The ability of insulin to protect neurons from
apoptosis was examined in differentiated R28 cells, a neural cell line
derived from the neonatal rat retina. Apoptosis was induced by serum
deprivation, and the number of pyknotic cells was counted. p53 and Akt
were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by
immunocytochemistry. Serum deprivation for 24 h caused ~20% of
R28 cells to undergo apoptosis, detected by both pyknosis and
activation of caspase-3. 10 nM insulin maximally
reduced the amount of apoptosis with a similar potency as 1.3 nM (10 ng/ml) insulin-like growth factor 1, which
acted as a positive control. Insulin induced serine phosphorylation of
Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition
of PI 3-kinase with wortmannin or LY294002 blocked the ability of
insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor
of NF-
B nuclear translocation, blocked the rescue effect of insulin,
but neither insulin or serum deprivation induced phosphorylation of
IB. These results suggest that insulin is a survival factor for
retinal neurons by activating the PI 3-kinase/Akt pathway and by
reducing caspase-3 activation. The rescue effect of insulin does not
appear to be mediated by NF-B or p53. These data suggest that
insulin provides trophic support for retinal neurons through a PI
3-kinase/Akt-dependent pathway.
 |
INTRODUCTION |
Insulin is an important survival factor for primary cerebellar
neurons because it can supply the trophic needs for these cells in
culture (1). Less is known about neurons from the retina, but it is
clear that the retina expresses abundant insulin receptors, which are
at the highest concentration in the inner plexiform layer, where many
neuronal projections are located (2, 3). Barres and co-workers (4) have
shown that insulin is among the growth factors that promote survival
and growth of primary retinal ganglion cells in culture.
The insulin receptor has multiple downstream targets that are conserved
in many cell types. It is generally thought that its antiapoptotic
mechanism signals primarily through PI 3- kinase to activate the
serine-threonine kinase, Akt (5). Akt inhibits many proapoptotic
targets by phosphorylating caspase-9, glycogen synthase kinase,
BCL-2-associated death promoter, and members of the forkhead
family (6-12). Insulin also inhibits the release of cytochrome
c from mitochondria and IB, which regulates NF-B translocation (13-16), similar to the
IGF-11 receptor-stimulated
pathway (17, 18).
NF-B is a transcription factor that is translocated from the
cytoplasm to the nucleus and has been implicated in neuronal survival
(19). NF-B may also be involved in insulin signaling to prevent
apoptosis. Insulin activates NF-B in Chinese hamster ovary
cells transfected with the insulin receptor, and the anti-apoptotic signal of insulin is blocked by overexpression of a dominant negative IB-
, which is the inhibitory peptide for NF-B (20). It is possible that the anti-apoptotic effect mediated by NF-B is due to
increased transcription of manganese-superoxide dismutase (21). Overexpression of the NF-B c-rel subunit in neurons blocked
apoptosis as potently as IGF-1, whereas overexpression of a
dominant-negative IB- enhanced apoptosis, suggesting that the
function of NF-B in neurons is primarily neuroprotective (22).
The tumor suppressor gene p53 is also strongly implicated in apoptosis
of neurons (23, 24). Although p53 is known to induce growth arrest, it
is likely that it induces apoptosis by an independent mechanism (25).
Overexpression of p53 in postmitotic neurons leads to apoptosis (26)
and involves activation of BAX and caspase-3 (27). p53 also
up-regulates glyceraldehyde-3-phosphate dehydrogenase in cerebellar
granule neurons during apoptosis (28). However, p53 may not be involved
in apoptosis induced by factors that do not lead directly to DNA
damage, such as low potassium (29, 30). The involvement of p53 in
apoptosis of retinal neurons is unclear, but it is elevated
after retinal ischemia (31).
Here, we tested the hypothesis that physiological concentrations of
insulin may act as a survival factor in R28 cells, which are a subclone
of E1A-NR.3 transfected rat retina cells (32, 33). R28 cells were
originally isolated from neonatal (P12) rat retina as a mixed cell type
and transfected with an E1A construct (34). They have been described as
having both neuronal and glial characteristics, expressing the
neuron-specific antigen Thy 1.1 and some glial cell markers (34, 35).
We first show that a combination of laminin substrate and cAMP induces
a neuronal phenotype in these cells (36). The ability of insulin to
rescue R28 cells from apoptosis induced by serum deprivation was
investigated. The data demonstrate that the PI 3-kinase/Akt pathway
mediates the survival effect of insulin and leads to inhibition of
caspase-3.
| |
EXPERIMENTAL PROCEDURES |
Materials and Reagents--
Insulin was from bovine pancreas
extract (Sigma, St. Louis, MO) 28 USP units/mg; recombinant
human IGF-1 (R3 mutant, resistant to IGF-binding proteins) was from
Upstate Biotechnology (Lake Placid, NY); cell culture medium and bovine
newborn serum were from Life Technologies, Inc. (Rockville, MD). All
dry chemicals were from Fisher and Sigma. Wortmannin, LY294002,
PD98059, and SN-50, an NF-B peptide translocation sequence
inhibitor, were from Biomol. Laminin and RNase were from Sigma. The
IGF-1 receptor inhibitor JB3 was a generous gift from Dr. Lois E. H. Smith, Boston Children's Hospital, Harvard University.
