From the Division of Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
Received for publication, August 2, 2002, and in revised form, October 8, 2002
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ABSTRACT |
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Hepatocyte growth factor (HGF) increases beta
cell proliferation and function in rat insulin promoter (RIP)-targeted
transgenic mice. RIP-HGF mouse islets also function superiorly to
normal islets in a transplant setting. Here, we aimed to determine
whether viral gene transfer of the HGF gene into mouse islets ex
vivo could enhance the performance of normal islets in a
streptozotocin-diabetic severe combined immunodeficient mouse marginal
islet mass model in which 300 uninfected or adenovirus (Adv)
LacZ-transduced islet equivalents were insufficient to correct
hyperglycemia. In dramatic contrast, 300 AdvHGF-transduced islet
equivalents promptly (day 1) and significantly (p < 0.01) decreased random non-fasting blood glucose levels, from 351 ± 20 mg/dl to an average of 191 ± 7 mg/dl over 8 weeks. At day 1 post-transplant, beta cell death was significantly (p < 0.05) decreased, and the total insulin content was significantly (p < 0.05) increased in AdvHGF-transduced islets
containing grafts. This anti-beta cell death action of HGF was
independently confirmed in RIP-HGF mice and in INS-1 cells, both
treated with streptozotocin. Activation of the phosphatidylinositol
3-kinase/Akt intracellular-signaling pathway appeared to be involved in
this beta cell protective effect of HGF in vitro. In
summary, adenoviral delivery of HGF to murine islets ex
vivo improves islet transplant survival and blood glucose control
in a subcapsular renal graft model in immuno-incompetent diabetic mice.
Recent clinical studies document that human islet transplantation
has the potential to replace pancreatic endocrine function in patients
with Type 1 diabetes (1, 2). One major drawback to this otherwise
exciting story is the requirement for islets derived from two to four
cadaver donors to treat a single patient with diabetes. Thus, an
enormous shortage of human islets is a barrier to the use of islet
transplantation on a larger scale. To solve this problem, one potential
approach is the use of factors that can increase beta cell
proliferation and/or function and/or protect against beta cell death.
Several groups have identified factors that can induce pancreatic islet
cells to expand, to augment their function, or to be more resistant to
cell death. Examples include insulin-like growth factors, glucagon-like
peptide 1, exendin, parathyroid hormone-related protein, placental
lactogen, and hepatocyte growth factor
(HGF)1 (3-11).
Recently, we have developed a transgenic mouse model that overexpresses
HGF in the pancreatic beta cell under the control of the rat insulin II
promoter (RIP) (10, 11). These RIP-HGF transgenic mice display
increased beta cell proliferation coupled with increased beta cell
function, the latter resulting from an increase in the expression of
insulin, glucokinase, and the glucose transporter, Glut-2. We have also
shown that RIP-HGF transgenic mouse islets are significantly more
effective than normal islets when transplanted into severe combined
immunodeficient (SCID) diabetic mice (11). Thus, a marginal number of
normal islets was insufficient to normalize blood glucose levels in
SCID diabetic mice. In contrast, the same number of islets from RIP-HGF
transgenic mice matched for size, protein, or DNA rapidly and
completely normalized the blood glucose (11).
These findings suggest that gene transfer of the HGF cDNA into
human islets could reduce the number of islets and donors required to
correct diabetes in a human transplant setting. In the present study,
we therefore have explored the delivery of HGF to mouse islets using an
adenoviral delivery system. Specifically, we sought to (i) investigate
the feasibility of adenoviral gene delivery of HGF in mouse islets
ex vivo and (ii) determine the effect of adenovirus-mediated
HGF overexpression on the performance of mouse islets in a marginal
mass model of islet transplant.
As anticipated, adenoviral delivery of HGF to mouse islets ex
vivo markedly improved islet transplant survival and performance. To our surprise, this protective effect was immediate and unlikely to
be explained simply by improved beta cell proliferation. Instead, this
rapid protective early effect was likely explained by a combination of
enhanced beta cell function in the graft induced by HGF, as occurs
in vivo in the RIP-HGF mice, and/or increased graft beta cell survival, a novel beta cell action of HGF. The ability of HGF to
enhance beta cell survival was directly documented using standard cell
death assays and was confirmed in RIP-HGF mice and the beta cell line,
INS-1. Mechanistically, the survival effect of HGF in beta cells
appeared to involve the PI3-kinase/Akt pathway and likely other
pathways. Collectively, our prior studies with RIP-HGF mice together
with the results reported in the present manuscript using adenoviral
HGF gene therapy suggest that HGF overexpression in the islet markedly
improves islet graft performance through a combination of effects
including improved beta cell survival, enhanced glucose sensing and
insulin secretion, and accelerated beta cell proliferation. Although
the precise quantitative definition of these effects at specific time
points in the post-transplant period will require additional study,
each would appear to be important. These results suggest that ex
vivo gene transfer of HGF into human islets may be particularly
useful in improving islet transplant performance and reducing the
number of islets required for humans with Type 1 diabetes.
Generation of Recombinant Adenovirus Vectors--
Adenovirus was
prepared according to the methods of Becker et al. (12)
using Ad.5 constructs generously provided by Dr. Christopher Newgard
(Duke University, Durham, NC). Complementary DNAs encoding
Islet Isolation and Gene Transfer--
Murine islets from adult
CD-1 mice were isolated as previously described (10). Briefly, the
pancreas was injected through the pancreatic duct with Hanks' buffered
saline solution containing collagenase P, removed, incubated for 17 min
at 37 °C, and then filtered through a 500-µm wire mesh. The
digested pancreas was rinsed with Hanks' buffered saline solution, and
islets were separated by density gradient in Histopaque (Sigma). After
several washes with Hanks' buffered saline solution, islets were
hand-picked under a microscope.
