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Glucose promotes pancreatic islet -cell survival
through a PI 3-kinase/Akt-signaling pathway
SHANTHI SRINIVASAN,
1
ERNESTO BERNAL-MIZRACHI,
2
MITSURU OHSUGI,
2
AND MARSHALL ALAN PERMUTT
2
Divisions of
1
Gastroenterology and
2
Endocrinology, Diabetes and Metabolism, Department of
Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
Received 29 April 2002; accepted in final form 17 June 2002
Srinivasan, Shanthi, Ernesto Bernal-Mizrachi, Mit-
suru Ohsugi, and Marshall Alan Permutt. Glucose pro-
motes pancreatic islet -cell survival through a PI 3-kinase/
Akt-signaling pathway. Am J Physiol Endocrinol Metab 283:
E784–E793, 2002. First published June 25, 2002; 10.1152/
ajpendo.00177.2002.—The concentration of glucose in
plasma is an important determinant of pancreatic -cell
mass, whereas the relative contributions of hypertrophy,
proliferation, and cell survival to this process are unclear.
Glucose results in depolarization and subsequent calcium
influx into islet -cells. Because depolarization and calcium
(Ca
2⫹
) influx promote survival of neuronal cells, we hypoth-
esized that glucose might alter survival of islet -cells
through a similar mechanism. In the present studies, cul-
tured mouse islet -cells showed a threefold decrease in
apoptosis under conditions of 15 mM glucose compared with
2 mM glucose (P⬍0.05). MIN6 insulinoma cells incubated in
25 mM glucose for 24 h showed a threefold decrease in
apoptosis compared with cells in 5 mM glucose (1.7 ⫾0.2 vs.
6.3 ⫾1%, respectively, P⬍0.001). High glucose (25 mM)
enhanced survival-required depolarization and Ca
2⫹
influx
and was blocked by phosphatidylinositol (PI) 3-kinase inhib-
itors. Glucose activation of the protein kinase Akt was dem-
onstrated in both insulinoma cells and cultured mouse islets
by means of an antibody specific for Ser
473
phospho-Akt and
by an in vitro Akt kinase assay. Akt phosphorylation was
dependent on PI 3-kinase but not on MAPK. Transfection of
insulinoma cells with an Akt kinase-dead plasmid (Akt-
K179M) resulted in loss of glucose-mediated protection,
whereas transfection with a constitutively active Akt en-
hanced survival in glucose-deprived insulinoma cells. The
results of these studies defined a novel pathway for glucose-
mediated activation of a PI 3-kinase/Akt survival-signaling
pathway in islet -cells. This pathway may provide impor-
tant targets for therapeutic intervention.
phosphatidylinositol 3-kinase; apoptosis; islet -cell mass;
depolarization
THE MASS OF INSULIN-PRODUCING islet -cells is deter-
mined by the combined rates of proliferation of existing
cells, by neogenesis, and by cell death (5). For example,
animals that are insulin resistant develop compensa-
tory hypertrophy and hyperplasia. Under such condi-
tions, growth factors and glucose are thought to be
important determinants of islet -cell mass and func-
tion (15). The importance of glucose in these responses
has been suggested by observing that animals sub-
jected to infusions of glucose over several days devel-
oped increased -cell mass (51). Similarly, glucose
treatment of insulinoma cells in culture resulted in
-cell hyperplasia (24). Pancreatic islet -cells are
unique in their responses to physiological changes in
glucose, converting metabolic energy into electrical ac-
tivity (26). Glucose results in closure of the ATP-acti-
vated potassium (K
ATP
) channels, leading to depolar-
ization of -cells and an influx of cytosolic calcium. The
importance of islet -cell depolarization in proper -cell
generation and proliferation was recently demon-
strated by disruption of the L-type calcium channel ␣
1D
subunit in mice (44). Knockout of this calcium channel
subunit resulted in a decrease in the number and size
of the islets due to a decrease in -cell generation.
These results highlighted the significance of depolar-
ization/Ca
2⫹
signaling for normal -cell development.
Sustained Ca
2⫹
overload in neurons is associated
with enhanced apoptosis, yet neurons deprived of de-
polarization and Ca
2⫹
also do not survive (53). This has
been demonstrated by the fact that promotion of sur-
vival of several types of neurons by depolarization can
be prevented by dihydropyridine calcium channel an-
tagonists (10, 17, 18, 34). Neurons and islet -cells
share many biochemical and molecular mechanisms.
We hypothesized that glucose might affect islet -cell
mass through similar mechanisms. Like neurons,
chronic depolarization and Ca
2⫹
overload could result
in apoptosis (42, 49); yet previous studies suggested
that glucose may enhance islet -cell viability. Isolated
rat islet -cells cultured in 10 mM glucose for 1 wk
exhibited marked enhancement of survival relative to
those in 3 mM glucose (23). Another study reported
increasing apoptosis when islet -cells were incubated
in ⬎12 mM glucose for 40 h; yet interestingly, this
study also showed a ⬎80% reduction in apoptosis when
-cells were incubated with glucose increasing from 2
to 12 mM (14). These studies provide preliminary evi-
dence that glucose in the 10–12 mM range, perhaps
Address for reprint requests and other correspondence: S. Srini-
vasan, Washington Univ. School of Medicine, Campus Box 8127, 660
S. Euclid Ave., St. Louis, MO 63110 (E-mail: ssriniva@im.wustl.edu).
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.
Am J Physiol Endocrinol Metab 283: E784–E793, 2002.
First published June 25, 2002; 10.1152/ajpendo.00177.2002.
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through activation of depolarization, may enhance sur-
vival of -cells by mechanisms similar to those ob-
served in neuronal cells.
