*
***
**
*
**
Low dose
Control
Low dose
Medium dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Vehicle
Low dose
Medium dose
High dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
At transplant
At nephrectomy
At transplant
At nephrectomy
At transplant
At nephrectomy
At transplant
At nephrectomy
At transplant
At nephrectomy
500
400
300
200
35
30
25
20
15
35
30
25
20
15
35
30
25
20
15
Blood glucose (mg/dL)Blood glucose (mg/dL)
Blood glucose (mg/dL)
Blood glucose (mg/dL)
Days
Days
Body weight (g)Body weight (g)Body weight (g)
35
30
25
20
15
Body weight (g)
35
30
25
20
15
Body weight (g)
100
0
600
500
400
300
200
100
0
600
500
400
300
200
100
0
600
500
400
300
200
100
0
Blood glucose (mg/dL)
600
500
400
300
200
100
0
Human islet
transplant
Human islet
transplant
Start of
treatment
Start of
treatment
Nephrectomy of
graft-bearing kidney
Nephrectomy of
graft-bearing kidney
Human islet
transplant
Start of
treatment
Nephrectomy of
graft-bearing kidney
Human islet
transplant
Start of
treatment
Nephrectomy of
graft-bearing kidney
Human islet
transplant
Start of
treatment
Nephrectomy of
graft-bearing kidney
0
0
−3
−1
1
1
2
6
8
9
13
Days
0
−3
1
4
9
12
15
19
24
29
32
Days
Days
0
−2
2
3
5
10
14
19
21
24
32
34
28
0
−2
2
1
5
7
9
12
14
16
19
23
21
4
7
11
14
18
21
25
27
29
Donor 2
Donor 3
Donor 4
Donor 5
Donor 5
Donor 4
Donor 3
Donor 2
Donor 1
B
*
**
Figure 1 Blood glucose and body weights of control and lixisenatide-treated mice engrafted with human islets.
Notes: Diabetic NSG RIP-DTR mice were transplanted with human islets from ve individual donors and injected sc twice daily with lixisenatide or vehicle control; n=5–6
per treatment group, mean ± SEM. *P,0.05, **P,0.01, and ***P,0.001. (A) Blood glucose levels of the mice were monitored on the days indicated. Donor 1, **medium
dose vs control; Donor 2, ***high dose vs control, low, and medium groups; Donor 4, day 3, **high dose vs control, *medium dose vs control; day 5, ***medium and high
doses vs control, *low dose vs control; Donor 5, *medium and high doses vs control. (B) Body weights were compared between the time of transplant and the day when
the islet graft-bearing kidney was removed.
Abbreviations: NSG RIP-DTR, nonobese diabetic–severe combined immunodeciency (NOD–scid) IL-2 receptor common gamma chain (IL-2rgnull) rat insulin promoter-
diphtheria toxin receptor; SEM, standard error of mean; sc, subcutaneously.

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Yang et al
Apoptosis and proliferation analyses of
human islet grafts recovered from control
and lixisenatide-treated mice
Treatment with GLP-1 receptor agonists has been reported
to protect rodent pancreatic beta cells from apoptosis.1 To
determine whether lixisenatide treatment modulates human
beta-cell apoptosis, the islet grafts were recovered on days 29,
32, and 21 (for Donor 3, 4, and 5 studies, respectively) and
examined by TUNEL staining (Figure 5). We found no signifi-
cant differences in beta-cell apoptosis between the control and
lixisenatide-treated groups at the time points examined (at
the end of each islet transplant study). In addition, although
there was a variability between donor islets, the average
percent beta-cell apoptosis observed at the end of each study
was very low (,1%).
100
50
0
0714
Control
Low dose
Medium dose
High dose
*** ****
*
Days post-transplant
Hyperglycemic mice (%)
21 28
Figure 2 Lixisenatide treatment improves recovery from hyperglycemia in human
islet-engrafted mice.