Mouse pancreatic islets were washed with RPMI 1640 medium and exposed
to purified adenovirus for 1 h at 37 °C. Islets were then
washed and incubated in 1 ml of RPMI 1640 medium containing 10% fetal
bovine serum, 5 mM D-glucose, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Twenty-four hours after the
infection, islets were harvested and used in the experiments described
below. At 24 h after infection, with 250 m.o.i. AdvLacZ under
these conditions, 29 ± 6% (n = 4) of islet cells
were stained for X-gal following the standard methods for staining
islets infected with AdvLacZ (13). This infection rate is comparable
with that reported previously with rat and human islets (13, 14).
RNA Isolation, Reverse Transcription, and Relative
Semiquantitative PCR Analysis of HGF mRNA Expression--
Total
DNA-free RNA was isolated from uninfected or adenovirus-transduced
islets by using the DNA-free RNA isolation kit (Ambion, Austin, TX),
according to the manufacturer's instructions. Reverse transcription
was performed as previously described (11). PCR was performed with 5 µl of cDNA in a final volume of 25 µl containing 1X
Taq buffer (Promega, Madison, WI), 2.5 mM
MgCl2 (Promega), 200 µM dNTPs (Promega), 0.5 µCi of [ Immunoblot Analysis of HGF Protein Expression--
Islet
extracts were made in freshly prepared ice-cold lysis buffer
(phosphate-buffered saline (PBS) with 1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM sodium orthovanadate, 100 µg/ml phenylmethylsulfonyl fluoride, and 57 µg/ml aprotinin), incubated for 30 min on ice, and sonicated, the
supernatant containing the cell lysate was separated, and protein
concentrations were measured using the MicroBCA assay (Pierce). Fifty
micrograms of protein from uninfected or adenovirus-infected islets
were resuspended in loading buffer containing Glucose-stimulated Insulin Secretion in Isolated
Islets--
Insulin release from uninfected and adenovirus-transduced
islets was measured in triplicate for each glucose concentration tested
as previously described (11), with some modifications. Briefly, islets
obtained 24 h after infection with AdvLacZ or AdvHGF and
uninfected islets were preincubated in Krebs-Ringer bicarbonate buffer
supplemented with 10 mM HEPES, 1% bovine serum albumin,
and 5.5 mM glucose for 1 h at 37 °C in a 5%
CO2 incubator. After washing the islets once with the same
solution, groups of 10 islets of similar size for each condition were
incubated in 1 ml of fresh Krebs-Ringer bicarbonate buffer plus 1%
bovine serum albumin and 5.5 or 22.2 mM glucose at 37 °C
in the 5% CO2 incubator. After a 30-min incubation, buffer
was removed and frozen at Islet Transplantation--
Murine CD-1 islets isolated and
transduced with AdvLacZ or AdvHGF as described above were transplanted
under the kidney capsule of streptozotocin (STZ)-induced diabetic, SCID
mice (BALB/cByJ) (The Jackson Laboratory, Bar Harbor, ME) as previously
described (11). Briefly, SCID mice were rendered diabetic by injecting 250 mg/kg of body weight of STZ intraperitoneally. Diabetes was confirmed by the presence of hyperglycemia (>300 mg/dl), polyuria, and
weight loss. Random non-fasted blood glucose was measured from the
snipped tail by a Precision Q.I.D. portable glucometer (Medisense,
Bedford, MA). After three consecutive days of hyperglycemia, SCID
diabetic mice were transplanted with either 300 uninfected or AdvLacZ-
or AdvHGF-transduced islet equivalents (IE) (1 IE = 125 µm
diameter islet) beneath the kidney capsule, as previously described
(11). Blood glucose levels were measured at days 1, 4, and 7 and then
weekly through day 56 after transplantation. At day 54, retroorbital
bleedings were performed to measure plasma insulin levels. At day 56, mice transplanted with 300 IE transduced with AdvHGF, AdvLacZ, or
uninfected islets underwent unilateral nephrectomy. The kidneys were
fixed in Bouin's solution, paraffin-embedded, sectioned, and stained
with the anti-insulin antibody, as previously reported (10).
Intraperitoneal Glucose Tolerance Test--
Forty-nine days
after islets were transplanted in STZ-induced SCID diabetic mice (see
above), glucose tolerance was analyzed in 16-h-fasted mice by
intraperitoneal injection of 2 g of glucose/kg of body weight as
previously reported (11). Blood samples were obtained from the snipped
tail and analyzed for glucose levels using the portable glucometer.
Determination of Insulin Content--
Insulin was extracted from
graft-containing kidneys as previously reported (10) with some
modifications. Briefly, graft-containing kidneys were washed in
ice-cold PBS, finely minced, passed through a 21-gauge needle,
resuspended in 5 ml of acid/ethanol (0.18 M HCl in 70%
ethanol), sonicated for 15 s, and extracted at Quantification of Beta Cell Death by Propidium Iodide
Staining--
Pancreata and graft-containing kidneys from four animals
per group in each case were fixed in Bouin's solution, embedded in paraffin, and sectioned. In the graft-containing kidneys, three serial
sections separated by 25 µm each were immunostained after deparaffinization and rehydration. Beta cell death was detected by
co-staining for insulin and propidium iodide (Sigma) as
previously reported (15). Immunostaining was carried out with a guinea pig anti-porcine insulin antibody (Zymed Laboratories
Inc., San Francisco, CA) as primary antibody followed by a
fluorescein isothiocyanate-conjugated rabbit anti-guinea pig IgG
secondary antibody (Zymed Laboratories Inc.). After
several washes with PBS, samples were incubated for 10 min at 37 °C
with 2 µg/ml propidium iodide and 100 µg/ml RNase I (Ambion) made
in PBS, washed several times with water, and cover-slipped using the
Prolong antifade kit (Molecular Probes, Inc., Eugene, OR).