Cell Culture and Protein Extraction--
R28 Cells were seeded
at 4 × 105 cells/cm2 on 60-mm dishes
coated with laminin and 2 × 105 cells/cm2
on glass coverslips for immunocytochemistry and apoptosis assays. They
were fed with Dulbecco's modified Eagle's medium supplemented with
10% newborn bovine serum and 250 µM pCPT-cyclic AMP, a
cell-permeable cAMP analogue (Sigma).
At the start of each experiment, all cells were rinsed in PBS and then
treated with fresh medium with or without serum, insulin, recombinant mutant IGF-1, or IGF-1 receptor inhibitor, JB3 (37). The
half-life of insulin in this medium was 49.7 h, measured by insulin enzyme-linked immunosorbent assay (Alpco, Windham, NH) in samples of culture medium taken from cells treated with 10 nM insulin for 48 h. The concentration of insulin in
the undiluted serum was estimated as 0.2 ng/ml by enzyme-linked
immunosorbent assay.
Quantification of Apoptosis--
Cells were grown to 90%
confluence before 24-h experimental treatment. The coverslips were
rinsed in PBS and fixed in 1% paraformaldehyde before staining with 1 µg/ml propidium iodide and 0.5 mg/ml DNase-free RNase A (Sigma).
Cells were viewed using the × 40 objective of an Olympus BH-2
fluorescence microscope mounted with a Sony 3CCD video camera attached
to an IBM personal computer running image analysis software
(Optimus, Media Cybernetics, Silver Spring, MD). Five visual
fields were randomly sampled from each coverslip and all the cells in
each field were counted as either pyknotic, mitotic, or viable. The
number of pyknotic cells was summated in the five sampled regions and
expressed as percentage of apoptosis per coverslip = (total number
of pyknotic cells) (total number of pyknotic cells + total number of
viable cells) × 100. A similar approach was used to estimate the
percentage of mitotic cells.
Immunoprecipitation and Immunoblotting--
The cells were
harvested as described previously (38) in Triton buffer (10 mM HEPES, 42 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 10 mM sodium pyrophosphate, 10 mM
benzamidine, 1% Triton X-100, and protease inhibitor tablet; Roche,
Mannheim, Germany). To detect receptor phosphorylation, an
immunoprecipitation buffer was used (50 mM HEPES, pH 7.3, 137 mM NaCl, 1 mM MgCl2, 1 mM Ca Cl2, 2 mM
Na3VO4, 10 mM sodium pyrophosphate,
10 mM NaF, 2 mM EDTA, 2 mM
phenylmethylsulfonyl fluoride, 10 mM benzamidine, 10%
glycerol, 1% Nonidet P-40, and 1 protease inhibitor tablet/10 ml). For
Akt immunoblotting, the following lysis buffer was used: 10 mM HEPES, 42 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM 10 mM sodium pyrophosphate, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 10 mM NaF,
10 mM benzamidine, 1% Triton, and 1 protease inhibitor
tablet/10 ml. Protein concentrations were measured with the Pierce BCA
reagent, and all samples were adjusted for equal protein before
SDS-polyacrylamide gel electrophoresis.
Immunoprecipitation was performed overnight at 4 °C with polyclonal
anti-insulin or anti-IGF-1 receptor-
subunits (Santa Cruz
Biotechnology, San Diego, CA), 1 µg/mg protein/ml, and 30 µl
protein A-Sepharose beads. Samples were mixed with 5× sample buffer
(0.32 M Tris, pH 6.8, 62.5% glycerol, 6.25%
-mercaptoethanol, 5% SDS, 5.6 mg/ml bromphenol blue) and boiled for
3 min before SDS-polyacrylamide gel electrophoresis. Phosphotyrosine
blotting was performed with mouse monoclonal anti-phosphotyrosine
(1:1000, Upstate Biotechnology, Inc.) followed by biotinylated
sheep anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ) and
alkaline-phosphatase-conjugated streptavidin (Life Technologies, Inc.).
Protein bands were detected by enhanced chemical fluorescence (Amersham
Pharmacia Biotech) and read with a Fluorimager (Molecular Dynamics).
Phosphotyrosine blots were washed overnight and reprobed with the
polyclonal anti-insulin receptor-, followed by horseradish
peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech), and
detected using enhanced chemical luminescence. Whole lysate
immunoblotting was performed using enhanced chemical fluorescence, as
described (38). p53 was detected with polyclonal rabbit anti-p53
(1:500, Santa Cruz Biotechnology, Santa Cruz, CA); Akt and Ser 473 phospho-Akt were detected with rabbit polyclonal antibodies (Cell
Signaling Technology, Beverly, MA). Densitometric analysis of enhanced
chemical fluorescence immunoblots was performed with ImageQuant
(Molecular Dynamics, Sunnyvale, CA), and analysis of ECL
immunoblots was performed with NIH Image.
