Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets

Abstract

Therapeutic strategies for transplantation of pancreatic islet cells are urgently needed to expand β-cell mass by stimulating
islet cell proliferation and/or prolonging islet cell survival. Control of the islets by different growth factors provides
a potential venue for augmenting β-cell mass. In the present study, we show the expression of the biologically active splice
variant-1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor in rat insulinoma (INS-1) cells as well as in rat and
human pancreatic islets. In studies in vitro of INS-1 cells, the GHRH agonist JI-36 caused a significant increase in cell
proliferation and a reduction of cell apoptosis. JI-36 increased islet size and glucose-stimulated insulin secretion in isolated
rat islets after 48–72 h. At the ultrastructural level, INS-1 cells treated with agonist JI-36 revealed a metabolic active
stimulation state with increased cytoplasm. Coincubation with the GHRH antagonist MIA-602 reversed the actions of the agonist
JI-36, indicating the specificity of this agonist. In vivo, the function of pancreatic islets was assessed by transplantation
of rat islets under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic-severe combined immunodeficiency
(NOD-SCID) mice. Islets treated with GHRH agonist JI-36 were able to achieve normoglycemia earlier and more consistently than
untreated islets. Furthermore, in contrast to diabetic animals transplanted with untreated islets, insulin response to an
i.p. glucose tolerance test (IPGTT) in animals receiving islets treated with agonist Jl-36 was comparable to that of normal
healthy mice. In conclusion, our study provides evidence that agonists of GHRH represent a promising pharmacological therapy
aimed at promoting islet graft growth and proliferation in diabetic patients.

Full text

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Agonist of growth hormone-releasing hormone as
a potential effector for survival and proliferation
of pancreatic islets
Barbara Ludwiga,b, Christian G. Zieglera, Andrew V. Schallyc,1, Claudius Richtera, Anja Steffena, Normund Jabsa,
Richard H. Funkd, Mathias D. Brendela,b, Norman L. Blockc, Monika Ehrhart-Bornsteina, and Stefan R. Bornsteina
aDepartment of Medicine III, University Hospital Carl Gustav Carus, 01307 Dresden, Germany;bPaul Langerhans Institute, 01307 Dresden, Germany;
cDepartments of Pathology and Medicine, Divisions of Endocrinology and Hematology-Oncology, Veterans Administration Medical Center, University of
Miami Miller School of Medicine, Miami, FL 33136; anddInstitute of Anatomy, University Hospital Carl Gustav Carus, 01307 Dresden, Germany
Contributed by Andrew V. Schally, April 16, 2010 (sent for review March 25, 2010)
Therapeutic strategies for transplantation of pancreatic islet cells
are urgently needed to expand β-cell mass by stimulating islet cell
proliferation and/or prolonging islet cell survival. Control of the
islets by different growth factors provides a potential venue for
augmenting β-cell mass. In the present study, we show the expres-
sion of the biologically active splice variant-1 (SV-1) of growth
hormone-releasing hormone (GHRH) receptor in rat insulinoma
(INS-1) cells as well as in rat and human pancreatic islets. In studies
in vitro of INS-1 cells, the GHRH agonist JI-36 caused a significant
increase in cell proliferation and a reduction of cell apoptosis. JI-36
increased islet size and glucose-stimulated insulin secretion in iso-
lated rat islets after 48–72 h. At the ultrastructural level, INS-1 cells
treated with agonist JI-36 revealed a metabolic active stimulation
state with increased cytoplasm. Coincubation with the GHRH an-
tagonist MIA-602 reversed the actions of the agonist JI-36, indicat-
ing the specificity of this agonist. In vivo, the function of pancre-
atic islets was assessed by transplantation of rat islets under
the kidney capsule of streptozotocin-induced diabetic non-obese
diabetic-severe combined immunodeficiency (NOD-SCID) mice. Is-
lets treatedwithGHRHagonistJI-36wereabletoachievenormogly-
cemia earlier and more consistently than untreated islets. Further-
more, in contrast to diabetic animals transplanted with untreated
islets, insulin response to an i.p. glucose tolerance test (IPGTT) in
animals receiving islets treated with agonist Jl-36 was comparable
to that of normal healthy mice. In conclusion, our study provides
evidencethatagonistsofGHRHrepresentapromisingpharmacolog-
ical therapy aimed at promoting isletgraft growthand proliferation
in diabetic patients.
diabetes|islet proliferation|regenerative therapies
T
current protocols, the main therapeutic goal that can be reliably
achieved is improved glycemic control and prevention of severe
hypoglycemic episodes. Insulin independence can only be ach-
ieved for a limited time after repeated transplantations (1) due
to insufficient islet mass and progressive loss of islets over time.