Terminal Deoxynucleotidyltransferase (TdT)-mediated dUTP Nick
End-labeling (TUNEL) and Insulin Staining in RIP-HGF Mice--
Twelve
hours after the treatment of four RIP-HGF transgenic mice and four
normal littermates (3-6 months old) with a single intraperitoneal
injection of streptozotocin (150 mg/kg of body weight), pancreata were
removed, fixed in Bouin's solution, embedded in paraffin, sectioned,
and immunostained after deparaffinization and rehydration. Cell death
was detected by enzymatic in situ labeling for DNA strand
breaks using the TUNEL method using the in situ cell death
detection kit (Roche Molecular Biochemicals) according to the
manufacturer's protocol. Staining was achieved using nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Vector Laboratories
Inc., Burlingame, CA) as substrate. Subsequent to TUNEL, sections were
stained with the anti-insulin antibody described above. Visualization
was achieved using antibody-coupled peroxidase and diaminobenzidine
tetrahydrochloride substrate (Biogenex, San Ramon, CA).
Cell Viability in STZ-treated INS-1 Cells--
Cell viability
was assessed by using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Sigma) assay and by cell counting. Viability tests were performed in
rat insulinoma cells (INS-1) cells kindly provided by Dr. Doris
Stoffers (University of Pennsylvania School of Medicine) and seeded at
a density of 4 × 104 cells/well onto 96-well plates
in RPMI 1640 medium containing 11 mM D-glucose
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM Phosphatidylinositol-3 Kinase (PI3-kinase) Assay--
INS-1
cells and mouse islets were serum-depleted for 24 h before the
addition of 25 ng/ml HGF. After a 10-min incubation, cells were rinsed
once with ice-cold PBS and twice with ice-cold buffer A (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2,
and 100 µM Na3VO4). Cells were
solubilized in buffer A containing 1% Nonidet P-40 and 10% glycerol,
cell extracts were centrifuged at 13,000 × g for 10 min, and the protein content in the supernatant was measured by the
Micro BCA method (Pierce). PI3-kinase activity was measured as
previously reported (17) with some modifications. Briefly, protein
extracts were incubated with either a 1:200 dilution of rabbit
anti-PI3-kinase p85 Protein Kinase B/Akt Activity--
The protein kinase
B/Akt immunoprecipitation and kinase assay was carried out with a
glycogen synthase kinase 3 phosphorylation kit (New England Biolabs,
Beverly, MA) following the manufacturer's procedure. Briefly, after
stimulation of the cells with 25 ng/ml HGF for 30 min, 200 µg of cell
lysate was incubated with protein G/Akt 1 monoclonal antibody complex
for 2 h at 4 °C. After washing twice with cell lysis buffer and
twice with kinase buffer, the immunoprecipitates were resuspended in
kinase buffer containing 200 µM ATP and 1 µg of
glycogen synthase kinase 3 fusion protein and incubated at 30 °C for
30 min. The reaction was stopped with SDS gel-loading buffer, and
samples were analyzed by immunoblotting (as shown above) with the
phospho-glycogen synthase kinase 3 Statistical Analysis--
Results are expressed as the
means ± S.E. Statistical differences were determined by
two-tailed unpaired Student's t test and analysis of
variance for repeated measurements. A p value less than 0.05 was considered statistically significant.
HGF Expression in Adenovirus-transduced Mouse Islets--
Murine
islet expression of HGF was analyzed using reverse transcription-PCR
and Western blot in adenovirus-transduced mouse islets harvested
24 h after infection. As shown in Fig.
1, HGF was highly expressed in
AdvHGF-transduced islets both in terms of mRNA (Fig. 1A)
as well as protein (Fig. 1B) as compared with non-transduced
or AdvLacZ-transduced islets. These results indicate that
AdvHGF-transduced murine islets effectively produce HGF.
Insulin Secretion by Adenovirus-transduced Mouse Islets--
To
determine whether insulin secretion by mouse islets was adversely
affected by adenovirus infection, glucose-stimulated insulin secretion
was examined 24 h after adenoviral transduction. Insulin release
was corrected for islet protein as described under "Materials and
Methods" and in the legend to Fig. 2.
The insulin secretory response from islets transduced with 250 m.o.i. of AdvLacZ or AdvHGF was similar to non-transduced islets (Fig.
2). This indicates that adenovirus-transduced islet function at
250 m.o.i. is not adversely affected. In contrast,
glucose-stimulated insulin secretion was markedly blunted in islets
transduced with 500 m.o.i. of either AdvLacZ or AdvHGF (Fig. 2).
These studies suggest that 250 m.o.i. is an optimal and perhaps
maximal m.o.i. for the studies described below.
Islet Transplantation of Adenovirus-transduced Mouse Islets into
Diabetic SCID Mice--
We next compared the performance of uninfected
and AdvLacZ- and AdvHGF-transduced islets in vivo using a
marginal mass islet renal transplant model in STZ-induced diabetic SCID
mice. As is clear in Fig. 3A,
300 uninfected IE (125-µm diameter) were insufficient to maintain
euglycemia during the 8-week period of this study. Similarly, 300 AdvLacZ-transduced IE were unable to sustain a blood glucose value
under 300 mg/dl (Fig. 3A). In marked contrast, however, 300 AdvHGF-transduced IE were able to immediately reduce blood glucose
concentrations in the diabetic SCID renal transplant model (Fig.
3A). Furthermore, this dramatic decrease in the blood glucose was sustained for the complete 8 weeks of the study. These glucose concentrations were significantly lower (p < 0.01) than in mice transplanted with 300 uninfected or
AdvLacZ-transduced IE. In addition, random, non-fasting plasma insulin
levels at day 54 after the transplant were significantly
(p < 0.025) increased in SCID mice transplanted with
AdvHGF-transduced islets (1.62 ± 0.24 ng/ml, n = 8) compared with mice transplanted with AdvLacZ-transduced islets
(0.90 ± 0.11 ng/ml, n = 10). After removal of the
kidney containing the AdvHGF islet grafts on day 56, blood glucose
levels immediately returned to pre-transplant diabetic levels,
confirming that the transplant was responsible for reducing the blood
glucose (Fig. 3A). Representative photomicrographs of kidney
sections containing the grafts and stained for insulin are shown in
Fig. 3B. Grafts were easily and abundantly visualized in
each of the eight kidneys containing AdvHGF-transduced islets.