Notes: Diabetic NSG RIP-DTR mice were transplanted with human islets and treated
with lixisenatide or vehicle control. Mice with blood glucose values .300 mg/dL were
considered hyperglycemic. Data shown are pooled from islet transplants of Donors 1, 3,
4, and 5; n=23–25 per treatment group. *P,0.05, ***P,0.001, ****P,0.0001.
Abbreviation: NSG RIP-DTR, nonobese diabetic–severe combined immunodeciency
(NOD–scid) IL-2 receptor common gamma chain (IL-2rgnull) rat insulin promoter-
diphtheria toxin receptor.
A
0.3
Donor 1
FastingGlucose stimulationStimulation index
Donor 1Donor 1
0.2
Human insulin (ng/mL)
Human insulin (ng/mL)
Stimulation indexGlucose stimulation index
0.1
0.0
1.0
P<0.05 P<0.01
P<0.05
0.8
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
0.8
0.4
0.6
0.2
0.0
0
0
1
2
3
4
5
2
4
6
Control
Low dose
Low
Vehicle
Medium dose
Medium
High dose
Donor 3 Donor 3Donor 3
Human insulin (ng/mL)
Human insulin (ng/mL)Human insulin (ng/mL)
Human insulin (ng/mL)
0.3
0.2
0.1
0.0
0.3
0.2
0.1
0.0
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Stimulation index
P<0.05
P<0.05
0
2
4
6
Donor 4Donor 4Donor 4
Control
Low dose
Medium dose
High dose
High
BC
Figure 3 Fasting and glucose-stimulated human insulin levels and stimulation indices in control and lixisenatide-treated mice engrafted with human islets.
Notes: Blood was collected at fasting and 15 minutes after ip glucose injection; plasma levels of (A) fasting human insulin and (B) glucose-stimulated human insulin are shown.
(C) Stimulation indices (glucose-stimulated insulin/fasting insulin) from vehicle control and lixisenatide-treated mice are shown; n=4 or 5 per group, mean ± SEM.
Abbreviations: ip, intraperitoneal; SEM, standard error of mean.

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Lixisenatide improves human islet grafts
To determine whether lixisenatide treatment induced
human beta (or alpha)-cell proliferation, the mice were sup-
plied with BrdU in their drinking water for 1 week prior to
recovery of the graft-bearing islets. The percentages of BrdU+
beta cells (Figure 6A) and BrdU+ alpha cells (Figure 6B) for
each human islet graft are shown for control and lixisenatide-
treatment groups from Donor 3, 4, and 5 transplant studies
combined. Consistent with previous reports,30–32 most islet
grafts had very low levels of proliferating human beta cells,
but neither beta- nor alpha-cell proliferation was significantly
affected by lixisenatide treatment (as measured by BrdU
incorporation during the last 7 days of each islet transplant
study).
Quantitation of total beta and alpha cells
in human islet grafts recovered from
control and lixisenatide-treated mice
Our proliferation and apoptosis analyses reflect lixisenatide
effects on the human islet grafts only as a “snapshot” at late
stages of engraftment. Therefore, to better interrogate the
effect of lixisenatide on human beta-cell survival throughout
the ∼30-day treatment period, we counted total beta- and
alpha-cell numbers within the recovered islet grafts. We
observed considerable donor-to-donor variability with regard
to the numbers of beta (Figure 7A) and alpha (Figure 7B)
cells in the recovered islet grafts, even though the same
numbers of IEQs were transplanted in each of the three
studies examined.
In the Donor 5 transplant study, medium- and high-dose
lixisenatide-treated mice displayed significantly greater beta-
cell numbers within the graft compared to control mice. In
this study, an average of ∼2,000 beta cells was detected in the
islet grafts of control mice compared to an average of ∼12,500
and ∼4,500 beta cells in the control groups of Donor 3 and
4 studies, respectively. Interestingly, Donor 5 control mice
remained hyperglycemic throughout the study, whereas
normoglycemia was restored in control group mice in the
Donor 3 and 4 studies. The beta (and alpha)-cell counts in the
low-dose lixisenatide-treated group from the Donor 5 study
were approximately twofold higher than the control group,
250
Control *
** Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose200
400
300
200
100
0
150
100
50
Minutes Minutes
Blood glucose (mg/dL)
Blood glucose (mg/dL)
Donor 3 Donor 4
0
0
15
30
60
90
120
0
15
30
60
90
120
Figure 4 GTTs in control and lixisenatide-treated mice engrafted with human islets.