The purpose of the present study was to attempt to
confirm previous observations on the survival-enhanc-
ing effects of glucose on islet -cells in culture and, if
so, evaluate potential mechanisms. Using cultures of
mouse islets and rodent insulinoma cell lines previ-
ously shown to secrete insulin in response to physio-
logical changes in glucose concentrations (2–25 mM)
(26), we determined that there were significant effects
of glucose at ⬎15 mM on survival relative to those in
glucose at ⬍5 mM during 24- to 96-h incubations. To
elucidate the mechanisms of glucose-mediated sur-
vival, we examined signal transduction pathways that
might be involved. The results suggested that glucose
promotes pancreatic islet -cell survival through depo-
larization and subsequent Ca
2⫹
activation of a phos-
phatidylinositol (PI) 3-kinase/Akt survival-signaling
pathway.
EXPERIMENTAL PROCEDURES
Antibodies
We used the following antibodies: Akt, phospho-Akt, and
cleaved caspase-3 antibodies (New England Biolabs); hemag-
glutinin (HA) antibody (Santa Cruz Biotechnologies); Alexa
488 goat anti-rabbit IgG antibody, and Alexa 594 goat anti-
mouse IgG antibody (Molecular Probes); CY3-conjugated Af-
finipure donkey anti-guinea pig antibody (Jackson Immu-
noresearch Lab), and anti-insulin antibody (Biogenex).
Cell Culture
MIN6 cells were cultured in Dulbecco’s minimal essential
medium (DMEM) containing 15% FCS and 25 mM glucose
(passage no. 20–40). INS-1 cells were cultured in RPMI
containing 10% FCS and 11.2 mM glucose and PC12 cells in
DMEM containing 10% FCS, 5% goat serum, and 25 mM
glucose. For detection of apoptosis, cells were plated at
1,000,000 per coverslip, and experiments were performed
24 h after plating at ⬃70–80% confluence. The protocol for
glucose experiments involved washing cells with phosphate-
buffered saline (PBS), followed by incubation for 24–96 h
with 2–40 mM glucose and other reagents such as mannitol,
diazoxide (DZ), PD-98059, wortmannin, LY-294002, and ni-
fedipine.
Isolation of mouse islets was accomplished by collagenase
digestion and Ficoll centrifugation, followed by hand picking
as described (39). Islets were cultured for 24 h in RPMI with
10% serum and 11.2 mM glucose.
Detection of Apoptosis
The TdT-mediated dUTP nick end labeling (TUNEL) tech-
nique was used to detect DNA strand breaks formed during
apoptosis (19). Cells on coverslips were fixed with 4% para-
formaldehyde for 45 min at room temperature and then
permeabalized with 1% Triton X-100. After a rinse with PBS,
cells were incubated with fluorescein isothiocyanate (FITC)-
labeled dUTP in the presence of enzyme TdT for1hat37°C.
Coverslips were mounted on glass slides in mounting me-
dium containing the counterstain propidium iodide (2.5 g/
ml) and visualized using a fluorescent microscope. Five hun-
dred cells were scored (in triplicate) in a blinded fashion to
determine the percentage of TUNEL-positive cells. In RE-
SULTS,nrepresents the number of independent experiments.
Activated caspase-3 was detected by immunohistochemis-
try using an antibody (1:100) to the cleaved caspase-3 (17
kDa) fragment as described (16). Alexa 488 goat anti-rabbit
IgG antibody (1:200) was used as a secondary antibody. Five
hundred cells were scored in a blinded fashion to determine
the percentage of cleaved caspase-3-positive cells by use of a
fluorescent microscope.
For detection of apoptosis in cultured islet cells, the islets
were isolated and cultured for 24 h in different glucose
concentrations (2 and 15 mM). After 24 h of incubation, the
islets were dispersed into single cells with dispase (46) and
were spun down with a Cytospin machine onto a slide. Cells
were fixed with 4% paraformaldehyde for 45 min at room
temperature and then permeabalized with 1% Triton X-100.
After a rinse with PBS, cells were incubated with FITC-
labeled dUTP in the presence of enzyme TdT for1hat37°C.
After a wash with PBS, cells were incubated with guinea pig
anti-insulin antibody (1:200) for 1 h followed by a 30-min
wash with PBS; finally, cells were incubated with secondary
antibody (CY3-conjugated donkey anti-guinea pig antibody)
for 1 h. After a 30-min wash with PBS, cells were covered in
mounting medium and visualized with a fluorescent micro-
scope. No propidium iodide counterstain was used in the
TUNEL method. Staining for insulin as described identified
-cells. Five hundred -cells were scored in a blinded fashion
to determine the percentage of FITC-positive (TUNEL-posi-
tive) -cells.
Detection of Necrosis
MIN6 cells were treated with 5 and 25 mM glucose for
24 h, and necrosis was assessed by trypan blue exclusion.
Five hundred cells were counted in a blinded fashion, and the
number of trypan blue-positive cells determined the percent-
age of necrosis (2).
Western Blot Analysis
MIN6 cells. MIN6 cells were plated in 100-mm dishes at a
seeding density of 5,000,000 cells per dish and cultured in
DMEM for 4 days. At ⬃80% confluence, cells were preincu-
bated in the absence of serum and with 5 mM glucose for
14 h, followed by no serum and no glucose for 4 h. Kinase
inhibitors were added 1 h before the addition of glucose. After
incubation with glucose (5 or 25 mM) for various time inter-
vals, cells were lysed and assayed on an acrylamide gel (10%)
for phospho-Akt as described (1). Protein (40 g) was loaded
per lane. Phospho-Akt was assessed by use of an antibody to
Akt phosphorylated at Ser
473
. The blot was stripped and
reprobed for total Akt.