Therefore, efforts to improve islet transplantation focus on im-
proving the exploitation of mechanisms governing β-cell pro-
liferation and growth as well as islet quality (2–4).
Several growth factors that may have potential for enhancing
β-cell mass have been identified (5). A natural growth factor-
mediated adaptation of islet cell mass occurs due to increased
demand during pregnancy as well as with obesity (6). In addition,
promotion of islet cell growth has been linked to glucagon-like
peptide 1 (GLP-1), obestatin, and ghrelin (4, 7, 8). Surprisingly,
little attention has been given to the possible role of growth hor-
mone-releasing hormone (GHRH) or its agonists. In his Nobel
lecture more than 60 y ago, Bernardo Houssay described the
critical role of the “hypophysis in carbohydrate metabolism and in
diabetes” (9). He observed that extracts of the anterior pituitary
ransplantation of pancreatic islet cells is a valid treatment
option for selected patients with brittle diabetes. Under
gland can produce a stimulation and hyperplasia of islets under
certain conditions. With the advent of stem cell biology and re-
generative medicine, there has now been a renewed interest in
elucidating the role of hypothalamic-pituitary growth factors in
islet cell regulation.
GHRH stimulates the release of growth hormone (GH) from
the pituitary and has been the focus of intense studies since its
structurewasdescribedin1982(10,11).Thefullbiologicalactivity
of GHRH resides in the N-terminal 1–29 amino acid sequence of
this peptide (12). GHRH and the pituitary type of GHRH re-
ceptor as well as its splice variants are expressed in many human
tissues (i.e., ovary, testis, pancreas, colon, esophagus, breast, kid-
ney, liver, prostate, lungs, and thymus) (13–15).
Recent study has shown that rat GHRH promoted survival of
cardiomyocytes in vitro and protected rat hearts from ischemia-
reperfusion injury (16). The detection of the GHRH receptor
(GHRH-R) on the cardiomyocyte sarcolemma supports the view
that GHRH may elicit direct signal transduction within the
heart, independent of the GH/IGF1 axis per se (17). Synthetic
GHRH agonists, such as JI-36 (GHRH-A), are more potent and
longer-acting than native GHRH (18, 19). Recently, we showed
that GHRH-agonist JI-36 has a favorable cardiac effect, atten-
uating infarct size as well as the progressive decrease of cardiac
structure and function following myocardial infarction (MI) (16).
Finally, GHRH has been shown to promote angiogenesis by in-
creasing vascular endothelial growth factors (VEGF) (20). VEGF
and vascularization play a crucial role in β- cell function and islet
regeneration (21, 22). In the present study, we show expression of
GHRH receptor splice variant-1 (SV-1) (23, 24) in rat insulinoma
INS-1 cells as well as in rat and human pancreatic islets. We also
analyzed the effect of a synthetic GHRH agonist on β- cell survival
and cell proliferation in vitro and in vivo. Inaddition, we tested the
effect of this agonist, JI-36, on β-cells before transplantation in
a diabetic animal model.
Results
Expression of Receptor for GHRH in Insulinoma Cells and in Rat Islet
Cells. RT-PCR analysis showed expression of GHRH receptor
(564 bp) in INS-1 cells and in rat islets. Rat pituitary was used as
a positive control (Fig. 1A). In addition, the protein of the bi-
ologically more-active splice variant SV-1 of GHRH receptor
Author contributions: B.L., A.V.S., R.H.F., M.D.B., M.E.-B., and S.R.B. designed research; B.L.,
C.G.Z., C.R., A.S., and N.J. performed research; A.V.S. contributed new reagents/analytic
tools; B.L., C.G.Z., C.R., A.S., N.J., N.L.B., M.E.-B., and S.R.B. analyzed data; and B.L., C.G.Z.,
A.V.S., and S.R.B. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: andrew.schally@va.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1005098107/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1005098107PNAS
| July 13, 2010
| vol. 107
| no. 28
| 12623–12628
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was detected in INS-1 and rat islets by Western blotting (39.5
kDa). Rat pituitary was used as a positive control (Fig. 1B).