Note: A GTT was performed in 5- to 6-hour fasted mice engrafted with islets at 4 weeks post-transplant; n=4 or 5 mice per group, mean ± SEM; *P,0.05, **P,0.01.
Abbreviations: GTT, glucose tolerance test; SEM, standard error of mean.
0.10
Donor 3 Donor 4 Donor 5
0.08
0.06
% insulin+ TUNEL+ cells
% insulin+ TUNEL+ cells
% insulin+ TUNEL+ cells
0.04
1.5 2.0
1.5
1.0
0.5
0.0
1.2
0.9
0.6
0.3
0.0
0.02
0.00
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Figure 5 Percent TUNEL-positive human beta cells in islet grafts recovered from control and lixisenatide-treated mice.
Notes: Human islet grafts were immunostained for insulin and TUNEL, and the percent of TUNEL-positive beta cells was determined; n=3 or 4 per group for Donor 3 and
4 studies and n=2 for Donor 5 study, mean ± SEM.
Abbreviations: SEM, standard error of mean; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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Yang et al
even though the blood glucose of these mice remained
elevated (∼400 mg/dL, Figure 1A). The blood glucose values
in the medium- and high-dose groups were significantly lower
than the control group, and these mice had approximately
threefold higher numbers of beta (and alpha) cells. In sup-
port of this, the islet grafts from three vehicle control mice
appear noticeably smaller than from lixisenatide-treated mice
(Figure 8), consistent with the significantly lower beta- and
alpha-cell counts in the control group.
Discussion
In this study, we demonstrated that lixisenatide treatment
significantly improved human beta-cell function and survival
in diabetic NSG RIP-DTR mice engrafted with human islets.
The human islets were derived from both male and female
islet donors, aged 30–61 years, with body mass index (BMI)
from 22.2 to 42.7 kg/m2; none was diagnosed as having
diabetes. Five independent islet transplant studies were
conducted, and mice were treated with three different doses
of lixisenatide (50, 150, and 500 µg/kg) and vehicle control.
A significantly accelerated recovery from diabetes was
observed in lixisenatide-treated mice compared to controls,
with the median time to a diabetes-free condition of 5, 3, and
4 days for low-, medium-, and high-dose lixisenatide groups,
respectively, compared to controls at 12 days. The numbers
of human islets transplanted were 2,500 IEQs in Donor 1 and
A
1.6
1.3
1.0
0.8
0.6
% insulin+ BrdU+ cells
% glucagon+ BrdU+ cells
0.4
0.2
0.0
Low dose
Control
Medium dose
High dose
Low dose
Control
Medium dose
High dose
1.0
1.2
0.8
0.6
0.5
0.4
0.3
0.2
0.1
0.0
B
Figure 6 Percent human beta- and alpha-cell proliferation in each islet graft recovered from control and lixisenatide-treated mice.
Notes: (A) Percent BrdU+ beta cells and (B) percent BrdU+ alpha cells are shown. Beta and alpha cells were identied by immunostaining for insulin and glucagon,
respectively. Each dot represents data obtained from a single islet graft recovered from one mouse. The data from Donor 3 (black dots), 4 (green dots), and 5 (red dots)
transplant studies were pooled. The bar represents the mean of n=10 or 11 individual islet grafts in each treatment group; .62,000 total beta cells and .24,000 total alpha
cells were counted in each group.
Abbreviation: BrdU+, bromodeoxyuridine positive.