Isolated islets. Isolated islets were cultured overnight in
complete medium (11.2 mM glucose and 10% FCS). Cells
were incubated in Krebs buffer for 1 h followed by exposure
to 2 or 5 mM glucose for 45 min. After incubation with
glucose (2 and 15 mM) for various time intervals, cells were
lysed and assayed on an acrylamide gel (10%) for phospho-
Akt and total Akt.
Akt Kinase Activity
Measurement of Akt kinase activity was performed using a
kit (Cell Signaling Technology, Beverly, MA). MIN6 cells
were stimulated as described and lysed in cell lysis buffer.
Cell lysates containing 300 g of total protein were immuno-
precipitated with immobilized Akt monoclonal antibody
slurry with gentle rocking for3hat4°C. After two washes
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with cell lysis buffer followed by two washes with kinase
buffer (25 mM Tris, pH 7.5, 5 mM -glycerophosphate, 2 mM
DTT, 0.1 mM Na
3
VO
4
, 10 mM MgCl
2
), the immunoprecipi-
tates were resuspended in 40 l of kinase buffer added with
200 M ATP and 1 g of GSK-3 fusion protein. Samples were
incubated for 30 min at 30°C. The reaction was terminated
with 20 lof3⫻SDS loading buffer, and then samples were
analyzed by immunoblotting with the phospho-GSK-3␣/
(Ser
21/9
) antibody (38).
Transient Transfection and Detection of Akt
by Immunofluorescence
Akt constructs were a gift of Dr. Philip Stahl (Washington
University School of Medicine). Plasmids containing three
HA-tagged Akt constructs were used: wild-type Akt (Akt-
WT), kinase-dead Akt (Akt-K179M), and a constitutively
active form of Akt (Akt-CA) (33). Transfection efficiency was
5%, determined by immunohistochemical detection of the HA
tags of the Akt constructs with an antibody to HA (1:200).
Cells were transfected with 1 g of DNA per dish with the
use of lipofectamine plus reagent under serum-free condi-
tions for 6 h, and then the cells were replenished with
complete medium for 24 h. Next, the cells were cultured for
24 h in different concentrations of glucose. The cells were
fixed and assayed for transfection and apoptosis. TUNEL
staining was performed first, followed by staining for the HA
tag of Akt. No propidium iodide counterstain was used in the
TUNEL method. The TUNEL-positive cells appeared green
and the HA tag appeared red due to the ALEXA 594 second-
ary antibody. Apoptosis in transfected cells was detected by
scoring yellow cells due to overlap of red and green fluores-
cence.
Statistical Analysis
Statistical analysis was performed using a two-tailed Stu-
dent’st-test. A Pvalue ⬍0.05 was considered significant.
RESULTS
Effects of Glucose on Survival of Insulinoma Cells
and Cultured Islet Cells
To assess whether glucose affected survival of islet
-cells, apoptosis was measured by the TUNEL method
in cultures of isolated mouse pancreatic islets and
insulinoma cells incubated in various glucose concen-
trations. The effects of glucose were assessed on sur-
vival of primary mouse islet -cells maintained in
culture for 24 h. -Cells were identified using a stain-
ing protocol for insulin in conjunction with the TUNEL
method, as described in EXPERIMENTAL PROCEDURES.
Mouse islet -cells cultured in the presence of 15 mM
glucose had a threefold reduction in apoptosis com-
pared with those cultured in 2 mM glucose [29 ⫾9.2 vs.
75 ⫾8.9% apoptosis (n⫽4 independent experiments),
P⫽0.03]. In Fig. 1A, the percentage of apoptosis is
shown relative to apoptosis in 15 mM glucose (2 mM
glucose: 258 ⫾30.68 vs. 15 mM glucose: 100 ⫾31.72,
P⫽0.03). MIN6 insulinoma cells cultured in 5 mM
glucose over 24 h showed 6.7 ⫾1% TUNEL-positive
cells at the end of the incubation (n⫽13). In contrast,
cells incubated in 25 mM glucose exhibited a threefold
decrease in TUNEL-positive cells [1.7 ⫾0.2% (n⫽11),
P⬍0.001]. In Fig. 1B, the percentage of apoptosis is
shown relative to apoptosis in 5 mM glucose (5 mM
glucose: 370 ⫾61.76 vs. 25 mM glucose: 100 ⫾17.54,
P⬍0.001). A representative photograph of MIN6 cells
is shown to illustrate TUNEL-positive yellow fluores-
cent cells (Fig. 1B). A similar effect of glucose on
apoptosis was also observed in another rodent insulin-
oma cell line, INS-1 [10 mM glucose: 2.69 ⫾0.4% (n⫽
6) vs. 2 mM glucose: 6.02 ⫾0.4% (n⫽6), P⬍0.001].
Measurement of apoptosis by the TUNEL method
was confirmed by detecting apoptosis by means of im-
munohistochemical detection of cleaved caspase-3 (17-
kDa fragment). Activation of caspase-3, one of the key
executioners of apoptosis, requires its cleavage into
two fragments (17 and 12 kDa). As shown in Fig. 1C,
MIN6 cells cultured for 24 h under conditions of 5 mM
glucose showed a 294.87 ⫾54.48% increase in apopto-
sis by cleaved caspase-3 staining compared with those
cultured in 25 mM glucose [100 ⫾14.86% (n⫽6), P⬍
0.001].
MIN6 cells are typically cultured in 25 mM glucose
(26). To determine whether the effects of lowering
glucose were due to differences in osmolarity, experi-
ments were performed with the addition of mannitol.