ImmunohistochemicalConfirmation of the Expression of GHRHReceptor
Protein in Insulinoma Cells and Rat and Human Islets. Immunohisto-
chemical analysis showed pronounced GHRH-R immunostaining
of INS-1 cells (Fig. 2 A and D), rat (Fig. 2 B and E), and human
(Fig. 2 C and F) islets. To confirm the localization of GHRH-R on
β-cells, costaining for insulin was performed (Fig. 2F).
Ultrastructural Analysis of Insulinoma Cells Before and After Incubation
with the GHRH Agonist. INS-1 cells under normal culture conditions
were characterized by secretory granules close to the cell mem-
brane (Fig. 3A). The cell surface itself extended long filopodia and
othermembraneprotrusions(Fig.3B).Treatmentofisletcellswith
10−6M JI-36 produced an enlargement of the cell membrane and
the volume of the cytoplasm. This was accompanied by the disap-
pearance of membrane protrusions (Fig. 3C). Furthermore, mito-
chondria and lysosomes were also enlarged; the latter contained
numerous vesicles, indicating intracytoplasmatic digestion of the
contents (peptides, proteins) of secretory vacuoles, after the vac-
uoles fuse with lysosomes. Additional changes became obvious in
the cell nucleus, demonstrating an increased amount of hetero-
chromatin as well as nucleoli (Fig. 3D). These morphological
changessuggestanincreasedactivemetabolicstateoftheisletcells.
Cell Proliferation Studies on Insulinoma Cells. Incubation of INS-1
cells with JI-36 (10−6to 10−9M) for 24–96 h caused a significant
and dose-dependent increase in cell proliferation rates. The most
effective concentration of the agonist was 10−6M, with a 50%
increase after 72 h (Fig. 4 A and C). Coincubation of INS-1 cells
with the GHRH agonist JI-36 (10−6M) and the GHRH antag-
onist MIA-602 (10−6M) for 72 h reversed the proliferation-
stimulating effect of the agonist.
Cell Apoptosis Studies on Insulinoma Cells. Incubation of INS-1 cells
with JI-36 (10−6-10−9M) for 24–96 h resulted in a significant de-
crease in degree of cell apoptosis as measured by the reduction of
activity of caspases 3 and 7. The maximal antiapoptotic effect was
seen after 72 h; the most effective concentration of the agonist
causing this effect was 10−6M (Fig. 4 B and C).
Determination of Islet Number and Islet Volume. Culturesofisolated
rat islets in the presence of JI-36 showed no relevant change in
number of islets over time compared with control islets (Fig. 5A).
Calculation of islet equivalents (IEQ) by relative conversion into
islets of 150 μm diameter showed a significant increase in IEQ/
islet ratio, indicating a relative “islet growth” after 48 h, and up to
72 h, following exposure to JI-36 (Fig. 5B).
Immunohistochemical staining of the islets, after 72 h in culture
with JI-36, for insulin and the proliferation marker Ki-67, showed
colocalizationofthetwomarkers,indicatinganinducedproliferation,
specifically although not exclusively in β-cells (Fig. 5 C and D).
Measurement of Islet Membrane Integrity.Rat islets were evaluated
by fluorescent microscopy using FDA/PI staining. We observed
no difference in islet viability between the groups after 24, 48,
and 72 h in culture (72-h time point: 93 ± 2.2% for control islets,
96 ± 3.3% for islets exposed to JI-36; n = 4). Morphological
appearance following dithizone staining also did not differ be-
tween treatment groups.
Effects of JI-36 on Glucose-Stimulated Insulin Secretion. In a static
model of glucose-stimulated insulin secretion, exposure to JI-36
INS-1 rat islets pituitary
INS-1 rat islets pituitary
A
B
Fig. 1.
detection by Western blots (B) was shown in INS-1 cells and primary rat islets.
Rat pituitary was used as a positive control.
The expression of GHRH-R based on mRNA levels (A) and receptor
INS-1 cells
Pancreatic Islets
Rat
Human
AB
D
E
C
F
Fig. 2.
islets (B and E), and human pancreatic islets (C and F). To show the presence
of GHRH-R protein on islet β-cells, costaining with insulin was performed (F).