This was in marked contrast to kidneys containing AdvLacZ-transduced
islets in which the islet graft was either undetectable despite
aggressive sectioning (8 of 10 mice) or minuscule (2 of 10). These
studies collectively indicate that AdvHGF-transduced islets, when
transplanted under the kidney capsule into SCID diabetic mice, clearly
improve blood glucose control and graft survival compared with mice
transplanted with uninfected or AdvLacZ-transduced islets.
To further define the function of the transplanted AdvHGF-transduced
islets, glucose tolerance tests were performed on day 49. As shown in
Fig. 4, basal fasting blood glucose
levels were significantly lower (p < 0.05) in SCID
mice transplanted with 300 AdvHGF IE (96 ± 6 mg/dl) as compared
with SCID mice transplanted with 300 uninfected (161 ± 20 mg/dl)
or AdvLacZ-transduced IE (160 ± 18 mg/dl). Importantly, after
intraperitoneal glucose injection, glucose tolerance was markedly and
significantly (p < 0.01) improved in SCID mice
receiving AdvHGF islets as compared with uninfected or AdvLacZ islets.
As compared with normal SCID mice, glucose tolerance in AdvHGF mice was
slightly impaired, although these differences were only significant at
the basal and 180-min time points (p < 0.01 and
p < 0.05, respectively) (Fig. 4). These results collectively indicate that at 7 weeks after transplant of an equivalent mass of islets, adenovirally HGF-enhanced islets lead to superior fasting glucose control and to superior glucose tolerance as compared with control islets and that glucose tolerance approaches that observed
in normal mice.
Beta Cell Death and Insulin Content in Adenovirally Transduced
Islet Grafts on Day 1 after Transplant--
The first few hours and
days after islet transplantation are characterized by substantial islet
cell dysfunction and death (18, 19). In our transplant studies,
improvement in blood glucose values was easily apparent on day 1 in
animals receiving AdvHGF-transduced islets. This temporally rapid
effect on graft survival suggested that in addition to the well
characterized effects of HGF to increase beta cell proliferation (10,
20-22) and glucose-stimulated insulin secretion (11), HGF may also confer a previously unrecognized survival benefit on beta cells in the
transplant setting. To address this question, we transplanted 300 AdvLacZ- or AdvHGF-transduced IE under the renal capsule of SCID
diabetic mice and sacrificed the animals 24 h later. The number of
dead beta cells in the grafts at 24 h was quantitated. As is
obvious in Fig. 5, A and
B, the number of condensed, pyknotic beta cell nuclei
24 h after transplant was significantly (p < 0.05) reduced in AdvHGF grafts compared with AdvLacZ grafts.
Furthermore, the insulin content in the AdvHGF grafts was also
~2.5-fold higher (p < 0.05) than in the AdvLacZ
grafts (Fig. 5C). These results demonstrate that
HGF-enhanced islets have a survival advantage as early as
day 1 after transplantation. In addition, the increase in insulin
content in AdvHGF-transduced islet-containing grafts might
also reflect an increase in insulin expression induced by HGF in the
beta cells of the graft, as previously observed in vivo in
RIP-HGF mice (10, 11).
Beta Cell Death Induced by STZ in RIP-HGF Transgenic
Mice--
This novel protective effect of HGF on beta cells was
confirmed independently using a different model system and a different inducer of cell death. We used our previously described RIP-HGF transgenic mouse model for this purpose (10). Fig.
6 shows the effect of intraperitoneal
injection of 150 mg/kg STZ on beta cell death in normal and
RIP-HGF islets. Pancreatic sections co-stained for insulin and TUNEL or
propidium iodide displayed numerous TUNEL-positive or condensed
pyknotic nuclei, respectively, in the islets of normal mouse pancreata
compared with RIP-HGF transgenic pancreata (Fig. 6, A and
C). Quantification of these TUNEL-positive beta cells or
condensed pyknotic beta cell nuclei showed that the index of beta cell
death was 3-5-fold higher (p < 0.01) in normal
littermates as compared with RIP-HGF transgenic mice (Fig. 6,
B and D). These observations independently
confirm that HGF overexpression confers a survival advantage on beta
cells and extend it to two types of cell death agonists,
ischemia/transplantation and STZ.
Cell Viability in INS-1 Cells Treated with STZ and HGF in
Vitro--
The cellular basis of the HGF protective effect against
beta cell death is difficult to study in vivo. We therefore
sought to develop an in vitro model of HGF-induced beta cell
cytoprotection in which to study the cellular mechanisms underlying
this phenomenon. We thus examined the ability of HGF to confer
protection of INS-1 cells in vitro against STZ-induced
cell-death. Cell viability was assessed in INS-1 cells treated with 25 ng/ml HGF and 0.5 or 1 mM STZ in complete medium for
24 h (Fig. 7, white
bars). As shown in the figures, STZ
at 0.5 and 1 mM dramatically reduced cell viability in
INS-1 cells as assessed by both MTT assay (Fig. 7A) and cell
number (Fig. 7B). However, HGF treatment significantly increased the number of viable INS-1 cells 24 h after the addition of 0.5 or 1 mM STZ (Fig. 7, A and B).
These results document the cytoprotective effect of HGF in a third
independent system and indicate that INS-1 cells may serve as a useful
model system for studying cellular mechanisms underlying the anti-cell
death effects of HGF in beta cells.
PI3-kinase Activity Is Induced by HGF in INS-1 Cells, and Its
Blockade Leads to Decreased Cell Viability--
HGF is known to
increase PI3-kinase activity in human islets and other cell types
in vitro (23-25). In addition, the PI3-kinase/Akt pathway
is involved in the survival effects of HGF in several non-beta cell
systems (26-29). To investigate whether the PI3-kinase/Akt pathway is
involved in the protective effect of HGF in the STZ-induced cell death
in INS-1 cells, we examined PI3-kinase activity in cell extracts from
INS-1 cells treated with HGF. As shown in Fig. 8A, after stimulation of INS-1
cells with 25 ng/ml HGF for 10 min, a significant increase in
PI3-kinase activity was observed. Furthermore, mouse islets incubated
for 10 min with 25 ng/ml HGF also exhibited an increase in PI3-kinase
activity (Fig. 8A). Moreover, transgenic HGF-overexpressing
islets from the RIP-HGF mouse also showed increased PI3-kinase activity
(Fig. 8A). PI3-kinase stimulation results in phosphorylation
and activation of Akt. Akt activity was therefore examined in INS-1
cells incubated with 25 ng/ml HGF for 30 min. As expected, HGF rapidly
increased protein kinase B activity (Fig. 8B). These results
confirm that PI3-kinase/Akt signaling pathway is activated by HGF in
pancreatic beta cells.