To maintain osmolarity, cells were incubated in 5 mM
glucose plus 20 mM mannitol. This treatment did not
change the percentage of apoptosis seen under condi-
tions of 5 mM glucose [%apoptosis; 25 mM glucose:
1.7 ⫾0.2 (n⫽11); 5 mM glucose: 6.7 ⫾1(n⫽13); 5
mM glucose ⫹20 mM mannitol: 6.3 ⫾1.2 (n⫽6), P⬎
0.05 vs. 5 mM glucose]. To decrease the rates of prolif-
eration to reduce this as a variable while assessing
apoptosis, experiments were performed in the presence
of low serum (1%). Even in the presence of low serum
concentrations, a three- to fourfold decrease in apopto-
sis was seen in cells cultured in the presence of 25 mM
glucose compared with those in 5 mM glucose [2.8 ⫾
0.29% (n⫽3) vs. 7.63 ⫾2.2% (n⫽3), P⬍0.05]. Min6
cells express predominantly GLUT2, a high-K
m
glucose
transporter (26, 27). To determine whether the protec-
tive effect of 25 mM glucose was a property of cells with
high-K
m
glucose transporters and glucokinase, the
same experiments were performed in a neuronal cell
line, PC12. After incubation for 24 h in 5 or 25 mM
glucose, no difference in apoptosis was observed [5 mM
glucose: 0.26 ⫾0.11% (n⫽6), 25 mM glucose: 0.56 ⫾
0.07% (n⫽6), P⬎0.05]. This result suggested that the
correlation between physiological levels of glucose and
the regulation of apoptosis might be unique to cells
with high-K
m
glucose transporters and hexokinases
like islet -cells.
Effect of Glucose on Necrosis in Insulinoma Cells
Cell survival can be determined by apoptosis or ne-
crosis. MIN6 insulinoma cells were treated with glu-
cose (5 or 25 mM) for 24 h. There was no difference in
necrosis noted in cells cultured in 5 vs. 25 mM glucose
[%necrotic cells, 5 mM: 2.1 ⫾0.3 (n⫽4); 25 mM: 2.3 ⫾
0.5 (n⫽6), P⫽0.7].
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Dose Dependency and Time Course of Glucose-
Regulated Islet

-Cell Survival
In nonislet -cells, absence of glucose induces apop-
tosis, whereas the protective effect on survival is max-
imal at ⬍2 mM (29). In contrast, in islet -cells, be-
cause of the high-K
m
, low-affinity glucose transporter
and hexokinase activities, the glucose effect on sur-
vival would be predicted to have a K
m
in the 5–15 mM
range (52). After 24 h of incubation, inhibition of apop-
tosis was present at 15 mM glucose and was not sig-
nificantly altered by glucose concentrations up to 40
mM [%apoptosis at various glucose concentrations: 0
mM: 4.64 ⫾1.1; 5 mM: 3.08 ⫾1.1; 10 mM: 3.04 ⫾1.1;
15 mM: 2.43 ⫾0.07; 25 mM: 2.08 ⫾0.6; 40 mM: 2.33 ⫾
0.55 (n⫽4 in each category), P⬍0.05 between 0 and
⬎15 mM]. These results indicate that glucose regula-
tion of islet -cell apoptosis occurs over a physiologi-
cally relevant glucose concentration range (5–15 mM).
To assess the time course of the glucose effect on
apoptosis, cells were examined at 24-h intervals for up
to 96 h of incubation. The rate of apoptosis increased
with time in both 5 and 25 mM glucose, whereas at
each time point cells incubated in 25 mM glucose had a
three- to fourfold reduced rate of apoptosis relative to
those in 5 mM glucose [%apoptosis at 24 h at 5 mM
glucose: 6.3 ⫾1(n⫽13) and 25 mM glucose: 1.7 ⫾0.3
(n⫽11), P⬍0.001; at 48 h in 5 mM glucose: 4.16 ⫾0.3
(n⫽8) and 25 mM glucose: 0.7 ⫾0.3 (n⫽8), P⬍0.001;
at 72 h in 5 mM glucose: 15.99 ⫾4(n⫽6) and 25 mM
glucose: 0.9 ⫾0.3 (n⫽4), P⬍0.0001; and at 96 h at 5
mM glucose: 34.4 ⫾2.4 (n⫽6) and 25 mM glucose:
13.6 ⫾4.3 (n⫽6), P⬍0.05]. The data in Fig. 2
represent the aforementioned results expressed as a
percentage of apoptosis at 24 h of incubation in the
presence of 25 mM glucose.
Fig. 1. Effect of glucose on survival in islet -cells and insulinoma
cells. A: apoptosis in mouse islets. Isolated mouse islets were treated
with 2 or 15 mM glucose for 24 h, and apoptosis was assessed in
-cells by the TdT-mediated dUTP nick end labeling (TUNEL)
method as described in EXPERIMENTAL PROCEDURES. Apoptosis was
higher in the presence of 2 mM glucose (258.62 ⫾30.68%) compared
with 15 mM glucose (100 ⫾31.72%). Results represent means ⫾SE;
*P⬍0.05. B: MIN6 insulinoma cells were treated with glucose for
24 h at the indicated concentrations and assessed for apoptosis using
the TUNEL method as described in EXPERIMENTAL PROCEDURES. Apop-
tosis was higher in the presence of 5 mM glucose (370 ⫾61.76%)
compared with 25 mM glucose (100 ⫾17.64%). Results represent
means ⫾SE; **P⬍0.001. A representative photograph to illustrate
TUNEL-positive yellow (stained with FITC and propidium iodide)
fluorescent cells is shown. C: caspase-3 activity assay. MIN6 insu-
linoma cells were treated with glucose for 24 h at the indicated
concentrations and assessed for cleaved caspase-3 staining as de-
scribed in EXPERIMENTAL PROCEDURES. The cleaved caspase-3-positive
cells were higher in the presence of 5 mM glucose (294 ⫾54.48%)
compared with 25 mM glucose (100 ⫾16.66%). Results represent
means ⫾SE; **P⬍0.001. Representative photograph of immuno-
histochemical detection of cleaved caspase-3 positive cell is shown.