Immunohistochemical staining of GHRH-R in INS-1 cells (A and D), rat
Fig. 3.
conditions show secretory granules frequently lining up at the cell membrane
as well as a substantial number of cell membrane protrusions and filopodia
(AandB).Incontrast,isletcellstreatedwithGHRHagonistJI-36(10−6M)revealan
enlargementofthecellmembraneanddisappearanceoffilopodia.Furthermore,
hyperplasia and enlargement of mitochondria as well asa conspicuous inclusion
of vesicles into lysosomes could be found. An increasing amount of heterochro-
matin and nucleoli in the cell nucleus was also documented. These signs suggest
a more-active metabolic state of the islet cells (C and D). (Scale bar: 1 μm.)
Ultrastructural analysis of INS-1 cells. These cells under normal culture
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for 48 h resulted in a slight increase of insulin release into the
culture media after 1 h at basal (3.3 mM) glucose concentration
as compared with control (2.3 ± 0.5 ng/mL vs. 1.7 ± 0.1 ng/mL;
n = 5). Upon stimulation with high levels of glucose (16.7 mM),
insulin release from treated islets was significantly increased (3.6-
fold) relative to insulin release at basal glucose concentration,
whereas untreated islets augmented insulin release only 1.5-fold
(8.2 ± 0.2 ng/mL compared with control 2.6 ± 0.2 ng/mL; n = 5;
P < 0.001; Fig. 6). Thus, treatment of rat islets in vitro more than
doubled total insulin release upon stimulation.
Performance of Islets Exposed to JI-36 in Vivo. For all islet prepa-
rations tested, animals transplanted with islets previously ex-
posed to agonist Jl-36 consistently performed better, with blood
glucose levels reaching the range of normal healthy mice. When
evaluated at day 25, five of six animals from the JI-36 group were
“cured,” and one animal showed partial graft function. In com-
parison, in the control group, only three animals were normo-
glycemic, two had impaired graft function, and one showed graft
failure (Fig. 7A). In the i.p. glucose tolerance test (IPGTT), the
JI-36 group showed an insulin response comparable to that of
normal healthy mice, whereas control islets in animals, classified
as cured on the basis of attaining normoglycemia before chal-
lenge, exhibited delayed and inadequate responses to glucose
challenge (Fig. 7B).
Islet grafts retrieved on day 27 after transplantation were immu-
nostained for insulin and showed stable graft integration (Fig. 7C).
Discussion
The main finding of the present study is that GHRH agonist JI-36
improves β-cell survival and growth as well as metabolic function.
We have shown the expression of mRNA and protein for GHRH
in both rodent and human islets. The GHRH agonist reduced
programmed cell death of β-cells. This was reversed by an an-
tagonist of GHRH. Finally, pretreatment with GHRH agonist
improved β-cell engraftment and metabolic function of islets
following transplantation under the kidney capsule in the strep-
tozotocin-induced diabetic mice. Furthermore, islets treated with
the GHRH agonist before transplantation into diabetic NOD-
SCID mice were able to produce normoglycemia in these mice
earlier and more consistently than islets sham-treated without JI-
36. In addition, JI-36 exposed islets showed a stronger response
upon glucose challenge compared with untreated islets in vitro
and in vivo
GH itself and IGF1, as well as GH-releasing peptides such as
ghrelin and other GH secretagogues, have been shown to increase
β-cell proliferation in transplanted human and fetal rat islets (25,
26). This study, however, shows the potential role of a GHRH
agonist in islet cell proliferation and survival. The detection of the
GHRH receptor on β-cells in rat and human islets supports the
view that GHRH may exert a direct signal transduction within the
pancreas independent and/or in addition to the effects mediated
by the GH/IGF1 pathways.
Though ghrelin and other GH secretagogues may have pleio-
tropic actions with potentially unexpected side effects, the admin-
istrationofGHRHmayofferamorephysiologicalapproachdueto
its direct actions. Our ultrastructural analysis shows an increase of
β-cell cytoplasm with a reduction of cell extensions and filopodia.
Interestingly,arecentstudyhasshownabeneficialeffectofanother
hypothalamic-releasing hormone, corticotrophin-releasing hor-
mone, on β-cell proliferation (27), further emphasizing an impor-
tant connection between the hypothalamic-pituitary axis and the
integrity of insulin-producing cells in the pancreas. Synthetic ago-
nists of GHRH such as JI-36 are more potent and longer acting
than native GHRH or other growth factors. This may open new
therapeutic options. Because there are millions of patients with
type 1 diabetes, and the availability of pancreatic islet donors is
extremely limited, reaching less than a few hundred per year, there
is a desperate need for the development of methods for increasing
the efficiency of β-cell function and islet cell mass. In vitro expan-
sion of islet cell function and mass by the use of growth factors is
therefore of great interest. If future studies can show that this
strategycanbesafelyappliedinvivo,treatmentwithGHRHanalog
may have a tremendous impact also on the prevention and treat-
ment of type 2 diabetes patients. A major feature of diabetes
mellitus type 2 is the progressive loss of β-cell mass over time, very
similar to the situation with transplanted human islets.
