From the Departments of Medicine and Pathology, Rhode Island Hospital, Brown Medical School, Providence, Rhode Island 02903
Received for publication, January 14, 2003 , and in revised form, March 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Previous experiments demonstrated that neuronal loss following ethanol exposure was mediated by apoptosis (810) or mitochondrial dysfunction (1012), and recent studies correlated these adverse effects of ethanol to inhibition of growth factor-stimulated survival signaling (9, 1113). In the developing CNS, insulin and insulin-like growth factor type 1 (IGF-1)1 receptors are abundantly expressed (1416), and the corresponding growth factor-stimulated responses are critical mediators of neuronal growth, viability, energy metabolism, and synapse formation. Because insulin and IGF-1 signaling pathways are among the important targets of ethanol neurotoxicity in immature nervous system (9, 13, 17, 18), neuronal loss associated with microencephaly in ethanol-exposed fetuses may be caused, in part, by ethanol inhibition of insulin/IGF-1stimulated survival mechanisms.
The stimulatory effects of insulin and IGF-1 are mediated through complex pathways, beginning with ligand binding and activation of intrinsic receptor tyrosine kinases (19, 20), which phosphorylate specific cytosolic molecules, including two of their major substrates, the insulin receptor substrate types 1 (IRS-1) and 2 (IRS-2) (21, 22). Tyrosyl-phosphorylated IRS-1 (PY-IRS-1) transmits intracellular signals that mediate growth, metabolic functions, and viability by interacting with downstream Src homology 2-containing molecules through specific motifs located in the C-terminal region of IRS-1 (21, 22). The 897YVNI motif of IRS-1 binds to the Grb2 (growth factor receptor-bound protein 2) adapter molecule (23, 24). The 1180YIDL motif binds to Syp protein tyrosine phosphatase, and the 613YMPM and 942YMKM motifs bind to the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) (25). Binding of PY-IRS-1 to p85 stimulates glucose transport (26) and inhibits apoptosis by activating Akt/protein kinase B (27, 28) or inhibiting glycogen synthase kinase-3 (GSK-3) (29). Akt kinase inhibits apoptosis by phosphorylating GSK-3 (29, 30) and BAD (31), rendering them inactive. Low levels of Akt kinase and high levels of GSK-3 activity or activated BAD are associated with increased neuronal death (3234). BAD inactivates anti-apoptotic Bcl family proteins, rendering the mitochondrial membrane more susceptible to pro-apoptotic molecules that promote membrane permeabilization, cytochrome c release, and caspase activation (35). Perturbations in mitochondrial membrane permeability increase cellular free radicals that cause mitochondrial DNA damage, impair mitochondrial function, and activate pro-apoptosis cascades (36, 37).
Our previous studies (11, 13) using in vitro or in vivo models demonstrated that ethanol profoundly inhibits insulin-stimulated survival and mitochondrial function in cultured neuronal cells. The present study was designed to examine the effects of chronic gestational exposure to ethanol on insulin-, IRS-1-, and PI3 kinase-mediated signaling in the intact brain to determine the extent to which abnormalities in these pathways correlate with ethanol-induced developmental defects in the CNS. PTEN expression and activity were also examined, because PTEN dephosphorylates and negatively regulates PI3 kinase function (38), and the effects of ethanol on phosphatase and tensin homolog deleted in chromosome 10 (PTEN) were unknown. Cerebellar tissue was studied, because it represents a major in vivo target of ethanol neurotoxicity (2, 39).
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EXPERIMENTAL PROCEDURES |
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In Situ Assays for ApoptosisThe terminal transferase dUTP end-labeling (TUNEL) assay was used to detect nicked or fragmented DNA in cryostat sections of cerebella. TUNEL assays were performed using fluorescein-labeled dUTP ([Fl]dUTP; Invitrogen) and terminal deoxynucleotide transferase (17). The labeled DNA was detected with biotinylated secondary antibody, alkaline phosphatase-conjugated Streptavidin, and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as the substrate. To detect nuclear pyknosis and fragmentation characteristic of apoptosis, adjacent sections were stained with Hoechst H33258 [GenBank] (1 µg/ml in phosphate-buffered saline) for 2 min at room temperature. The slides were rinsed thoroughly in phosphate-buffered saline, cover-slipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and examined by fluorescence microscopy. Adjacent sections were immunostained with polyclonal antibodies to activated (cleaved) caspase-3 (Cell Signaling, Beverly, MA). Histological sections were prepared for immunostaining according to the manufacturer's protocol. Immunoreactivity was detected with biotinylated secondary antibody, alkaline phosphatase-conjugated Streptavidin, and the BCIP/NBT substrate.