Immunocytochemistry--
R28 cells grown on laminin-coated
coverslips were deprived of serum and treated for 24 h with or
without 10 nM insulin or 1.3 nM IGF-1-3R,
which has decreased affinity for IGF-binding proteins relative to
native IGF-1 (39). The cells were fixed in 1% paraformaldehyde and
then incubated with a rabbit polyclonal antibody against activated
caspase-3 (CM-1, Idun Pharmaceuticals, La Jolla, CA) or a rabbit
anti-rat antibody against p65 of NF-B (a generous gift of Dr.
Shao-Cong Sun, Pennsylvania State University) (both antibodies at
1:1000). The secondary antibody was rhodamine red X-conjugated donkey
anti-rabbit (1:2000, Jackson Immunologicals, West Grove, PA). Cells
were simultaneously stained with the nuclear dye bisbenzimide (Hoechst
dye 33258, 0.5 µg/ml, Sigma).
Transfections--
R28 cells were transfected using
LipofectAMINE PLUSTM reagent (Life Technologies, Inc.) with
2.5 µg of either the parental pCMV4 or the pCMV4-HA-IB S36A
expression vector and seeded on coverslips. The parental vector pCMV4
and the cDNA expression vector encoding the hemagglutinin
(HA)-tagged IB with a serine to alanine substitution at
residue 36 (S36A) (pCMV4-HA-IB S36A) were generous gifts from Dr.
Shao-Cang Sun from Department of Microbiology and Immunology, Pennsylvania State University College of Medicine (Hershey, PA) (40).
24 h after transfection, the cells were either left in serum or
deprived of serum with or without 10 nM insulin and
incubated for another 24 h. The cells were fixed with 1%
paraformaldehyde and blocked at room temperature for 1 h in PBS
containing 0.1% Triton and 10% donkey serum (PBST). They were then
incubated at room temperature for 1 h in PBST with rabbit anti-HA
polyclonal antibody (1:200, CLONTECH, Palo Alto,
CA). The cells were washed and incubated at room temperature for 1 h with rhodamine red X-conjugated anti-rabbit IgG (1:2000) and 0.5 µg/ml Hoechst 33258.
Statistical Analysis--
Statistical comparisons were made by
one-way analysis of variance with post hoc Student-Newman-Keuls
multiple comparisons test (Instat 2.0, GraphPad Software). Statistical
significance was accepted if p < 0.05.
| |
RESULTS |
Induction of a Neuronal Phenotype in R28 Cells--
R28 cells have
been described as expressing both neuronal and glial cell markers (34).
In order to induce an entirely neuronal phenotype, the cells were
seeded on laminin and fed with medium containing cell-permeable
cAMP. Under these conditions, the R28 cells adopt a neuronal
morphology, with neurite-like projections (Fig.
1, A and B), and
their rate of division is reduced. Immunoblotting showed that the
differentiated R28 cells contain the neuronal markers, neuron-specific
enolase (Fig. 1C), and 200-kDa neurofilament (both
phosphorylated and nonphosphorylated; Fig. 1D) but do not have detectable levels of glial fibrillary acidic protein (Fig. 1E; no band could be detected when the brightness and
contrast settings on the blot image were increased to the upper limits of sensitivity). Immunoreactivity for neuron-specific enolase was also
abundant, measured by immunocytochemistry, and distributed homogeneously throughout the entire cell population (data not shown).
Immunocytochemistry also revealed immunoreactivity for phosphorylated
neurofilament but not glial fibrillary acidic protein or the
endothelial cell antigen von Willebrand factor (data not shown). Thus,
R28 cells can be differentiated in this way to provide a useful
in vitro model of retinal neurons.
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Fig. 1.
Characterization of a neuronal phenotype in
R28 cells. R28 cells were grown on laminin coated slides with cAMP
(250 µM) added to the medium. A, the cells
developed neurite-like projections and their growth rate was reduced
(100×). B, enlargement of A (200×). Western
blots on whole cell homogenates and rat retinas were probed for
neuronal and glial cell-specific antigens. C, both the
retina (lane 1) and R28 cells (lane 2) were
positive for neuron-specific enolase. D, R28 cells also
contained the 200-kDa heavy neurofilament (lane 1) and
phosphorylated neurofilament (lane 2). E, the
retina sample (lane 1) was positive for the glial-specific
antigen glial fibrillary acidic protein, whereas the R28 cells
(lane 2) had no detectable glial fibrillary acidic
protein.
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|
Insulin Is a Survival Factor for Retinal Neurons--
Insulin
mediates cell survival in primary cell cultures of cerebellar neurons
and retinal ganglion cells (1, 4). To determine whether insulin can
also act as a survival factor for the R28 model of retinal neurons, the
effect of insulin on apoptosis in serum-deprived cells was quantified.
A time course study showed that the rate of apoptosis peaked at 16-18
h after serum withdrawal (data not shown). Cells were deprived of serum
for 24 h and treated with either 10 nM insulin or 1.3 nM recombinant mutant IGF-1 (3R), which served as a
positive control. The number of apoptotic cells was counted after
propidium iodide staining. Between 10 and 20% of cells were pyknotic
after 24 h of serum deprivation, whereas less than 1% were
pyknotic in control conditions (Fig.