ADonor 3
20,000
10,000
8,000
6,000
4,000
2,000
0
10,000
7,500
2,500
5,000
0
10,000
P<0.05
P<0.05
7,500
2,500
5,000
3,000
2,000
1,000
0
5,000
0
10,000
8,000
4,000
4,000
2,000
6,000
0
15,000
10,000
5,000
0
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Control
Low dose
Medium dose
High dose
Donor 3
Insulin+ cells/graft
Insulin+ cells/graft
Insulin+ cells/graft
Glucagon+ cells/graft
Glucagon+ cells/graft
Glucagon+ cells/graft
Donor 4
Donor 4
Donor 5
Donor 5
B
Figure 7 Total beta- and alpha-cell counts in human islet grafts recovered from control and lixisenatide-treated mice.
Notes: Human islet grafts of control and lixisenatide-treated mice were serially sectioned through the entire graft and (A) insulin+ beta cells and (B) glucagon+ alpha cells
were counted; n=3 or 4 per group, mean ± SEM.
Abbreviation: SEM, standard error of mean.

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Lixisenatide improves human islet grafts
2 studies, and 3,000 IEQs in studies with Donors 3, 4, and 5.
Although human islets from nondiabetic donors can comprise
as few as 28% or as many as 75% beta cells,33 in our experi-
ence, ∼3,000 human IEQs is typically sufficient to restore
normoglycemia in the NSG RIP-DTR model.
Plasma levels of human insulin and C-peptide in fed or
fasted islet-engrafted mice were variable or not significantly
different between the control and lixisenatide-treatment
groups. In contrast, mice treated with medium- and high-dose
lixisenatide (compared to control) had significantly increased
glucose-stimulated plasma levels of human insulin and glucose-
stimulation indices for each of the three donors that were ana-
lyzed. This is consistent with human studies in T2D patients and
nondiabetic subjects, in whom lixisenatide stimulated insulin
secretion when blood glucose levels were increased, but not at
normoglycemia.34 In support of this, GTTs were significantly
improved in mice treated with lixisenatide compared to control
mice engrafted with the same donor islets. Similarly, in a study
with T2D patients insufficiently controlled on metformin, lix-
isenatide treatment provided a significantly greater reduction in
postprandial plasma glucose than in placebo control.35
To measure the proliferation of human beta and alpha cells
in the islet grafts, control and lixisenatide-treated mice were
given BrdU in their drinking water 7 days before removal
of the graft-bearing kidney. No significant differences were
seen between lixisenatide treatment and control groups,
although individual islet grafts derived from Donors 4 and
5 tended to have more BrdU+ cells in control mice compared
to lixisenatide-treated animals engrafted with the same islets.
Of note, control mice in the Donor 4 and 5 transplant stud-
ies had higher blood glucose levels than lixisenatide-treated
mice, and we and others have reported that even mild hyper-
glycemia induces human beta-cell proliferation in human
islet-engrafted mice.31,32 Nonetheless, beta cells from humans
have a very low proliferation rate compared to rodents, and
hyperglycemia induction results in only a ∼0.5% proliferative
rate in human beta cells.30–32
GLP-1 and its receptor agonists have been reported to
inhibit beta-cell apoptosis in short-term culture of insuli-
noma cells and freshly isolated human and rodent islets.8,26,36
At recovery of the human islet grafts ∼4 weeks post-transplant,
we observed no significant difference between lixisenatide-
treated and control groups in beta-cell apoptosis (as measured
by TUNEL staining). However, it is possible that lixisenatide
may have had an anti-apoptotic effect on the islet grafts at
earlier stages in the post-transplant period. Indeed, it has been
estimated that up to 70% of islet mass may be lost in the early
post-transplant period, even in immunodeficient or syngeneic
transplant models.37,38 Thus, it is likely that lixisenatide may
have modulated beta-cell survival at earlier post-transplant
time periods. In support of this, GLP-1 receptor agonist
treatment of diabetic mice engrafted with syngeneic islets
reversed the loss of both the number and mass of islets grafts
at 1 and 3 days post-transplant.39 With longer GLP-1 receptor
agonist treatment (2–3 weeks), both Ins2Akita (Akita) and Leprdb
mice had increased islet mass and elevated pancreatic insulin
Low dose
Insulin/glucagon/DAPI Insulin/glucagon/DAPI
Medium dose
Control human islet grafts
High dose
Figure 8 Photomicrographs of human islet grafts from control and lixisenatide-treated mice.