Western Blot Analysis and Immunoprecipitation StudiesFor Western blot analysis, tissue from individual cerebella were polytron homogenized in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 2 mM EGTA) containing protease and phosphatase inhibitors (1 mM NaF, 1 mM Na4P2O7, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, pepstatin A, and leupeptin) (41). For immunoprecipitations, homogenates were prepared in Triton lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors as indicated. Cellular debris was pelleted by centrifuging the samples at 14,000 x g for 15 min at 4 °C, and supernatant fractions were used in the studies. Protein concentration was measured with the bis-chloracetate (BCA) assay (Pierce). 60- or 100-µg protein aliquots were used for Western blot analysis (17, 42, 43), and 500-µg samples were used for immunoprecipitation/Western immunoblotting or kinase assays (11, 17). Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibody and SuperSignal enhanced chemiluminescence reagents (Pierce). Immunoreactivity was quantified using the Eastman Kodak Co. Digital Science Imaging Station (PerkinElmer Life Sciences).
Kinase AssaysPI3 kinase activity was measured in p85
immunoprecipitates (42)
obtained from individual 500-µg protein samples using rabbit polyclonal
anti-p85 (1 µg/ml) and protein A-Sepharose (Amersham Biosciences).
Immunoprecipitates complexed with protein A-Sepharose were suspended in 50
µl of TNE buffer (10 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 5 mM EDTA), and reactions were initiated by sequentially
adding 20 µg of sonicated phosphatidylinositol (10 µl), 10 µl of 100
mM MgCl2, and 5 µl of [-32P]ATP
working solution composed of 0.88 mM [
-32P]ATP
(30 µCi of [
-32P]ATP/3000 Ci/mmol), 20 mM
MgCl2, and 150 mM cold ATP. Reactions were incubated for
10 min at 37 °C and 300 rpm and stopped by adding 15 µl of 6
N HCl. Phospholipids extracted with chloroform/methanol were
analyzed by thin layer chromatography using plates pre-coated with 1%
potassium oxalate (Merck). PI3 kinase activity was detected by film
autoradiography and quantified using the Kodak Digital Science Imaging
Station.
To measure Akt and GSK-3 kinase activities, corresponding
immunoprecipitates captured onto protein A-Sepharose beads
(41) were suspended in 10
µl of 5x assay dilution buffer (ADB; 100 mM MOPS, pH 7.2,
125 mM
-glycerol phosphate, 5 mM EGTA, 5
mM sodium orthovanadate, and 5 mM dithiothreitol).
Reactions were performed by sequentially adding 10 µl each of
Mg2+/ATP mixture (100 mM non-radioactive ATP,
75 mM MgCl2 in ADB), [
-32P]ATP
(diluted to a final concentration of 1 µCi/µl using
Mg2+/ATP mixture), and 10 nM synthetic
peptide substrate. Crosstide (Upstate Biotechnology, Inc., Lake Placid, NY)
was used to measure Akt activity, and cAMP-response element-binding protein
(New England Biolabs, Beverly, MA) was used as the substrate for GSK-3
(Upstate Biotechnology). Reactions were incubated for 10 (GSK-3) or 15 (Akt)
min at 30 °C and 300 rpm and then terminated by adding 5 µl of 0.5
M EDTA. 10 µl of each reaction were spotted in duplicate onto
P81 phosphocellulose. Nonspecific counts were removed by washing the P81 three
times in 0.85% phosphoric acid
(41) followed by 95% ethanol.
[
-32P]ATP incorporation was measured in a TopCount machine
(Packard Instrument Co., Meriden, CT).