2A). Treatment with insulin
significantly reduced the frequency of pyknosis (Fig. 2B;
p < 0.05). Higher concentrations of insulin had no
greater effect on apoptosis, indicating that the maximal effect
was at 10 nM (Fig. 2C). Insulin treatment had no
effect on the frequency of mitosis (Fig. 2C). The IGF-1
inhibitor JB3 (37) prevented IGF-1 from rescuing cells by 50% but did
not block the survival effect of insulin (data not shown). There was no
greater effect of adding insulin and IGF-1 together (data not shown).
These results suggest that insulin blocks apoptosis in retinal neurons
by increasing cell survival rather than proliferation.
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Fig. 2.
Insulin rescues retinal neurons from
apoptosis. R28 cells were grown without serum for 24 h and
stained with propidium iodide. Half the samples were treated with 10 nM insulin during the entire period of serum deprivation.
The number of pyknotic nuclei was counted and expressed as a percentage
of the total number of cells. A, serum withdrawal
((-)serum) increased the number of pyknotic nuclei compared
with control cells ((+)serum). Addition of insulin
((+)insulin) reduced the number of pyknotic cells after
serum deprivation, (400X). B, the number of pyknotic cells
was measured as the percentage of pyknotic nuclei out of the total
number of cells counted (average from five randomly sampled visual
fields in three coverslips). There were almost no pyknotic cells in the
control cultures. Addition of 10 nM insulin to control
cells had no effect on pyknosis. Serum withdrawal significantly
increased the number of pyknotic cells (*, p < 0.001 compared with all other groups). Insulin significantly reduced the
number of pyknotic cells compared with the untreated serum deprived
cultures (*, p < 0.001; n = 3 coverslips). 1.3 nM IGF-1reduced apoptosis to a similar
degree. C, rescue by insulin was dose-dependent.
1 nM insulin did not significantly reduce the number of
pyknotic cells compared with the untreated serum deprived cultures,
whereas 10 nM insulin significantly reduced the number of
pyknotic cells (**, p < 0.001; n = 3 coverslips). No further reduction in the number of pyknotic cells was
observed with 50 or 100 nM insulin. The number of mitotic
bodies was also counted and expressed as a percentage of the total
number of cells. Insulin treatment did not alter the number of mitotic
cells in any of the culture conditions. n.s., no significant
difference. All comparisons were by analysis of variance and the
Student-Newman-Keuls test.
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|
Differentiated R28 Cells Express Functional Insulin and IGF-1
Receptors--
The insulin receptor signaling system in R28 cells was
explored by immunoblotting for the -subunit of the insulin receptor. R28 cells express both the 125-kDa isoform of the -chain, similar to
the most abundant form in liver (41), as well as the 115-kDa isoform
that is typical of the adult retina (Fig.
3A). By immunoprecipitation of
the -subunits of the insulin or IGF-1 receptors, followed by
phosphotyrosine immunoblotting, it was determined that the insulin
receptors were tyrosine-phosphorylated by 10 nM insulin, whereas the IGF-1 receptors were not (Fig. 3B). By two-point
binding assay with 0.5 nmol of 125I-bovine insulin, the
concentration of insulin receptors in the R28 cells was 110 pmol/mg
total protein, which is comparable to that measured in liver plasma
membranes and hepatocytes (42). The content of IGF-1 receptors in R28
cells was also similar to that of whole retina, measured by immunoblot.
The content of insulin receptors in R28 cells was also comparable to
that of equivalent protein samples of whole retina and liver lysates
(data not shown). These data show that the survival effect of 10 nM insulin is mediated through the insulin receptor and not
IGF-1.
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Fig. 3.
Insulin and IGF-1 receptors are activated by
their respective ligands without cross-activation. Lysates
of R28 cells, retina, and liver were immunoblotted for the -subunit
of the insulin receptor. To confirm that the insulin and IGF-1
receptors of R28 cells respond to their respective ligands, the
-subunits of these receptors were immunoprecipitated and blotted for
phosphotyrosine after 2 min or 24 h of incubation with insulin (10 nM) or IGF-1 (1.3 nM). A, R28 cells
express both the 115-kDa and the 125-kDa isoform of the insulin
receptor -subunit (lane 1) compared with equivalent
protein loading of retina (lane 2) and liver (lane
3) samples. B, R28 cells were incubated with insulin or
IGF-1, and the -subunits of the insulin and IGF-1 receptor were
immunoprecipitated. Samples were resolved in parallel by
SDS-polyacrylamide gel electrophoresis followed by phosphotyrosine
immunoblotting to determine autophosphorylation of the respective
receptors. Insulin added for 2 min induced tyrosine phosphorylation of
the insulin receptor but not the IGF-1 receptor. IGF-1 treatment for 2 min also induced some weak phosphorylation of the insulin receptor and
strongly phosphorylated the IGF-1 receptor. There was no detectable
basal phosphorylation of either receptor. Whereas phosphorylation of
the insulin receptor was reduced after 24 h of insulin treatment,
phosphorylation of the IGF-1 receptor was more attenuated. Immunoblots
are typical of three replications.