We and others have previously shown that by improving the
quality of islets and by a careful quality control of the islets before
transplantation, the results can be substantially improved (2).
Furthermore, multiple studies performed recently have clearly
shown that β- cells are able to replicate under basal conditions
and that β-cell mass can be augmented in response to a variety of
physiological and/or pathophysiologicalstimuli(28).Indeed,ithas
become obvious that the major source of new β-cells during adult
lifeismorelikelyduetotheproliferationofpreexistingβ-cellsthan
the differentiation of progenitor or stem cells in the pancreas (29).
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stimulated cell proliferation significantly (50% increase compared with
control) after 72 h in culture (n = 3). (B) Apoptosis as indicated by activity of
caspases 3 and 7 was significantly reduced by 20% after treatment with JI-36
(10−6M) for 72 h (n = 3). (C) JI-36 treatment dose-dependently increased cell
proliferation and conversely decreased the rate of apoptosis in INS-1 cells
with the maximum effect at 10−6M. ***P < 0.001; **P < 0.01; *P < 0.05.
In vitro effects of GHRH agonist JI-36 on INS-1 cells. (A) JI-36 (10−6M)
Ludwig et al. PNAS
| July 13, 2010
| vol. 107
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| 12625
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Therefore, improving β-cell function and replication in vivo may be
an important therapeutic strategy for both the prevention and the
cure of diabetes mellitus. Although our study was mainly performed
in rodents, we have also shown expression of the receptor in human
islet cells. On the basis of previous studies with other growth factors,
it is appropriate to extrapolate that human islets will have the same
potentialtoexpandandimproveisletcellmassinafashionsimilarto
the results observed in our animal models. In addition to refining
quality of islet cells and islet cell function before transplantation, it
may be possible to improve islet engraftment and reduce the
number of islets needed for a successful outcome by using
a short-term in vivo exposure to the agonist. Previous work has
shown that temporary systemic administration of growth factors
such as hepatocyte growth factor (HGF) may improve graft sur-
vival and blood glucose control in vivo (30). This, however, re-
quires further study in vivo to adequately address safety issues
and the risk of uncontrolled proliferation and tumorigenesis.
In summary, the current long-term efficacy of clinical islet trans-
plantationis rather low.One ofthe major underlyingfactors for this
outcome is the loss of islet mass over time. Therefore, the explo-
ration of mechanisms promoting islet proliferation and growth is
critically important for further progress in the field. The application
of synthetic GHRH agonist for islet proliferation in vitro as well as
graft function and survival in vivo in therapies of diabetes, and our
study showing the importance of local autocrine and paracrine
GHRH in β-cell regulation and growth, suggest a promising re-
generative therapeutic potential for patients with diabetes.
Materials and Methods
Peptide Analogs Preparation. GHRH agonist Jl-36 and GHRH antagonist MIA-
602 were synthesized in the laboratory of author A.V.S. (17, 19, 20).
Rat Insulinoma Cell Line. Rat insulinoma cells (INS-1) were cultured in RPMI
medium1640(PAA)supplementedwith2mML-glutamine,10%FBS,1mMNa-
pyruvate, 50 μM 2-mercaptoethanol, and 100 U/mL penicillin-streptomycin
(Gibco) in a humidified 5% CO2/95% O2atmosphere at 37 °C. The culture
medium was changed every other day. Cells were grown for 72 h before ex-
perimentation. GHRH agonist JI-36 (10−6to 10−9M) and GHRH antagonist
MIA-602 (10−6to 10−7M) were used for 24–96 h, respectively.
Isolation of Rat Pancreatic Islets. Pancreatic islets were isolated from male
Wistar rats according to guidelines established by the University of Dresden
Fig. 5.
between treatment group and control. The bars represent the percentage of islet number compared with islet yield right after isolation (t0). (B) When
converted to islet equivalents (IEQ), a significant difference between JI-36-treated islets and controls was seen after 24 h and continued to increase over time.
