Divisions of 1 Gastroenterology and 2 Endocrinology, Diabetes and Metabolism, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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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 (Ca2+) influx promote survival
of neuronal cells, we hypothesized that glucose might alter survival of
islet
-cells through a similar mechanism. In the present studies,
cultured 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
Ca2+ influx and was blocked by phosphatidylinositol (PI)
3-kinase inhibitors. Glucose activation of the protein kinase Akt was
demonstrated in both insulinoma cells and cultured mouse islets by
means of an antibody specific for Ser473 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
enhanced 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 important targets for therapeutic intervention.
phosphatidylinositol 3-kinase; apoptosis; islet -cell
mass; depolarization
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INTRODUCTION |
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THE MASS OF
INSULIN-PRODUCING islet -cells is determined by the combined
rates of proliferation of existing cells, by neogenesis, and by cell
death (5). For example, animals that are insulin resistant
develop compensatory hypertrophy and hyperplasia. Under such
conditions, growth factors and glucose are thought to be important
determinants of islet
-cell mass and function (15). The
importance of glucose in these responses has been suggested by
observing that animals subjected to infusions of glucose over several
days developed 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 activity (26). Glucose
results in closure of the ATP-activated potassium (KATP)
channels, leading to depolarization of
-cells and an influx of
cytosolic calcium. The importance of islet
-cell depolarization in
proper
-cell generation and proliferation was recently demonstrated
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 depolarization/Ca2+ signaling for normal
-cell development.
Sustained Ca2+ overload in neurons is associated with
enhanced apoptosis, yet neurons deprived of depolarization and
Ca2+ also do not survive (53). This has been
demonstrated by the fact that promotion of survival of several types of
neurons by depolarization can be prevented by dihydropyridine calcium
channel antagonists (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
Ca2+ 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 evidence that glucose in the
10-12 mM range, perhaps through activation of depolarization, may
enhance survival of
-cells by mechanisms similar to those observed
in neuronal cells.
The purpose of the present study was to attempt to confirm previous
observations on the survival-enhancing effects of glucose on islet
-cells in culture and, if so, evaluate potential mechanisms. Using
cultures of mouse islets and rodent insulinoma cell lines previously
shown to secrete insulin in response to physiological 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 survival,
we examined signal transduction pathways that might be involved. The
results suggested that glucose promotes pancreatic islet
-cell
survival through depolarization and subsequent Ca2+
activation of a phosphatidylinositol (PI) 3-kinase/Akt
survival-signaling pathway.
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EXPERIMENTAL PROCEDURES |
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Antibodies
We used the following antibodies: Akt, phospho-Akt, and cleaved caspase-3 antibodies (New England Biolabs); hemagglutinin (HA) antibody (Santa Cruz Biotechnologies); Alexa 488 goat anti-rabbit IgG antibody, and Alexa 594 goat anti-mouse IgG antibody (Molecular Probes); CY3-conjugated Affinipure donkey anti-guinea pig antibody (Jackson Immunoresearch 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 nifedipine.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) technique was used to detect DNA strand breaks formed during apoptosis (19). Cells on coverslips 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 fluorescein isothiocyanate (FITC)-labeled dUTP in the presence of enzyme TdT for 1 h at 37°C. Coverslips were mounted on glass slides in mounting medium containing the counterstain propidium iodide (2.5 µg/ml) and visualized using a fluorescent microscope. Five hundred cells were scored (in triplicate) in a blinded fashion to determine the percentage of TUNEL-positive cells. In RESULTS, n represents the number of independent experiments.Activated caspase-3 was detected by immunohistochemistry 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 for
1 h at 37°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 microscope. 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-positive)
-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 percentage 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 preincubated 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 intervals, cells were lysed and assayed on an acrylamide gel (10%) for phospho-Akt as described (1). Protein (40 µg) was loaded per lane. Phospho-Akt was assessed by use of an antibody to Akt phosphorylated at Ser473. 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 µg of total protein were immunoprecipitated with immobilized Akt monoclonal antibody slurry with gentle rocking for 3 h at 4°C. After two washes with cell lysis buffer followed by two washes with kinase buffer (25 mM Tris, pH 7.5, 5 mMTransient 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 µg of DNA per dish with the use of lipofectamine plus reagent under serum-free conditions 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 secondary antibody. Apoptosis in transfected cells was detected by scoring yellow cells due to overlap of red and green fluorescence.Statistical Analysis
Statistical analysis was performed using a two-tailed Student's t-test. A P value < 0.05 was considered significant. ![]() |
RESULTS |
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Effects of Glucose on Survival of Insulinoma Cells and Cultured Islet Cells
To assess whether glucose affected survival of islet
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Measurement of apoptosis by the TUNEL method was confirmed by detecting apoptosis by means of immunohistochemical 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 apoptosis 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, experiments 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 conditions 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
proliferation 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 apoptosis 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-Km glucose
transporter (26, 27). To determine whether the protective
effect of 25 mM glucose was a property of cells with
high-Km 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-Km glucose transporters and hexokinases
like islet -cells.