The cleaved caspase-3-positive cells appear green due to Alexa 488-
conjugated secondary antibody.
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Glucose-Regulated Survival is Dependent on RNA
Synthesis, Depolarization, and Ca
2
⫹
Influx
Having established a model system where reproduc-
ible effects of glucose on -cell survival could be ob-
served, we next sought mechanisms. In neuronal cells,
apoptosis induced by growth factor withdrawal is de-
pendent on RNA synthesis (43). Therefore, MIN6 cells
were treated for 24 h with actinomycin D (1 g/ml) to
inhibit RNA synthesis in the presence of 5 or 25 mM
glucose. This treatment completely attenuated the en-
hanced apoptosis seen in 5 mM glucose [5 mM glucose:
6.3 ⫾1% (n⫽13); 5 mM glucose ⫹actinomycin D:
2.06 ⫾0.6% (n⫽5), P⫽0.01]. No change in apoptosis
was seen when actinomycin D was added under condi-
tions of 25 mM glucose [25 mM glucose: 1.7 ⫾0.2%
(n⫽11); 25 mM glucose ⫹actinomycin D: 1.11 ⫾0.3%
(n⫽6), P⬎0.05 vs. 25 mM glucose]. In Fig. 3A, these
data are represented as percentages relative to apop-
tosis at 24 h in the presence of 25 mM glucose. These
results suggested that the increased rate of apoptosis
observed in the presence of 5 mM glucose is dependent
Fig. 3. Glucose-regulated islet -cell survival is dependent on RNA
synthesis, depolarization, and Ca
2⫹
influx. A: effect of actinomycin
D. MIN6 cells were treated for 24 h at the indicated glucose concen-
trations in the presence or absence of actinomycin D (1 g/ml) to
inhibit RNA synthesis, and apoptosis was assessed by the TUNEL
method. Low glucose-induced apoptosis was attenuated in the pres-
ence of actinomycin D (%apoptosis relative to 25 mM glucose; 5 mM
glucose: 370 ⫾61%; 5 mM glucose ⫹actinomycin D: 121 ⫾35%; 25
mM glucose: 100 ⫾17%; 25 mM glucose ⫹actinomycin D: 65 ⫾17%).
Results represent means ⫾SE; **P⬍0.001 (n.s., not significant, P⬎
0.05.) B: effect of diazoxide. MIN6 cells were treated for 24 h at the
indicated glucose concentrations in the presence or absence of dia-
zoxide, and apoptosis was assessed by the TUNEL method. The 25
mM glucose suppression of apoptosis was lost in the presence of
diazoxide (%apoptosis relative to 25 mM glucose; 5 mM glucose:
370 ⫾61; 5 mM glucose ⫹diazoxide: 345 ⫾35; 25 mM glucose: 100 ⫾
17; 25 mM glucose ⫹diazoxide: 267 ⫾58). Results represent
means ⫾SE; **P⬍0.001. C: effect of nifedipine. MIN6 cells were
treated for 24 h with glucose at the indicated concentrations in the
presence or absence of nifedipine, and apoptosis was assessed by the
TUNEL method. Results are all given relative to those in 25 mM
glucose and represent means ⫾SE; ***P⬍0.0001.
Fig. 2. Time course of glucose-regulated survival in insulinoma cells.
MIN6 cells were treated with glucose at the indicated concentrations
and time points. Apoptosis was assessed using the TUNEL method
as described in EXPERIMENTAL PROCEDURES. Percent apoptosis relative
to apoptosis at 24 h in 25 mM glucose; 24 h at 5 mM glucose: 370 ⫾
61 (n⫽13) and 25 mM glucose: 100 ⫾17 (n⫽11), P⬍0.001; at 48 h
in 5 mM glucose: 244 ⫾17 (n⫽8) and 25 mM glucose: 41 ⫾17 (n⫽
8), P⬍0.001; at 72 h in 5 mM glucose: 940 ⫾235 (n⫽6) and 25 mM
glucose: 52 ⫾17 (n⫽4), P⬍0.0001; and 96 h at 5 mM glucose:
2,020 ⫾141 (n⫽6) and 25 mM glucose: 880 ⫾31 (n⫽6), P⬍0.05.
Results represent means ⫾SE; *P⬍0.05, **P⬍0.001, ***P⬍
0.0001.
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on synthesis of new RNA, whereas the protective effect
of 25 mM glucose is not.
To determine whether the glucose effect on survival
required membrane depolarization, experiments were
performed using DZ, an agent that activates K
ATP
channels and blocks glucose-mediated depolarization
of the -cell (50). The protective effect conferred by 25
mM glucose was lost in the presence of DZ (0.6 mM),
suggesting that depolarization is essential for glucose-
mediated protection of the -cell [25 mM glucose: 1.7 ⫾
0.3% (n⫽11); 25 mM glucose ⫹DZ: 4.54 ⫾0.75% (n⫽
6), P⫽0.001 vs. 25 mM glucose; 5 mM glucose 6.3 ⫾
1% (n⫽13), P⬍0.001 vs. 25 mM glucose; 5 mM
glucose ⫹DZ: 5.88 ⫾0.6% (n⫽6), P⬎0.05 vs. 5 mM
glucose]. In Fig. 3B, these data are represented as the
percentage of apoptosis relative to that seen at 24 h in
the presence of 25 mM glucose.