Gray bars represent control group (n = 4), black bars represent JI-36-treated islets (n = 4). *P < 0.05. (C and D) Immunostaining of islet serial sections for insulin
(C, brown staining) and Ki-67 (D, brown staining) showed colocalization (arrowheads) of the proliferation marker within β-cells.
Effect of JI-36 on islet number and islet size in vitro (n = 4). (A) The number of islets decreased slightly over time in culture, with no difference
n
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3
4
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6
7
8
9
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.
1
6 . 3
* * *
Secreted Insulin (ng/ml)
normalized to DNA content
l a
u
s
m i t S
a B
d e t a l
Fig. 6.
After equilibration at 3.3 mM glucose, islets were stimulated with high glucose
concentration of 16.7 mM for 1 h. Exposure to JI-36 did not cause a relevant
differenceininsulin secretion at basal conditions. Glucose challenge resultedin
asignificantlyincreasedinsulinrelease3.6-foldrelativetoinsulinreleaseatbasal
glucose concentration when compared with untreated islets that increased in-
sulin release 1.5-fold relative to insulin release at basal glucose concentration
(n = 5). Overall, pretreatment with JI-36 resulted in a more than double insulin
release upon glucose stimulation compared with control (***P < 0.001).
Effect of GHRH agonist JI-36 on glucose-stimulated insulin secretion.
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Institutional Animal Care and Use Committee. Animals were anesthetized by
3% isoflurane; digestion solution (Collagenase V; Sigma-Aldrich) was injected
in situ via the pancreatic common bile duct. Islets were purified by centrifu-
gation on a discontinuous Ficoll gradient (Mediatech). Purified islets were
maintainedinculturemedia(CMRL1066;Mediatech)supplementedwith10%
FBS at 37 °C in a 5% CO2incubator. Volume and purity were determined by
microscopic sizing after staining with dithizone (Sigma-Aldrich).
Isolation of Human Pancreatic Islets. Human pancreata from cadaver donors
were obtained through Eurotransplant following consent for research use
obtained from the next of kin and authorization by the German Foundation
for Organ Transplantation. Islets were isolated using a modification of the
automated Ricordi method (31). Briefly, collagenase NB1, neutral protease
(Serva Electrophoresis), and DNase (Roche) were infused into the main pan-
creatic duct. Islets were separated from exocrine tissue by centrifugation on
acontinuous-densityBiocollgradient(Biochrom)inaCOBE2991cellprocessor.
For determination of purity and islet yield, islet samples were stained with
dithizone (Sigma-Aldrich) and sized using an eyepiece reticle and inverted
microscope. Islets were cultured in CMRL 1066 (Mediatech) containing 2.5%
humanserumalbuminat37°Cina5%CO2incubatorbeforeexperimentation.
Islet Equivalent Determination. Triplicate samples of 100–300 islets were
stained with dithizone (Sigma-Aldrich), which binds zinc ions present specif-
ically in islet β-cells, and sized using an eyepiece reticle and inverted micro-
scope(32).Allisletswithadiameter>50μmweredividedintoclassesof50-μm
increments (i.e., 50–100, 100–150, 150–200, etc.) for calculation of islet
equivalents (IEQ). Each diameter class was converted into the mean volume
of 150-μm diameter islets by a relative conversion factor. These factors allow
converting the total islet number from any preparation into IEQ.
Exposure of INS-1 Cells and Rat and Human Islets to GHRH Analogs. INS-1 cells
were grown for 72 h before experimentation; islets were collected immediately
after the isolation procedure and divided into three treatment groups: (i) culture
mediawithvehicle(DMSO)asacontrolgroup,(ii)culturemediacontainingGHRH
agonistJI-36(10−6M),and(iii)culturemediawithJI-36plusGHRHantagonistMIA-
602(10−6M).Mediachangeandadditionoftheanalogswasperformedafter24h
and 48 h in islet cultures and every other day in INS-1 cell cultures.
Fluorescein Diacetate-Propidium Iodide Viability Staining. Small aliquots of
islets were transferred in PBS-containing Petri dishes. Fluorescein diacetate
(FDA) and propidium iodide (PI) were added to the samples at a final con-
centration of 0.5 and 75 μM, respectively. Using a fluorescence microscope,
100 islets were assessed for cell viability by estimating the percentage of
viable cells (green) vs. nonviable cells (red) within each islet. The percentage
of viable cells was then calculated (33).