Effect of Glucose on Necrosis in Insulinoma Cells
Cell survival can be determined by apoptosis or necrosis. MIN6 insulinoma cells were treated with glucose (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].Dose Dependency and Time Course of Glucose-Regulated Islet -Cell
Survival
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.
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Glucose-Regulated Survival is Dependent on RNA Synthesis, Depolarization, and Ca2+ Influx
Having established a model system where reproducible effects of glucose on
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To determine whether the glucose effect on survival required membrane
depolarization, experiments were performed using DZ, an agent that
activates KATP 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 Ca2+ influx was required for glucose
inhibition of apoptosis, cells were incubated in the presence
of the L-type Ca2+ channel blocker nifedipine (10 µM). As
shown in Fig. 3C, the protective effect conferred by 25 mM
glucose was lost in the presence of nifedipine, indicating that
glucose-dependent Ca2+ influx 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 relative to
apoptosis in 25 mM glucose; 5 mM glucose: 343 ± 55 (n = 3), P < 0.0001; 5 mM glucose + nifedipine: 942 ± 136 (n = 6),
P < 0.0001]. Thus it appears that
Ca2+ influx following glucose-induced depolarization is
playing a critical role in survival and that perhaps the further
increase in survival in 25 mM is due to the enhanced Ca2+
influx known to occur with depolarization.
Signal Transduction Pathways Involved in Glucose-Regulated Survival
Having determined that glucose-induced depolarization and Ca2+ influx are important mediators of survival, the signal transduction pathways involved in this process were examined. In islet
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A major downstream target of activated PI 3-kinase is the
serine-threonine kinase Akt, and activation of this enzyme by
phosphorylation of Ser473 has been associated with
inhibition of apoptosis (13, 32). To determine
whether glucose-regulated survival observed in MIN6 cells was
associated with glucose activation of Akt, cells were treated with
glucose as indicated, and protein extracts were immunoblotted with an
antibody specific for Ser473 phospho-Akt (57 kDa) as a
measure of Akt activation (54). At 120 min after addition
of 25 mM glucose, there was marked Ser473 phosphorylation
of Akt (n = 4; Fig.
5A), whereas addition of 5 mM
glucose showed no phosphorylation at similar time points (data not
shown). In separate experiments, 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. Addition of kinase inhibitors
indicated that glucose activation of Akt was PI 3-kinase dependent and
MAPK independent (n = 3; Fig. 5B).
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To determine whether similar treatment of primary cultures of isolated mouse islets would also result in Ser473 phosphorylation of Akt, cells were incubated in 2 or 15 mM glucose for 45 min and immunoblotted with the antibody specific for Ser473 phospho-Akt (57 kDa). As shown in Fig. 5C, although phospho-Akt was detected 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 software Scion Image was used to determine the intensity of bands on the Western blots.