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|
Retinal Cell Survival Is Mediated by PI 3-kinase and Akt--
Many
growth factors have antiapoptotic effects via activation of PI 3-kinase
and Akt/protein kinase B in various cell types (6), so the
involvement of these pathways was investigated in differentiated R28
cells. Cells were treated with the PI 3-kinase inhibitors wortmannin
(100 nM) or LY294002 (10 µM) or with the MEK
kinase inhibitor PD98059 (25 µM), dissolved in dilute
Me2SO. Inhibition of PI 3-kinase activity with
wortmannin (100 nM) or the more specific PI 3-kinase
inhibitor LY294002 (10 µM) blocked the ability of both
insulin and IGF-1 to rescue R28 cells from apoptosis induced by serum
deprivation (Fig. 4). The MEK1 inhibitor PD98059 also blocked insulin-mediated survival, but the magnitude of
inhibition was not as great as that induced by the PI 3-kinase inhibitors. Therefore, the PI 3-kinase pathway has a primary role in
survival mediated by insulin.
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Fig. 4.
PI 3-kinase pathway plays a dominant role in
neuroprotection mediated by insulin and IGF-1. Differentiated R28
cells were serum-deprived and treated with the PI 3-kinase inhibitors
wortmannin or LY294002, with the MEK1 inhibitor PD098059, or with
0.53% Me2SO for 30 min, followed by addition of 10 nM insulin or 1.3 nM IGF-1. The cells were
stained with propidium iodide after 24 h of treatment, and the
pyknotic nuclei were counted. A, serum deprivation
significantly elevated the number of pyknotic cells, as before.
Addition of the PI 3-kinase inhibitors, the MEK inhibitor, or
Me2SO vehicle in the absence of serum did not alter the
number of pyknotic cells. B, insulin significantly reduced
the number of pyknotic cells compared with untreated serum-deprived
cells. The effect of insulin was reversed by wortmannin and LY294002,
but PD098059 did not block the significant reduction in pyknotic cells
caused by insulin treatment. C, IGF-1 also reduced the
number of pyknotic cells compared with untreated serum-deprived cells;
this effect was reversed by wortmannin and LY294002 but not PD098059.
*, p < 0.05; **, p < 0.01; ***,
p < 0.001; n.s., not significant.
n = 3 coverslips per experiment).
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Activation of the downstream target of the PI 3-kinase pathway, Akt,
was investigated by immunoblotting with antibodies to total Akt and to
Ser 473-phosphorylated Akt. Total Akt content was unchanged after serum
deprivation, and there was a low basal level of Akt phosphorylation.
The amount of Ser 473-phosphorylated Akt increased significantly within
10 min of insulin stimulation in the absence of serum (Fig.
5A). Akt phosphorylation was
also induced by insulin in cells grown with serum (data not shown). Wortmannin and LY294002 both blocked the Akt serine phosphorylation induced by insulin when serum was removed (Fig. 5B). The
reduction in Akt phosphorylation was statistically significant (Fig.
5C; p < 0.05). Similar results were
obtained after stimulation with IGF-1. These data suggest that insulin
induces Akt phosphorylation through PI 3-kinase in retinal neurons.
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Fig. 5.
Insulin activates Akt in R28 cells. R28
cells were immunoblotted for total Akt and serine-phosphorylated Akt
(pAkt) using total and phosphorylation-specific antibodies.
Serum-deprived cells were treated with 10 nM insulin for 30 min after blocking PI 3-kinase with wortmannin (100 nM) or
LY294002 (10 µM). A, insulin did not increase
the total amount of Akt (top panel), but it did increase the
content of serine-phosphorylated Akt (bottom panel). Similar
results were obtained with 1.3 nM IGF-1. B,
serum deprivation did not alter the amount of phosphorylated Akt
compared with serum-fed cells. Addition of insulin again increased the
amount of phosphorylated Akt without altering the amount of total Akt.
Both wortmannin and LY294002 blocked the Akt phosphorylation induced by
insulin. C, the amount of pAkt was elevated ~3-fold above
control levels by insulin stimulation in serum-deprived cells
(mean ± S.E. from three replications).
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Insulin Inhibits Caspase-3 Activation--
Caspase-3 is a cysteine
protease that becomes active during the later stages of apoptosis.
Previous studies have shown that activated caspase-3 can be detected by
immunocytochemistry using the antibody CM-1 (43). Immunocytochemistry
for activated caspase-3 was performed on serum-deprived R28 cells using
the CM-1 antibody. The cells were counterstained with Hoechst 33258 dye
to reveal nuclear morphology. In cells grown with serum, there was
little CM-1 immunoreactivity, and almost all cells had normal nuclear morphology (Fig. 6). In contrast, many of
the serum-deprived cells were immunoreactive for caspase-3. Those cells
with caspase-3 immunoreactivity also tended to have pyknotic nuclei.
Both insulin and IGF-1 reduced CM-1 immunoreactivity and the number of
pyknotic cells, whereas LY294002 blocked these effects. Thus, insulin
inhibits the activation of caspase-3 by a PI
3-kinase-dependent mechanism.
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Fig. 6.