To determine whether Ca
2⫹
influx was required for
glucose inhibition of apoptosis, cells were incubated in
the presence of the L-type Ca
2⫹
channel blocker nifed-
ipine (10 M). As shown in Fig. 3C, the protective effect
conferred by 25 mM glucose was lost in the presence of
nifedipine, indicating that glucose-dependent Ca
2⫹
in-
flux is essential for the glucose-mediated protection of
the -cell at 25 mM glucose [%apoptosis relative to
apoptosis in 25 mM glucose; 25 mM glucose: 102 ⫾15
(n⫽3); 25 mM glucose ⫹nifedipine: 996 ⫾161 (n⫽6),
P⬍0.0001 vs. 25 mM glucose]. Addition of nifedipine
to cells incubated in 5 mM glucose resulted in an
almost threefold increase in apoptosis [%apoptosis rel-
ative to apoptosis in 25 mM glucose; 5 mM glucose:
343 ⫾55 (n⫽3), P⬍0.0001; 5 mM glucose ⫹nifedi-
pine: 942 ⫾136 (n⫽6), P⬍0.0001]. Thus it appears
that Ca
2⫹
influx following glucose-induced depolariza-
tion is playing a critical role in survival and that
perhaps the further increase in survival in 25 mM is
due to the enhanced Ca
2⫹
influx known to occur with
depolarization.
Signal Transduction Pathways Involved in Glucose-
Regulated Survival
Having determined that glucose-induced depolariza-
tion and Ca
2⫹
influx are important mediators of sur-
vival, the signal transduction pathways involved in
this process were examined. In islet -cells, glucose-
mediated depolarization and Ca
2⫹
influx have previ-
ously been shown to result in activation of several
serine-threonine kinases, including MAP kinases and
PI-3 kinase, and these have been shown to be involved
in the survival of other cells (22, 31). MIN6 cells were
cultured in either 5 or 25 mM glucose in the presence
or absence of the MAPK inhibitor PD-98059 (50 M) or
the PI 3-kinase inhibitors wortmannin (200 nM) or
LY-294002 (50 M). Kinase inhibitors were added 1 h
before the change in the glucose concentration of the
medium. As shown in Fig. 4, the MAPK inhibitor did
not alter the protective effect of 25 mM glucose on
apoptosis [%change in apoptosis relative to apoptosis
in 25 mM; 25 mM: 101 ⫾10 (n⫽6); 25 mM ⫹
PD-98059: 123 ⫾1(n⫽6), P⫽0.5; Fig. 4]. However,
both PI 3-kinase inhibitors decreased the protective
effect of 25 mM glucose, indicating this effect to be PI
3-kinase dependent [%change in apoptosis relative to
apoptosis in 25 mM; 25 mM: 101 ⫾10 (n⫽6); 25 mM ⫹
wortmannin: 311 ⫾29 (n⫽3), P⫽0.001; 25 mM ⫹
LY-294002: 286 ⫾73 (n⫽6), P⫽0.008; Fig. 4]. No
significant change in apoptosis was noted when kinase
inhibitors were added to cells cultured in 5 mM glucose
compared with apoptosis in 5 mM glucose alone (data
not shown).
A major downstream target of activated PI 3-kinase
is the serine-threonine kinase Akt, and activation of
this enzyme by phosphorylation of Ser
473
has been
associated with inhibition of apoptosis (13, 32). To
determine whether glucose-regulated survival ob-
served in MIN6 cells was associated with glucose acti-
vation of Akt, cells were treated with glucose as indi-
cated, and protein extracts were immunoblotted with
an antibody specific for Ser
473
phospho-Akt (57 kDa) as
a measure of Akt activation (54). At 120 min after
addition of 25 mM glucose, there was marked Ser
473
phosphorylation of Akt (n⫽4; Fig. 5A), whereas addi-
tion of 5 mM glucose showed no phosphorylation at
similar time points (data not shown). In separate ex-
periments, it was shown that the activation of Akt by
glucose was first observed at 15 min and peaked at 120
min. Under these conditions, there was no change in
Akt protein detected by an antibody to total Akt. Ad-
dition of kinase inhibitors indicated that glucose acti-
vation of Akt was PI 3-kinase dependent and MAPK
independent (n⫽3; Fig. 5B).
To determine whether similar treatment of primary
cultures of isolated mouse islets would also result in
Ser
473
phosphorylation of Akt, cells were incubated in
2 or 15 mM glucose for 45 min and immunoblotted with
the antibody specific for Ser
473
phospho-Akt (57 kDa).
As shown in Fig. 5C, although phospho-Akt was de-
tected in cells incubated in 2 mM glucose, similar
treatment with 15 mM glucose resulted in a threefold
increase (3.05 ⫾0.23, P⫽0.01) in phospho-Akt. The
Fig. 4. Signal transduction pathways involved in glucose-regulated
islet -cell survival. MIN6 cells were cultured for 24 h at the indi-
cated glucose concentrations in the presence or absence of the indi-
cated inhibitors, and apoptosis was assessed by the TUNEL method.
Results represent means ⫾SE; **P⬍0.001.
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software Scion Image was used to determine the inten-
sity of bands on the Western blots.
To confirm that Ser
473
phosphorylation of Akt was
associated with Akt kinase activity, we measured the
Akt kinase activity under similar conditions. In this
assay, immunoprecipitates of Akt are incubated with a
substrate GSK-3 fusion protein in the presence of ATP
and kinase buffer. Akt phosphorylates GSK-3, which is
then detected by Western blot analysis using a phos-
pho-GSK-3␣/antibody. MIN6 cells were incubated in
25 mM glucose overnight and then removed from me-
dium and replaced with Krebs buffer containing glu-
cose or potassium chloride as indicated. After 15 min of
treatment, protein was extracted and Akt kinase activ-
ity measured with a GSK-3 substrate. As shown in Fig.