To confirm that Ser473 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 phospho-GSK-3/
antibody. MIN6 cells were incubated in 25 mM glucose overnight and then removed from medium and replaced with Krebs buffer containing glucose
or potassium chloride as indicated. After 15 min of treatment, protein
was extracted and Akt kinase activity 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).
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Akt Activity is Both Necessary and Sufficient for Glucose-Mediated Survival
The previous observations demonstrated that glucose-mediated reduction of apoptosis in insulinoma cells was associated with PI 3-kinase-dependent activation of Akt. To attempt to demonstrate a causal relationship between Akt activation and inhibition of apoptosis, MIN6 cells were transfected with cytomegalovirus promoter-based expression vectors encoding either 1) wild-type HA-tagged Akt (Akt-WT), 2) a catalytically inactive mutant (kinase dead) form of HA-tagged Akt (Akt-K179M) with dominant-inhibitory activity 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 glucose. The cells transfected with the kinase-dead plasmid 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 constitutively 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].
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DISCUSSION |
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The results of the present study provide evidence for a
glucose-mediated PI-3-kinase/Akt-dependent survival pathway in
pancreatic islet -cells. We have demonstrated glucose-mediated
activation of Akt in isolated mouse islets and in the MIN6 insulinoma
cell line. The results of this study confirm previous observations that
glucose activates Akt in insulinoma cells (8). Our
findings contribute to the similarities in properties of
-cells and
neurons, since activation of Akt in neuronal cells has been shown to
enhance survival and to be induced by neurotransmitter-mediated
depolarization, Ca2+ influx, and PI 3-kinase activation
(13). In neurons, survival mediated both by growth factors
such as insulin and by depolarization through neurotransmitters seems
to be converging on this common survival pathway (56).
Certainly, the effects of targeted 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 resistant to streptozotocin-induced diabetes. Although increased
-cell mass was shown to be accounted for, at least in part, by increased proliferation, further experiments 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 effects of glucose and
Ca2+ 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 Ca2+
influx (28, 45). As in neurons, the effects of prolonged sustained hyperglycemia on islet
-cells may result in
Ca2+ excess and subsequent apoptosis. Some studies
have reported 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 apoptosis at 5 mM
glucose was inhibited by addition of actinomycin D, suggesting the
requirement for transcription of proapoptotic proteins. This is
similar to the growth factor withdrawal-induced apoptosis in neurons 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 depolarization and Ca2+
influx are rapidly activating a signal transduction pathway mediating survival, perhaps 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/Ca2+-mediated 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 >107 M insulin did
result in increased survival (S. Srivivasan, unpublished observations). Recent studies have suggested that glucose
regulation of insulin and L-pyruvate kinase gene
transcription are mediated through glucose-stimulated insulin secretion
in an autocrine/paracrine fashion (11). Our results are
consistent with this hypothesis, and this important question needs to
be examined by further studies.
What could the possible significance of glucose-mediated 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 period, 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 restriction
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 apoptosis have been reported (7, 42). Studies
in rats transplanted with insulinoma cells have demonstrated increased
apoptosis in the endogenous islet
-cells in the animals transplanted with insulinomas. These animals have hyperinsulinemia and
hypoglycemia. There was a 67 ± 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 hypoglycemia 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 consequences for understanding the etiology
of diabetes and that may suggest new means of therapeutic intervention
as well.
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ACKNOWLEDGEMENTS |
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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 Shornick for technical support.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16746 (M. A. Permutt), a Howard Hughes Medical Institute Biomedical Research grant (S. Srinivasan), and the Diabetes Research and Training Center at the Washington University School of Medicine.
Address for reprint requests and other correspondence: S. Srinivasan, Washington Univ. School of Medicine, Campus Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: ssriniva{at}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.
June 25, 2002;10.1152/ajpendo.00177.2002
Received 29 April 2002; accepted in final form 17 June 2002.
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