Insulin prevents activation of caspase-3 by a
PI 3-kinase-dependent pathway. R28 cells were deprived
of serum and treated with insulin or IGF-1 after addition of the PI
3-kinase inhibitor LY294002 (10 µM). Activated caspase-3
was detected by immunofluorescent histochemistry with the polyclonal
antibody CM-1 (left panel of each group) and counterstained
with Hoechst 33258 to reveal the nuclear morphology (right
panel of each group). In the serum-fed group, there was no
detectable immunoreactivity for caspase-3, and all cells had normal
nuclear morphology (top left panels). Many cells deprived of
serum were immunoreactive for caspase-3 and had pyknotic nuclei
(top right panels; arrows indicate the same
cell). Insulin reduced the number of caspase-3 immunoreactive cells
(middle left panels). The effect of insulin was blocked by
addition of LY294002 (middle right panels). Similar results
were obtained with IGF-1 (bottom panels). All photographs
taken with a × 40 objective.
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Insulin Signaling and NF-B--
Insulin induces activation of
the nuclear transcription factor NF-B in sympathetic and cerebellar
neurons (22, 44, 45). In order to test whether translocation of p65
NF-B is also required for rescue by insulin in R28 cells, four
approaches were used. First, the peptide inhibitor of the NF-B
nuclear localization sequence, SN50, was tested. SN50 is a
cell-permeable antisense peptide that binds to the nuclear
translocation region of NF-B and prevents it from being transported
to the nucleus (46). SN50 alone increased the number of pyknotic cells
in serum-deprived cultures (Fig.
7A), whereas the control
peptide, SN50M, which is mutated to prevent its peptide binding
ability, had no effect on apoptosis. SN50 blocked the ability of
insulin to rescue R28 cells from apoptosis and also increased the
number of pyknotic cells significantly more than serum deprivation
(Fig. 7B). In contrast, SN50 blocked the ability of IGF-1 to
prevent apoptosis but did not increase the amount of apoptosis any more
than serum deprivation (Fig. 7C). The mutated peptide,
SN50M, did not block the effects of insulin or IGF-1. These data
suggest that NF-B translocation may be involved in cell survival
induced by insulin.
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Fig. 7.
Blocking the NF-B
nuclear translocation sequence inhibits the rescue induced by
insulin. R28 cells were serum-deprived for 24 h in the
presence of SN50 (75 nM), a peptide blocker of the nuclear
translocation sequence in NF-B, or SN50M, a mutated peptide
incapable of blocking the nuclear translocation sequence. The number of
pyknotic cells was counted as before. A, serum deprivation
increased the number of pyknotic cells, as before. SN50 significantly
increased the number of pyknotic cells beyond serum deprivation alone,
whereas SN50M had no effect. B, 10 nM insulin
reduced the number of pyknotic cells induced by serum deprivation, as
before. SN50 prevented the reduction induced by insulin and
significantly elevated the number of pyknotic cells compared with serum
deprivation alone, whereas SN50M had no effect on the reduction in
pyknotic cells induced by insulin. C, IGF-1 also reduced the
number of pyknotic cells induced by serum deprivation. SN50 blocked the
effect of IGF-1 but did not elevate the number of pyknotic cells beyond
that of serum deprivation alone. SN50M did not change the effect of
IGF-1. *, p < 0.05; **, p < 0.01;
***, p < 0.001; n.s., not significant. All
statistical comparisons were relative to the serum-deprived group;
n = 3 coverslips per experiment.
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Second, to examine nuclear translocation of the p65 subunit of NF-B
directly, we used immunocytochemistry in R28 cells treated with or
without insulin. R28 cells were grown in the presence of insulin for 6 or 24 h, fixed, and labeled with an antibody to the p65 subunit of
NF-B. No nuclear localization of p65 was observed after any of the
treatment conditions (Fig. 8), suggesting that insulin does not induce nuclear translocation of NF- in R28
cells.
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Fig. 8.
Insulin does not cause nuclear translocation
of the p65 subunit of NF-B. R28 cells
were grown in the presence of 10 nM insulin for either 6 or
24 h. The cells were stained with Hoechst 33258 to identify nuclei
(top panels) and labeled with an antibody to the p65 subunit
of NF-B (bottom panels). Cells treated with insulin for
24 h are shown (right panels) compared with untreated
cells (left panels). Positive immunoreactivity for p65 was
limited to the cytoplasm of all cells, whereas the nuclei had little or
no immunoreactivity. The distribution of p65 immunoreactivity was
unaltered by treatment with insulin.
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|
Third, R28 cells were transfected with a dominant negative construct of
IB, which cannot be activated by phosphorylation. About 20% of the
cells were transfected successfully, indicated by positive HA
immunoreactivity. However, none of the transfected cells contained
pyknotic nuclei or caspase-3 immunoreactivity after serum deprivation
(data not shown). These data suggest that transfection of a dominant
negative IB caused the cells to be resistant to apoptosis induced by
serum deprivation.
In the fourth and final approach, we examined the
phosphorylation of IB. In order for NF-B to be translocated to
the nucleus, IB must be phosphorylated. To test whether this occurs
during apoptosis in R28 cells, immunoblotting with a phospho-specific antibody to IB was carried out on lysates of cells that had been serum-deprived and treated with insulin, IGF-1, or LY294002. The cell
lysates were also immunoblotted for Ser 473-phosphorylated Akt and
compared with lysates of HeLa cells as positive controls. Compared with
HeLa cells, there was little IB phosphorylation in R28 cells
deprived of serum for 24 h. Treatment with insulin, IGF-1, or
LY294002 also had little effect on IB phosphorylation (Fig.