We next investigated the potential involvement of the PI3-kinase/Akt
pathway in the protective effect of HGF in INS-1 cells by inhibiting
this pathway with wortmannin. Fig. 7 (black bars) demonstrates that the treatment of INS-1 cells with 1 µM
wortmannin completely abolishes the protective effect of HGF. Indeed,
wortmannin enhanced the degree of cell death observed even under basal
conditions as well as after STZ treatment in the absence of HGF (Fig.
7). This suggests that basal cellular PI3-kinase/Akt activity present in INS-1 cells (Fig. 8) may play a role in beta cell survival. Together, these results suggest that the protective effect of HGF
against STZ-induced cell death in INS-1 cells involves the PI3-kinase/Akt pathway.
The studies of Shapiro and co-workers (1, 2) demonstrate the
efficacy of human islet transplantation for Type 1 diabetes. They have
also highlighted an acute shortage of human beta cells available for
transplantation into the millions of Type 1 diabetics who could benefit
from such therapy. We and others previously demonstrated that HGF
induces islet proliferation in vitro and also in
vivo in the RIP-HGF transgenic mouse (10, 20-22). We have also
demonstrated that islets of RIP-HGF mice 1) are larger and more
plentiful than normal, 2) display increased expression of three
proteins critical to beta cell differentiation and function (insulin,
glucokinase, and the Glut-2 transporter), 3) sense glucose and secrete
insulin more robustly than control islets, 4) therefore imbue on their
recipients enhanced glucose tolerance in vivo as assessed
using an intraperitoneal glucose tolerance test, and 5) function in a
fashion superior to normal islets when transplanted into diabetic mice
(10, 11). In short, HGF, both in vitro and in a transgenic
setting, simultaneously enhances both the proliferation and function of
beta cells and does so in a quantitatively very significant fashion.
These observations suggested to us that ex vivo delivery of
HGF gene might have therapeutic efficacy in enhancing islet transplant outcomes.
In the studies described herein, we demonstrate two broad types of
novel observations. First, we demonstrate that adenoviral gene delivery
of HGF to completely normal murine islets before transplantation
markedly improves graft performance in the diabetic recipient. If
applicable to human islet transplantation, this approach could
significantly reduce the number of human islets required to correct
diabetes in patients with Type 1 diabetes. We believe this is the first
successful example of beta cell gene therapy with a growth factor
leading to normalization of blood glucose in the setting of islet
transplantation. Second, we demonstrate a novel and striking additional
beneficial effect of HGF on the beta cell; in addition to its ability
to stimulate proliferation and induce key glucose-sensing and insulin
secretory proteins in beta cells, it also has a rapid, potent and
beneficial effect to enhance beta cell survival in the immediate
post-transplant period.
In these experiments, we employed adenovirus to deliver HGF to islets.
We selected adenovirus because it is easy to prepare and is highly
efficient (12). In rat and human islets, the infection rate has been
reported to vary between 20 and 50% depending on the m.o.i. and the
detection method used (13, 14). In the present studies, the infection
rate in mouse islets (29%), as assessed by standard X-gal-staining
methods in islets infected with AdvLacZ, was in the range of those
previously reported (13, 14). It is important to note that HGF is a
secreted paracrine factor and likely influences a greater number of
islet cells than the ~30% that have been targeted by the adenovirus.
It likely exerts its effects in islet cells that have HGF receptors
such as the beta cell (21).
As shown in Fig. 1, Western blot analysis of HGF expression in
AdvHGF-transduced islets reveals the presence of two forms of HGF, the
single chain precursor and heterodimeric mature HGF. HGF is produced as
an inactive precursor that awaits activation by extracellular
proteases. Several proteases have been reported to date to activate HGF
in vitro, including blood coagulation factor XIIa,
urokinase, tissue-type plasminogen activator, and a serum-derived
serine protease named HGF activator (31-34). In the rat endocrine
pancreas, tissue-type plasminogen activator has been detected
preferentially in somatostatin cells (35). Furthermore, it has been
shown that HGF activation is up-regulated specifically in injured
tissues (36). Taken together, these studies suggest that in a
transplant setting in which the islets are implanted into a hostile
environment (hypoxia, and nutrient deprivation), inactive HGF might
likely be activated by proteases inside or surrounding the graft.
We employed SCID mice as recipients, for we anticipated using this
model for human islet xenografts in the future. An additional benefit
of SCID mice is that they should not display immune intolerance to
adenovirus, potentially allowing for prolonged expression of HGF in the
graft. Although we have not tested this hypothesis, others have
demonstrated the presence of adenovirus in islet grafts up to 20 weeks
after islet transplant in syngeneic immunocompetent mice (37). Thus, in
our studies the model system was designed as a "proof of principle"
model to ask the question, Can HGF, delivered to normal islets by gene
transfer before transplantation, enhance the engraftment and
performance of normal islets in diabetic recipients? We also employed a
model of marginal beta cell transplant mass so that improvements over a
grossly inadequate base line could be observed. The results of these
studies are very clear; that is, adenoviral delivery of HGF to
otherwise normal CD-1 murine islets dramatically improves islet
performance in a transplant setting, resulting in better diabetes
control in their diabetic SCID mouse hosts.