6, exposure of cells to 3 mM glucose resulted in a
minimal increase in kinase activity, whereas exposure
to 25 mM glucose for 15 min resulted in a marked
increase in Akt kinase activity similar to that seen
with depolarization with 50 mM potassium chloride
(Fig. 6).
Akt Activity is Both Necessary and Sufficient
for Glucose-Mediated Survival
The previous observations demonstrated that glu-
cose-mediated reduction of apoptosis in insulinoma
cells was associated with PI 3-kinase-dependent acti-
vation of Akt. To attempt to demonstrate a causal
relationship between Akt activation and inhibition of
apoptosis, MIN6 cells were transfected with cytomeg-
alovirus promoter-based expression vectors encoding
either 1) wild-type HA-tagged Akt (Akt-WT), 2) a cat-
alytically inactive mutant (kinase dead) form of HA-
tagged Akt (Akt-K179M) with dominant-inhibitory ac-
tivity toward wild-type Akt kinase activity, or 3)a
constitutively active HA-tagged Akt mutant (Akt-CA)
that lacks amino acids 4–129 and that has a 14-amino
acid src myristoylation signal on the amino terminus of
Akt that targets Akt to the membrane (33). Because
our rate of transfection was ⬃5%, as determined by
immunohistochemistry using an antibody to HA, we
used immunohistochemical staining techniques to
identify transfected cells and study apoptosis in these
cells. This method has been used successfully in the
neuronal literature (13). The constitutively active Akt
targets Akt to the membrane, demonstrated by an
antibody to the HA tag (data not shown). Figure 7
demonstrates the percentage of apoptosis relative to
wild-type Akt-transfected cells cultured in 25 mM glu-
cose. The cells transfected with the kinase-dead plas-
mid Akt-K179M had increased rates of apoptosis
[246 ⫾57% increase vs. Akt-WT, 25 mM glucose (n⫽
8), P⬍0.05; Fig. 7]. Transfections with constitutively
active Akt plasmids in 25 mM glucose reduced the rate
of apoptosis [31 ⫾7.3% vs. Akt-WT in 25 mM glucose
(n⫽8), P⬍0.05; Fig. 7]. Transfections with constitu-
tively active Akt plasmids in 5 mM glucose reduced the
rate of apoptosis [31 ⫾7.3% reduction vs. Akt-WT in 5
mM glucose (n⫽8), P⬍0.05].
Fig. 6. Glucose activates Akt-kinase activity in insulinoma cells.
Min6 cells were cultured overnight in 25 mM glucose and 15% FCS.
Cells were then preincubated in Krebs buffer for 1 h (i.e., 0 time
point) and then treated with Krebs buffer containing glucose or KCl
as indicated. Akt kinase activity was measured as indicated in
EXPERIMENTAL PROCEDURES with a GSK-3 substrate at the indicated
times.
Fig. 5. Glucose activates Akt in insulinoma cells and isolated islets.
A: assessment of Ser
473
phospho-Akt (P-Akt). Cells were preincu-
bated in the absence of serum and with 5 mM glucose for 14 h,
followed by no serum and no glucose for 4 h and then treatment with
25 mM glucose for the indicated time. Lysates were prepared as
described in EXPERIMENTAL PROCEDURES. Protein (40 g) was loaded
in each lane and assayed on an acrylamide gel (10%). Activation of
Akt by glucose and total Akt was assessed by use of antibodies
specific for Ser
473
phospho-Akt and Akt, respectively. B: Akt activa-
tion is phosphatidylinositol (PI) 3-kinase dependent. Cells were
treated and assessed as described in A. Kinase inhibitors were added
1 h before addition of 25 mM glucose. The effect of glucose on
phospho-Akt and total Akt in the presence or absence of MAPK
inhibitor PD-98059 (50 M) or PI-3 kinase inhibitor wortmannin
(200 nM) was assessed. C: Akt activation in primary mouse islets.
Isolated mouse islets were preincubated in Krebs buffer for1hand
then treated with glucose for the indicated times and concentrations.
Lysates were prepared as described in EXPERIMENTAL PROCEDURES.
Protein (75 g) was loaded in each lane and assayed on an acryl-
amide gel (10%). Activation of Akt by glucose and total Akt was
assessed using antibodies specific for Ser
473
phospho-Akt and Akt.
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DISCUSSION
The results of the present study provide evidence for
a glucose-mediated PI-3-kinase/Akt-dependent sur-
vival pathway in pancreatic islet -cells. We have dem-
onstrated glucose-mediated activation of Akt in iso-
lated mouse islets and in the MIN6 insulinoma cell
line. The results of this study confirm previous obser-
vations that glucose activates Akt in insulinoma cells
(8). Our findings contribute to the similarities in prop-
erties of -cells and neurons, since activation of Akt in
neuronal cells has been shown to enhance survival and
to be induced by neurotransmitter-mediated depolar-
ization, Ca
2⫹
influx, and PI 3-kinase activation (13). In
neurons, survival mediated both by growth factors
such as insulin and by depolarization through neuro-
transmitters seems to be converging on this common
survival pathway (56). Certainly, the effects of tar-
geted disruption of the insulin receptor (35) and insulin
receptor substrates (36, 57) in islet -cells, along with
the present studies, suggest that the depolarization
and growth factor survival pathways may be playing a
role in -cell survival, thus affecting its mass and
function.
The novel finding in this study is the nutritional
regulation of a survival pathway in islet -cells
through activation of Akt. The importance of the role of
glucose and Akt in regulating -cell mass is seen in the
transgenic mice expressing constitutively active Akt in
islet -cells. Expression of constitutively active Akt
linked to an insulin gene promoter in transgenic mice
resulted in increased islet -cell mass by altering -cell
size and number (4, 9, 55). These mice are also resis-
tant to streptozotocin-induced diabetes. Although in-
creased -cell mass was shown to be accounted for, at
least in part, by increased proliferation, further exper-
iments will be needed to determine whether these
animals have decreased islet -cell apoptosis as well.