9). These data suggest that IB
phosphorylation does not occur in R28 cells under these conditions.
View larger version (43K):
[in this window]
[in a new window]
|
Fig. 9.
Serum deprivation and insulin treatment do
not induce IB phosphorylation. R28 cells
were deprived of serum for 3 h and stimulated with 10 nM insulin for the indicated periods. LY294002 was added to
some plates 15 min prior to insulin treatment. Cell lysates were
immunoblotted for total or phospho-IB (Ser32). Total cell
extracts from HeLa cells treated with or without tumor necrosis
factor (Cell Signaling Technology) served as controls.
A, immunoblot for total IB. Total IB was not
changed with serum deprivation or insulin treatment in R28 cells.
B, immunoblot for phospho-IB. R28 cells do not contain
the phosphorylated form of IB, whereas IB was increased in
HeLa cells after tumor necrosis factor stimulation (lane
b) compared with no stimulation (lane a). C,
the same lysates shown in A and B were blotted
for phospho-Akt. Insulin induced phosphorylation of Akt after 5 min of
stimulation; this effect was maintained for longer than 15 min.
Similar results were obtained with 1.3 nM IGF-1.
|
|
In total, the data from these four experiments suggest that NF-B
does not mediate the rescue effect of insulin and that SN50 may block
insulin signaling by an unknown mechanism in R28 cells.
Insulin Does Not Induce Survival by Altering p53
Expression--
The tumor suppressor protein p53 has been implicated
as a mediator of apoptosis in some cell types, but its involvement is determined by the type of apoptotic stimulus (30, 47). The PI 3-kinase
pathway can block p53-mediated apoptosis (48). To examine its
involvement in apoptosis of retinal neurons, p53 was quantified by
immunoblotting R28 cells after serum deprivation with or without
insulin (Fig. 10A). There
was a transient increase in p53 content that reached a peak after 2-4
h of serum deprivation, which fell to almost undetectable levels after
24 h (Fig. 10B). Treatment with 10 nM
insulin did not alter the transient elevation of p53 in serum-deprived
cells (Fig. 10C). Therefore, insulin may induce survival of
R28 cells by affecting a target either downstream or independent of
p53.
View larger version (25K):
[in this window]
[in a new window]
|
Fig. 10.
p53 is transiently activated by serum
deprivation and is not affected by insulin. R28 cells were
serum-deprived for between 1 and 24 h and immunoblotted for p53.
In a similar experiment, the cells were serum-deprived and treated with
10 nM insulin. A, p53 peptide resolved as a
single band in equal protein-loaded samples. B,
quantification relative to 15 ng of p53 peptide showed that the total
content of p53 was transiently elevated after 3 h of serum
deprivation. By 24 h, the total content of p53 was diminished to
less than control levels. C, 10 nM insulin did
not alter the change in p53 expression in serum-deprived cells.
|
|
| |
DISCUSSION |
The data presented here demonstrate that physiological
concentrations of insulin can protect retinal neurons from apoptosis induced by serum deprivation in culture. This concept has been demonstrated in in vitro models of neurons from other parts
of the nervous system, including sensory nerves (49), cortex (50), cerebellar granule cells (51), spinal motor neurons (52), and
PC12 cells (53). In this study, we used differentiated R28 cells to
model retinal neurons, and the results further support previous
observations that both insulin and IGF-1 are trophic factors for the
survival of retinal neurons (4). To the best of our knowledge, this is
the first study to examine the signaling mechanisms for retinal neurons
in response to insulin. The potency of insulin to act as a survival
factor in vitro has led to the suggestion that insulin alone
is sufficient for neuronal survival, at least at high concentration
(1). However, high concentrations of insulin can also activate the
IGF-1 receptor. In the present study, lower concentrations of insulin
were used. Insulin had a maximal effect in reducing apoptosis at a
concentration of 10 nM, which is within the physiological
range for plasma insulin and does not stimulate the IGF-1 receptor.
Therefore, we conclude that the protective effect of insulin was
specific for the insulin receptor.
Although it is not likely that neurons of the retina are directly
exposed to plasma when the blood-retinal barrier is intact, the
concentration of insulin in the ocular fluids
vitreous and aqueous
humorshas been estimated to be in the physiological range (54, 55).
Insulin may be transported into the retina across the blood-retinal
barrier similar to the blood-brain barrier (56, 57) or may arise by
de novo synthesis within the retina, as indicated by the
presence of preproinsulin mRNA in retina (58). Although the source
of insulin is not known, it is clear that both insulin and its receptor
are abundant in the retina, especially in the inner plexiform layer,
which is mostly formed by neuronal projections (3, 59, 60). There is
strong evidence that insulin has biological activity in the central
nervous system. The insulin receptor is expressed in the brain (61).
Insulin-like peptides may also be released from cultured
neuronal cells, synaptosomes, and astrocytes (62-64). Therefore, it is
reasonable to suggest that insulin signaling in retinal neurons is
active in vivo with canonical physiological consequences.