Notes: Human islets from a single donor (Donor 5) engrafted in three vehicle control mice (top panel) and representative islet grafts from low-, medium-, and high-dose
lixisenatide-treated mice (bottom panel, 1 of 3 islet graphs from each lixisenatide treatment group is shown) are shown; red, green, and blue indicate insulin, glucagon, and
DAPI staining, respectively.
Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

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Yang et al
content compared with controls.7,8 Similarly, we found that
lixisenatide treatment dramatically improved total beta- and
alpha-cell numbers in human islet grafts from Donor 5.
In the Donor 5 transplant study, the human islets failed to
lower blood glucose levels in the control group, and mice in
the low-dose lixisenatide group also remained hyperglycemic.
Remarkably, the insulin+ (beta) and glucagon+ (alpha) cell
counts in the islet grafts of mice treated with low-dose lixisen-
atide were twofold greater than in the control group, and mice
treated with medium- and high-dose lixisenatide had threefold
greater numbers of alpha and beta cells. Because the low rate of
human beta-cell proliferation cannot account for these differ-
ences in beta-cell numbers, these data are consistent regarding
the role of lixisenatide to preserve human beta-cell survival.
It is possible that lixisenatide’s effect to maintain beta-
cell viability may be secondary to its ability to increase
insulin secretion and lower blood glucose, thus preventing
glucotoxicity. However, in Akita mice, the protective effect
of GLP-1 receptor agonist on pancreatic islet mass was found
to be independent of lowered blood glucose levels.7 Similarly,
in our Donor 5 study, the low-dose lixisenatide group showed
a twofold increase in beta-cell number compared to the
control group, even though mice in this group remained
hyperglycemic throughout the study. These data suggest that
lixisenatide may have additional islet-protective effects on
human beta cells, such as that previously reported in rodent
diabetes models, in which GLP-1 receptor agonists acted to
reduce beta-cell endoplasmic reticulum stress.3,7–9 Taken
together, our data are consistent with a role for lixisenatide
to preserve human beta-cell function and survival in vivo, in
particular when numbers of functional beta cells are limiting,
as would be found in individuals with T2D.
Acknowledgment
Sanofi-Aventis funded this research.
Author contributions
CY, ML, AJ, LDS, DLG, DMH, and RB contributed to the
conception and design of the study and interpretation of the
data. CY, AJ, NP, and LL performed experiments and ana-
lyzed the data. All authors contributed toward data analysis,
drafting and critically revising the paper and agree to be
accountable for all aspects of the work. None of this work
has been published or submitted elsewhere.
Disclosure
Dr Loehn is an employee of Sanofi-Aventis. The authors
report no other conflicts of interest in this work.
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100,000
High dose (500 µg/kg)
Medium dose (150 µg/kg)
Low dose (50 µg/kg)
Vehicle control
10,000
1,000
100
10
1
Time (hours)
Lixisenatide plasma conc
(pg/mL)
0
0.083
0.25
0.5
1
2
4
8
24
Figure S1 Pharmacokinetic analysis of control and lixisenatide treatments.
Notes: NSG mice were given a single sc injection with vehicle control or three different doses of lixisenatide; plasma levels of lixisenatide were measured at the time points
indicated; n=3 mice per group at each time point (n=96 mice total + 3 untreated mice at time 0). The data from one low dose mouse at the 24-hour time point were deemed
a technical failure and removed from analysis.
Abbreviations: NSG, nonobese diabetic–severe combined immunodeciency (NOD–scid) IL-2 receptor common gamma chain (IL-2rgnull); sc, subcutaneously; conc,
concentration.
Supplementary material