Measurement of Insulin Secretion by Static Challenge with Glucose. For static
insulinsecretioninresponsetoglucosechallenge,isletsweretransferredintoPetri
dishescontainingoxygenatedKrebs–Ringerbicarbonatebuffer(137mMNaCl,4.7
mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4-7 H2O, 2.5 mM CaCl2-2 H2O, 25 mM
NaHCO3,0.25%BSA)andpreincubatedin3.3mMglucoseat37°C(5%CO2)for30
min.Groupsof8–10isletsfromtheequilibrationculturesweretransferredtofresh
oxygen-saturated media containing either 3.3 or 16.7 mM glucose and then in-
cubated an additional 60min in a37 °C water bath withgentleshaking.Secreted
insulin in the media was measured by ELISA (Millipore) and values normalized to
extracted islet DNA (Quant-iT PicoGreen; Invitrogen).
In Vivo Islet Functional Assessment. NOD-SCID mice (MTZ breed) with induced
diabetes were used as islet recipients following guidelines established by the
University of Dresden Institutional Animal Care and Use Committee. Diabetes
was induced by a single i.p. injection of 180 mg/kg streptozotocin (Sigma-
Aldrich). Serum glucose was then measured daily using an Ascensia Elite
glucometer(Bayer).Micewereconsidereddiabeticifnonfastingbloodglucose
was >350 mg/dL for 2 or more consecutive d. Rat islet preparations were used
for transplantation. Islets from each preparation were divided into two
groups, and JI-36 (10−6M) or vehicle (DMSO) was added to the culture media.
Islets were cultured for 48 h before transplantation. After culture, samples of
300IEQwerewashedintransplantmedia(Ringeracetatewith5%glucoseand
10% FBS) and transplanted to beneath the left kidney capsule. The animal
experiments andhousing were inaccordance with institutionalguidelines and
German animal regulations.
Posttransplant Follow-Up. The mice were observed for 30 d after trans-
plantation. The nonfasting blood glucose levels were measured daily during
thefirstweek andtwiceaweekthereafter. On day25,micewere subjected to
an IPGTT. Two days later, grafts were removed. This led to a recurrence of the
diabetic state. This suggests that restoration and maintenance of normo-
glycemia was to the result of islet graft function.
Definition of Metabolic Control. On follow-up, sustained nonfasting blood
glucose levels of ≤10 mM (≤180 mg/dL) were defined as “cure,” 10–18 mM
(180–320 mg/dL) as “partial function” of transplanted islets, and levels above
18 mM (>320 mg/dL) as “graft failure.”
Glucose Tolerance Test. Mice were fasted overnight (at least 6 h) before exami-
nation.Aglucosesolutionwasgivenat3g/kgbodyweighti.p.,andbloodglucose
wasrecordedbeforeinjectionand15,30,45,60,90,and120minfollowingglucose
injection. Nontransplanted mice were used as controls and tested concurrently.
-4-20246810 12 14 16 18 20 22 24 26 28 30
Days post Tx
0
5
10
15
20
25
30
35
Control (n=6)
JI-36 (n=6)
T
T
G
P
I
y
m
o
t c
e
r
h
p
e
N
) l / l o
m
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d
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B
0 10 20
Time post Glucose Injection (min)
30 4050 6070 8090 100 110 120
0
5
10
15
20
25
Control (n=3)
JI-36 (n=5)
) l / l o
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m
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o
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B
A
B
C
Fig. 7.
of streptozotocin- induced diabetic NOD-SCID mice. (A) After transplantation,
the control group showed a delayed decrease in blood glucose levels, and only
threeofsixanimalsshowedstablenormoglycemiaduringthefollow-upperiod.
Animals receiving a graft of islets pretreated with JI-36 showed rapid and
persistent recovery from diabetes (five of six animals). (B) On day 25 following
islettransplantation,animalswithnormal glucose controlweresubjected toan
IPGTT. Thoughcontrol animals respondedina delayed and insufficient manner
to the glucose challenge, the group treated with JI-36 was able to revert initial
hyperglycemia to normal ranges within 2 h. Shaded area highlights normal
range of blood glucose. (C) Pancreatic islets were treated with JI-36 before
transplantation beneath the kidney capsule. Representative immunostained
section for insulin (green) shows stable graft integration after 27 d.
Islet transplantation (300islet equivalents) beneaththe kidneycapsule
Ludwig et al. PNAS
| July 13, 2010
| vol. 107
| no. 28
| 12627
MEDICAL SCIENCES