The R28 cells used here as a model of retinal neurons were derived from
mixed retinal cells but have been used successfully in other studies of
neuronal apoptosis (34, 65). Under the culture conditions employed
here, these cells had a neuronal phenotype with no evidence to suggest
glial or endothelial cell characteristics. When laminin is used as a
substrate for other neuronal cell lines, such as PC12 cells, it causes
them to develop extensive neurite outgrowth (66). This effect may be
modulated by protein kinase C, but a direct regulatory role has not
been established. The role of cAMP in further differentiating R28 cells
is also important to consider. cAMP elevation promotes survival of
spinal motor neurons in vitro (67), possibly by elevating
the recruitment of the neurotrophin TrkB receptor to the plasma
membrane (68). It is not clear whether cAMP has a similar effect on R28
cells. Although the E1A transfection of R28 cells causes their
persistent proliferation, we have shown that the addition of laminin
and cAMP provides a more stable model of retinal neurons.
The characteristics of the insulin receptor in the retina differ from
those seen in peripheral tissues such as liver, muscle, and fat. The
predominant insulin receptor in the central nervous system is less
glycosylated on both and subunits (41). The differentiated R28
cells express both the 125- and 115-kDa species of the -subunit of
the insulin receptor. The -subunits of the insulin receptors were
tyrosine-phosphorylated by 10 nM insulin, but the IGF-1
receptor was not. Thus, differentiated R28 cells respond to insulin
stimulation with receptor tyrosine phosphorylation. This is in
agreement with other data showing that exogenous physiological concentrations of insulin also cause receptor tyrosine phosphorylation in intact retinas (69).
Insulin reduced the activation of caspase-3 in R28 retinal neurons, and
this effect was blocked by the PI 3-kinase inhibitors. These data imply
that insulin regulates the activation of caspase-3 through the PI
3-kinase/Akt pathway. Akt can directly phosphorylate caspase-9, which
is at the head of the caspase protease cascade (7). Therefore,
caspase-3 activation may be blocked by insulin receptor signaling in
retinal neurons through Akt phosphorylation of caspase-9. This implies
that reduction of insulin stimulation could lead to apoptosis mediated
by caspase-3 activation in retinal neurons.
Previous reports have suggested that the PI 3-kinase pathway activates
NF-B in response to insulin, to promote neuronal survival. We used
four approaches to test this hypothesis in R28 cells. First, the
peptide inhibitor of p65 nuclear translocation, SN50, blocked the
protective effect of insulin. These data suggested that translocation
of NF-B to the nucleus is required for insulin to rescue the R28
cells. However, additional experiments did not support this initial
interpretation. Transfection of a dominant negative IB mutant
blocked the apoptosis induced by serum deprivation, rendering this
approach inoperable. Therefore, effects of insulin on IB
phosphorylation were examined. Serum deprivation or insulin did not
induce detectable IB phosphorylation despite a relative abundance of
IB protein compared with HeLa cells. Because phosphorylation of
IB is generally considered to be required for NF-B translocation, it appears that this may not be an important insulin signaling mechanism in R28 cells. Finally, immunohistochemistry revealed no
nuclear translocation of the p65 subunit in response to insulin. Although SN50 augmented apoptosis, it may not be specific for NF-B, because it also blocks other transcription factors with nuclear localization sequences by competing for binding to components of the nuclear transport machinery (46, 70). Taken together, these data
suggest that another transcription factor requiring nuclear
translocation may be involved. Therefore, the rescue effect of insulin
in R28 cells deprived of serum may not occur by activation of
NF-B.
In conclusion, our data show that insulin can act as a survival factor
for R28 cells by stimulating the PI 3-kinase, Akt/protein kinase
B pathway and inhibiting the activation of caspase-3. Although p53 is transiently invoked during this form of cell death, the survival
effect of insulin does not alter p53 expression, implying that it
modulates downstream effectors of p53. Insulin may regulate multiple
systems in retinal tissue, and it is likely that part of its function
is to act as a survival signal for retinal neurons. Recent evidence
shows that experimental diabetes induces retinal neurodegeneration by
increasing apoptosis of inner retinal neurons (71). Apoptosis increases
soon after the onset of streptozotocin diabetes and is reversed by
exogenous insulin. This cell death gives rise to a chronic
neurodegeneration in which ~10% of retinal ganglion cells is lost
after 7.5 months. Insulin also increases glutamine synthetase activity
in the retinas of diabetic rats (72-74). Acute insulin treatment also
reverses the effects of diabetes on the expression of glial fibrillary
acidic protein and the tight junction protein, occludin, in diabetic
rats (75). Taken together these findings raise the possibility that
defective insulin signaling could contribute to retinal
neurodegeneration in diabetes.
We thank Dr. Lois E. H. Smith for the
gift of JB3 and Dr. Shao-Cong Sun for the gift of IB and NFB
antibodies and dominant negative IB constructs, along with his
invaluable advice concerning the transfection experiments.
The abbreviations used are:
IGF, insulin-like growth factor;
HA, hemagglutinin;
PI, phosphatidylinositol;
PBS, phosphate-buffered saline.
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§,
,
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ABSTRACT
INTRODUCTION
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