Thus Akt activation may be of importance in both islet
-cell proliferation and survival.
Although the present studies demonstrated the ef-
fects of glucose and Ca
2⫹
influx on the short-term
survival of -cells, they do not address the question of
reduced -cell survival that may be associated with
chronic hyperglycemia and excessive Ca
2⫹
influx (28,
45). As in neurons, the effects of prolonged sustained
hyperglycemia on islet -cells may result in Ca
2⫹
ex-
cess and subsequent apoptosis. Some studies have re-
ported increased apoptosis in rodent islets incubated in
glucose at ⬎20 mM for periods as short as 2–4 days.
However, we did not observe increased apoptosis in
Min6 cells cultured in 40 mM glucose for 3 days. The
effects of prolonged hyperglycemia on -cells need to be
further addressed.
In the present studies, the increased rate of apopto-
sis at 5 mM glucose was inhibited by addition of acti-
nomycin D, suggesting the requirement for transcrip-
tion of proapoptotic proteins. This is similar to the
growth factor withdrawal-induced apoptosis in neu-
rons that is also dependent on transcription of new
RNA (41). The effects of high glucose on survival in
MIN6 cells, however, appeared to be independent of
RNA synthesis. This suggests that glucose-induced de-
polarization and Ca
2⫹
influx are rapidly activating a
signal transduction pathway mediating survival, per-
haps through activation of a serine-threonine kinase.
In fact, the results of the present studies in which
glucose rapidly activates Akt are consistent with these
data. Certain possible mechanisms for this could be
through the well known effect of Akt activation on
inhibition of proapoptotic transcription factors of the
forkhead family (40) or inactivation of BAD (12). The
downstream targets of activated Akt and their possible
effects on -cell survival can now be assessed.
The results of the present studies do not rule out the
possibility that the glucose/depolarization/Ca
2⫹
-medi-
ated survival pathway noted in these experiments is
dependent on insulin secretion, perhaps acting in an
autocrine/paracrine fashion. Insulin has been shown to
inhibit apoptosis in a number of cells (3, 20), and
treatment of MIN6 cells with ⬎10
⫺7
M insulin did
result in increased survival (S. Srivivasan, unpub-
lished observations). Recent studies have suggested
that glucose regulation of insulin and L-pyruvate ki-
nase gene transcription are mediated through glucose-
stimulated insulin secretion in an autocrine/paracrine
fashion (11). Our results are consistent with this hy-
pothesis, and this important question needs to be ex-
amined by further studies.
What could the possible significance of glucose-me-
diated survival be to the normal physiology of islet
-cells? Glucose regulation of islet -cell survival may
play a critical role in the neonatal and postnatal pe-
riod, a time during which the rates of -cell apoptosis
are high and thus influence the ultimate islet -cell
mass in the adult (21, 48). Epidemiological studies
correlate low birth weight and fetal malnutrition with
the onset of non-insulin-dependent diabetes later in
life (47). In animal models, maternal protein restric-
Fig. 7. Akt activity is both necessary and sufficient for glucose-
mediated survival. MIN6 cells were transfected with 1 g of either
wild-type Akt (Akt-WT), a catalytically inactive mutant (kinase
dead) form of Akt (Akt-K179M), or a constitutively active mutant
(Akt-CA) that targets Akt to the membrane and cultured in 25 mM
glucose. Apoptosis was assessed by the TUNEL method in trans-
fected cells at 24 h. Results are expressed as percent apoptosis
relative to Akt-WT cultured in 25 mM glucose and represent
means ⫾SE; *P⬍0.05, ***P⬍0.0001.
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tion imposed during fetal life and suckling resulted in
lower serum glucose levels and a reduction in -cell
mass in the offspring (6, 37). Examples of animal
models where hypoglycemia is associated with apopto-
sis have been reported (7, 42). Studies in rats trans-
planted with insulinoma cells have demonstrated in-
creased apoptosis in the endogenous islet -cells in the
animals transplanted with insulinomas. These ani-
mals have hyperinsulinemia and hypoglycemia. There
wasa67⫾13% reduction in islet cell volume in
insulinoma-transplanted animals, with a reduction in
-cell size (7). In the -cell-specific Kir6.2-dominant-
negative transgenic mouse, the neonatal mice develop
hypoglycemia (42). These mice have enhanced -cell
apoptosis at 2 wk of age. Subsequent to this, these mice
develop hyperglycemia, believed to be secondary to a
reduction in -cell mass. These results suggest that
glucose may be important not only in the regulation of
insulin synthesis and secretion but also in the survival
of the islet -cell. Increased -cell apoptosis has been
noted in patients with hyperinsulinism-induced hypo-
glycemia compared with age-matched controls (30).
The results of the present study define a molecular
mechanism for glucose regulation of an islet -cell
survival pathway that could have important conse-
quences for understanding the etiology of diabetes and
that may suggest new means of therapeutic interven-
tion as well.
We thank Dr. Philip D. Stahl for the Akt constructs; Dr. J.
Milbrandt for the PC12 cells; Drs. Eugene M. Johnson, Burton Wice,
and David Holtzman for their helpful advice; and Gary Skolnick for
preparation of the manuscript. We also acknowledge Michael Shor-
nick for technical support.
This work was supported in part by National Institute of Diabetes
and Digestive and Kidney Diseases Grant DK-16746 (M. A. Per-
mutt), a Howard Hughes Medical Institute Biomedical Research
grant (S. Srinivasan), and the Diabetes Research and Training
Center at the Washington University School of Medicine.
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