These observations raise several new questions. One key question was,
Why is the beneficial effect of HGF gene delivery so apparent at the
very earliest time point examined, day 1 post-transplant? It has been
shown recently that the early failure of islet cell grafts is due to
rapid cell death of the large majority of transplanted beta cells in
the immediate post-transplant period (18, 19). Thus, the very rapid
normalization of blood glucose observed in Fig. 3A suggests
that HGF may have an additional beneficial effect on beta cells to
enhance beta cell survival in the immediate post-transplant period. As
described below, this proves to be correct. Although HGF has been
demonstrated in other tissues to have pro-survival and
anti-apoptotic effects (26-29), this has not been demonstrated in
beta cells previously. In addition to this pro-survival effect, it is
also possible that the enhanced glucose-stimulated insulin secretion we
have previously described in RIP-HGF islets also contributes to this
early enhancement of graft function. Future studies are required to
define the relative contribution of the several beneficial effects of
HGF on the beta cell (increased proliferation versus
improved function versus enhanced survival) to the improved
islet graft performance induced by AdvHGF.
A second key question is, What is the cellular mechanism responsible
for the protective effect of HGF on beta cells? HGF has been shown to
induce anti-apoptotic actions via the PI3-kinase/Akt signaling pathway
in several different cell types subjected to a variety of cell death
agonists (26-29). In addition, overexpression of active Akt1 in the
pancreatic beta cell in transgenic mice confers complete resistance to
experimental diabetes induced by STZ (38-39). In our studies, we found
that HGF increases PI3-kinase and protein kinase B/Akt activities in
INS-1 cells in vitro. Importantly, the increased survival
effect induced by HGF in INS-1 cells treated with STZ was completely
blunted when these cells were treated with wortmannin, indicating that
activation of PI3-kinase and protein kinase B/Akt seems to be essential
for HGF to induce its protective effect on beta cells. These results
demonstrate for the first time that HGF through PI3-kinase/Akt
activation increases pancreatic beta cell survival. Additional studies
exploring the potential targets in the cell death pathways
activated/inactivated by HGF in pancreatic beta cells through the
PI3-kinase/Akt signaling pathway are in progress.
A third key question is, Would this approach, which is effective in a
highly artificial immunodeficient renal graft mouse model, have similar
efficacy in a more authentic setting that includes an intact immune
system and the vicissitudes of immunosuppression and autoimmune and
alloimmune considerations that would apply to human islet transplant
and portal delivery of islets as is also employed in humans?
Preliminary studies indicate that AdvHGF is similarly effective in a
rat allograft setting as
well.2
A fourth key question is, What applicability does this have to human
islets and humans with diabetes? Hayek and co-workers (20, 21, 40) show
that HGF is indeed a potent stimulator of proliferation in human beta
cells. This together with the RIP-HGF mouse data (10, 11) and the data
described herein suggest that such a beneficial effect may be observed
in human islets as well. In part, the reason we employed the SCID mouse
model is so that we could adapt these experiments to human islets. On balance, there is no obvious reason why this approach could not be used
with human islets or, indeed, for other beta cells or beta cell
surrogates derived from stem cell approaches to engineering islets (30,
41, 42). If strictly translatable to the Edmonton protocol setting,
these approaches would reduce by at least 50% the number of human
pancreata required to transplant a single patient with diabetes.
Indeed, because this is the early stage of AdvHGF therapy, one can
envision approaches that might further enhance the outcomes such that
one donor pancreas could eventually serve multiple diabetic recipients.
In summary, our studies demonstrate that adenoviral delivery of HGF to
murine islets ex vivo improves islet transplant survival and
performance in STZ-induced diabetic mice. The in vivo beta cell survival effect induced by HGF appears to play an important role
in preventing early failure of the islet graft. In vitro, the PI3-kinase/Akt pathway seems to be important for HGF-mediated survival effect in pancreatic beta cells. Gene delivery of HGF to human
islets may improve islet transplant outcomes and reduce the numbers of
islets required for successful islet transplant in humans.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase (Invitrogen) and murine HGF (10) were ligated into
the cytomegalovirus promoter-containing, adenovirus-based plasmid, pACCMV.pLpA (12). They were then co-transfected with a
second plasmid (pJM17) (12), which contained an additional 4 kilobases
of the pBR plasmid interrupting the E1 region and DNA from a
replication-defective adenovirus, into HEK-293 cells (ATCC, Manassas,
VA) using Superfect (Qiagen Inc., Valencia, CA), according to the
manufacturer's instructions. After recombination and lysis of the
cells, the medium was collected, and the remaining cells were lysed by
freezing and thawing. The cell debris was pelleted, and the viral
supernatant was saved for subsequent experiments. Adenovirus DNA was
sequenced to confirm the correct orientation and sequence of
-galactosidase and mouse HGF cDNAs. Adenovirus was purified on a
CsCl/Tris gradient, separated into aliquots, and stored at
80 °C
until use. m.o.i. was determined by A260 and plaque assays (12).
-32P]deoxycitidine triphosphate (3000 Ci/mmol, Amersham Biosciences), 400 nM HGF primer pair
(11), 4 nM actin primer pair (Ambion), 16 nM
actin competimers (Ambion) that allow actin RNA to be used as an
internal control, and 1.25 units of Taq DNA polymerase
(Promega). Tubes were placed in a thermal cycler, and the following
program was applied: 3 min at 94 °C followed by 25 cycles at
94 °C for 30 s, 58 °C for 1 min as annealing temperature,
and 72 °C for 1 min. The PCR products (actin, 293 bp, and HGF, 146 bp) were separated on a 6% polyacrylamide gel in Tris borate-EDTA
buffer, and the gel was dried and exposed to x-ray film.
-mercaptoethanol, boiled for 5 min, and analyzed using a 10% SDS-polyacrylamide gel.
Proteins were transferred to Immobilon-P membrane (Millipore, Bedford,
MA) using standard techniques. After blocking for 1 h at room
temperature with 5% nonfat milk in PBS containing 0.1% Tween 20, blots were incubated overnight at 4 °C with primary antisera against
the
chain of HGF (polyclonal rabbit anti-HGF antibody at 1:200
dilution, Santa Cruz Biotechnology, Inc.) or against actin (rabbit
polyclonal antibody at 1:400 dilution, Sigma). After several washes
with PBS containing 0.1% Tween 20, blots were incubated with the
corresponding peroxidase-conjugated secondary antibodies for 1 h
at room temperature. Chemiluminescence was detected using the ECL
system (Amersham Biosciences).
20 °C until insulin measurement by
radioimmunoassay (Linco Research, St. Louis, MO). Islets were then
washed 3 times with PBS and digested overnight in 1 ml of 0.1 N NaOH at 37 °C. After neutralization with HCl, protein
was measured by the Bradford method. Results are expressed as a
percentage of insulin concentration obtained with uninfected islets
incubated at 5.5 mM glucose.
20 °C. Tubes
were then centrifuged at 2500 rpm for 10 min at 4 °C, and the
supernatant was stored at
20 °C. After neutralization, the insulin
content in the extracts was measured by insulin radioimmunoassay (Linco).
-mercaptoethanol. Forty-eight hours later, cells
were refed with fresh medium containing vehicle or different agents
(HGF, STZ, and/or wortmannin). Cell viability was measured 24 h
after the addition of the tested agents. Cell counting was performed in
the presence of trypan blue to detect non-viable cells. MTT assay was
performed by incubating the cells with 0.5 mg/ml MTT for 2 h at
37 °C in 5% CO2, as previously described (16). Formazan
produced in the cells was dissolved with isopropanol, and absorbance
was read at 570 and 690 nm.
antiserum (Upstate Biotechnology, Lake Placid,
NY) (mouse islets and INS-1 cells) or 5 µg of rabbit polyclonal
anti-c-Met antibody (Santa Cruz) at 4 °C for 16 h.
Immunocomplexes were immunoprecipitated with recombinant protein
G-agarose (Invitrogen) for 2 h at 4 °C. Immunoprecipitates were
washed once with PBS containing 1% Nonidet P-40 and 100 µM Na3VO4, twice with 100 mM Tris-HCl containing 500 mM LiCl and 100 µM Na3VO4, and twice with
PI3-kinase buffer (10 mM Tris-HCl, 100 mM NaCl,
1 mM EDTA, and 100 µM
Na3VO4). Pellets were then resuspended in
PI3-kinase buffer containing 11.6 mM MgCl2, 20 µg of phosphatidylinositol, 50 µM ATP, and 30 µCi of [
-32P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences)
and incubated for 10 min at 22 °C with constant agitation. After
extraction with chloroform:methanol (1:1), reaction products were
resolved using thin layer chromatography and visualized by autoradiography.
/
(Ser-21/9) antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (41K):
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Fig. 1.
HGF expression in murine pancreatic islets
after transduction with AdvHGF. The expression of HGF was assessed
by relative semiquantitative reverse transcription-PCR (A)
and by Western blot (B). Murine islets were isolated and
exposed to different m.o.i. as indicated of AdvLacZ or AdvHGF for 60 min. Twenty-four hours after infection, islets were harvested, and RNA
or protein was extracted. Reverse transcription-PCR and immunoblot
analysis were performed as described under "Materials and Methods."
Actin was used in both assays as internal control. Uninf.,
uninfected.
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Fig. 2.
Glucose-stimulated insulin secretion in
uninfected and AdvLacZ- and AdvHGF-transduced islets. Murine
islets were isolated and exposed to 250 or 500 m.o.i. of AdvLacZ
or AdvHGF for 60 min. Twenty-four hours after infection, groups of 10 islets were incubated with 5.5 or 22.2 mM glucose for 30 min, and insulin was measured by radioimmunoassay. Results are the
means ± S.E. of 4-6 different experiments performed in
triplicate. The data are presented as percentage above control, where
insulin secretion by uninfected islets exposed to 5.5 mM
glucose (541 ± 113 pg of insulin/µg of protein/30 min) is
considered 100%. *, p < 0.05 versus the
corresponding value at 5.5 mM glucose.
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Fig. 3.
Immediate and sustained reversal of blood
glucose in STZ-induced diabetic SCID mice after renal
subcapsular transplantation of a marginal mass of AdvHGF-transduced
IE. A, twenty-four hours after infection, 300 uninfected or AdvLacZ- or AdvHGF-transduced IE were transplanted under
the kidney capsule of STZ-induced diabetic SCID mice. Non-fasting blood
glucose was measured by glucometer. Results are the means ± S.E. of 4 transplants with 300 uninfected IE, 10 transplants with
300 AdvLacZ IE, and 8 transplants with 300 AdvHGF IE. The
post-transplant blood glucose in mice receiving AdvHGF-transduced
islets was significantly lower (p < 0.01) than the
blood glucose values obtained after the transplant of either uninfected
or AdvLacZ-infected islets, as determined using analysis of variance
for repeated measures. Unilateral nephrectomy (UNX) was
performed in SCID mice transplanted with 300 AdvHGF-transduced IE at
day 56 post-transplant, and the blood glucose immediately returned to
pre-transplant diabetic levels. B, representative
photomicrographs (both at 100× magnification) of kidney sections
containing the grafts (day 56 after transplant) and stained for insulin
as described under "Materials and Methods." The transplant could be
identified in only 2 of 10 AdvLacZ kidneys and was small and difficult
to find. In contrast, grafts were large and easily identified in all
eight AdvHGF kidneys.
View larger version (25K):
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Fig. 4.
Glucose tolerance in normal and STZ-induced
diabetic SCID mice transplanted with uninfected or AdvLacZ- or
AdvHGF-transduced islets. At day 49 after the transplant, mice
were fasted for 16 h and then injected intraperitoneally with
glucose (2 g/kg of body wt). Blood glucose levels were measured from
the snipped tail at the time points indicated in the figure with a
portable glucometer. Results are the means ± S.E. Blood glucose
values were significantly lower (p < 0.01) in both
mice transplanted with AdvHGF-transduced islets and normal SCID mice
than in mice transplanted with either uninfected or AdvLacZ-infected
islets, as determined using analysis of variance for repeated measures.
Blood glucose values were not significantly different in normal mice
and mice transplanted with AdvHGF-transduced islets except at basal and
180 min (p < 0.01 and p < 0.05, respectively).
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Fig. 5.
Beta cell death and insulin content in
AdvLacZ grafts and AdvHGF grafts obtained at day 1 after
transplant. A, representative photomicrograph of renal
subcapsular grafts obtained at day 1 after transplant with AdvLacZ- or
AdvHGF-transduced islets. Sections were stained with propidium iodide
(red) to detect condensed pyknotic nuclei and insulin
(green) to identify beta cells. Notice that the graft with
AdvHGF-transduced islets contained fewer condensed pyknotic nuclei
(arrowheads) as compared with the grafts composed of
AdvLacZ-transduced islets. B, the percentage of pyknotic
condensed beta cell nuclei found in renal subcapsular
grafts containing AdvLacZ- (n = 4) or AdvHGF-transduced
islets (n = 4) obtained at day 1 after the transplant.
At least 600 beta cells were counted per section. C, insulin
content of AdvLacZ grafts and AdvHGF grafts harvested at day 1 after
transplant. Insulin content was significantly higher in grafts
containing AdvHGF-transduced islets than in grafts composed of
AdvLacZ-transduced islets. This finding correlates with the decreased
beta cell death observed in the grafts with AdvHGF-transduced islets.
*, p < 0.05.
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Fig. 6.
Overexpression of HGF in RIP-HGF transgenic
mice increases survival of pancreatic beta cells after treatment with
streptozotocin (STZ) in vivo. Adult mice (3-6
months old) were injected intraperitoneally with STZ (150 mg/kg of body
weight), and 12 h later pancreata were removed, fixed, embedded,
and stained as described under "Materials and Methods."
Representative photomicrographs of pancreatic sections from RIP-HGF
transgenic mice (TG) and normal littermates (NL)
stained for TUNEL (purple nuclei) and insulin
(brown) (A) or propidium iodide (PI,
red) and insulin (green) (C) are
shown. There are fewer TUNEL or pyknotic condensed beta cell nuclei in
the islets from RIP-HGF transgenic mice than in the islets from normal
littermates. Quantitation of STZ-induced beta cell death in
vivo in normal (n = 4) and RIP-HGF transgenic
(n = 4) mice by TUNEL and insulin (B) or
propidium iodide and insulin staining (D) reveals that the
index of beta cell death was significantly (p < 0.01)
reduced in RIP-HGF transgenic mice as compared with normal littermates.
At least 1000 beta cell nuclei were counted per pancreatic
section.
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Fig. 7.
HGF increases survival of INS-1 cells treated
with STZ in vitro. Cells were treated with 25 ng/ml HGF and 0.5 or 1 mM STZ in complete medium for
24 h. Cell viability was assessed by MTT assay (A) and
cell number (B) as described under "Materials and
Methods." STZ treatment significantly and
dose-dependently reduced cell viability at 0.5 and 1 mM. HGF treatment significantly increased (*,
p < 0.05) the number of viable INS-1 cells 24 h
after treatment with STZ. To investigate the involvement of the
PI3-kinase/Akt pathway in the protective effect of HGF, INS-1 cells
were treated with 1 µM wortmannin, a specific inhibitor
of PI3-kinase. Wortmannin was added to the cultures 10 min before the
addition of HGF. Wortmannin significantly (+,
p < 0.01) enhanced the cell death observed under basal
conditions and abrogated the protective effect of HGF. The & symbol indicates p < 0.01, and the $ symbol
indicates p < 0.05, both versus the
corresponding control (with or without wortmannin). #,
p < 0.05 versus 0.5 mM STZ.
Values are the means ± S.E. of five experiments performed in
triplicate.
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Fig. 8.
HGF induces PI3-kinase and Akt activities in
INS-1 cells and isolated mouse islets. A, PI3-kinase
activity was measured in INS-1 cells and mouse islets serum-deprived
for 24 h and stimulated with 25 ng/ml HGF (H25) for 10 min and in normal (NL) and RIP-HGF transgenic mouse islets
serum-deprived for 24 h. Equivalent amounts of protein extracts
were immunoprecipitated (IP) with antibodies to p85 and
c-Met. PI3-kinase assays were performed as described under "Materials
and Methods." Shown are representative autographs of
n = 3. B, Akt activity was measured in INS-1
cells serum-deprived for 24 h and stimulated with 25 ng/ml HGF for
30 min. The level of glycogen synthase kinase (GSK) 3
/
phosphorylation was used to measure Akt activity as described under
"Materials and Methods." A representative immunoblot
(IB) of two independent experiments using phospho-glycogen
synthase kinase 3
/
(Ser-21/9) antibody is shown. Fold activation
is expressed relative to the corresponding untreated control
(C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Darinka Sipula and Sophia Masters for superb technical assistance. We also thank Dr. Martinez-Brocca for help in performing these studies.
![]() |
FOOTNOTES |
---|
* This work was supported by NIDDK, National Institutes of Health Grants DK 55023 and DK 47168, the Juvenile Diabetes Foundation International, and the American Diabetes Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Junior Faculty Award by the American Diabetes
Association. To whom correspondence should be addressed: BST E-1140,
Division of Endocrinology, University of Pittsburgh School of Medicine,
3550 Terrace St., Pittsburgh, PA 15213. Tel.: 412-648-9770; Fax:
412-648-3290; E-mail: ocana@msx.dept-med.pitt.edu.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M207848200
2 J.-C. Lopez-Talavera, K. K. Takane, A. García-Ocaña, I. Cozar, and A. F. Stewart, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
HGF, hepatocyte
growth factor;
RIP, rat insulin promoter;
SCID, severe combined
immunodeficient;
PI3-kinase, phosphatidylinositol-3 kinase;
m.o.i., multiplicity of infection;
5-X-gal, bromo-4-chloro-3-indolyl--D-galactopyranoside;
PBS, phosphate-buffered saline;
STZ, streptozotocin;
IE, islet equivalents;
TUNEL, terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick
end-labeling;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide;
Adv, adenovirus.
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REFERENCES |
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