PTEN StudiesPTEN expression was measured by Western blot analysis and real-time quantitative reverse-transcribed (RT)-PCR assays. In addition, PTEN phosphatase activity was measured in PTEN immunoprecipitates using the Biomol Green Reagent according to the manufacturer's protocol. Phosphatase inhibitors were omitted from the lysis buffer. For the real-time RT-PCR studies, total RNA was isolated from cerebellar tissue homogenates using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Samples containing 2 µg of RNA were reverse-transcribed using the AMV First Strand cDNA synthesis kit (Roche Molecular Biochemicals) and random oligodeoxynucleotide primers. Highly conserved regions of PTEN and 18 S cDNAs isolated from rat cerebellar tissue by RT-PCR were cloned into the PCRII vector (Invitrogen) and used to generate standard curves for determining transcript abundance. PCR amplifications were performed using 25-µl reaction volumes containing 20 ng of RT product, 0.4 µM each of forward and reverse primers (Table I), and SYBR Green I PCR reagent (Applied Biosystems, Foster, CA). The amplified signals were detected continuously with the Bio-Rad iCycler and iCycler iQ MultiColor Real Time PCR Detection System (Hercules, CA). The following real-time PCR amplification protocol was used: 1) initial denaturation, 95 °C for 10 min; 2) a three-segment amplification and quantification program consisting of 40 cycles of 95 °C x 60 s, 60 °C x 45 s and 72 °C for 30 s; and 3) a cooling step down to 4 °C.
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In Vitro ExperimentsIn vitro experiments were used to examine the effects of ethanol on PTEN expression, phosphorylation, and phosphatase activity in CNS neurons and to determine whether the responses observed in vivo were mediated by impaired insulin or IGF-1 signaling. Primary neuronal cultures were generated with cerebellar tissue harvested from P2 pups (44). Fluorescence-activated cell sorting demonstrated that greater than 95% of cells isolated from control or ethanol-exposed pup cerebella were neuronal as evidenced by the immunoreactivity with antibodies to HuC/HuD neuron-specific RNA binding protein (45) (Molecular Probes, Eugene, OR). Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 2 mM glutamine, 10 mM non-essential amino acid mixture (Invitrogen), 25 mM KCl, and 9 g/liter glucose. After overnight seeding, cells were treated with 6 µM cytosine arabinoside to inhibit DNA synthesis. Cultures were exposed to 50 mM ethanol or nothing for 2 days using sealed humidified chambers (17, 42), after which they were serum-starved for 12 h and then stimulated with 50 nM insulin (Humulin; Eli Lilly & Co., Indianapolis, IN) or 25 nM IGF-1 for 0, 15, 30, 60, or 120 min in the presence or absence of 50 mM ethanol. PTEN protein was detected by Western blot analysis. PTEN phosphorylation was evaluated by PTEN Western blot analysis of anti-phospho-serine/phospho-threonine immunoprecipitates. PTEN phosphatase activity was measured in PTEN immunoprecipitates using the Biomol Green Reagent (Cell Signaling, Beverly, MA).
To examine the effects of ethanol on insulin-stimulated viability and kinase activity, primary neuronal cultures were generated with cerebella harvested from control or ethanol-exposed P1 pups. However, to mimic the in vivo model in which the ethanol exposure was discontinued after birth, the cultured cells were not further exposed to ethanol. Micro-cultures (96-well plates) in which 5 x 104 viable cells (determined by Trypan Blue exclusion) were seeded per well were used for viability assays, and 60-mm Petri dish cultures were used for kinase or PTEN phosphatase assays. To measure insulin-stimulated responses, after 1 day in culture, the cells were serum-starved for 12 h and then stimulated with 50 nM insulin (Humulin; Eli Lilly & Co., Indianapolis, IN). Viability was measured after 24 h of insulin stimulation using the crystal violet assay (13, 17, 42). IRS-1-associated PI3 kinase, total PI3 kinase, Akt kinase, GSK-3 kinase, and PTEN phosphatase activities were measured in corresponding immunoprecipitates from cells stimulated with insulin for 0, 5, 15, or 30 min.
Source of ReagentsMonoclonal antibodies to phospho-tyrosine
(PY20) and phospho-serine were purchased from Transduction Laboratories
(Lexington, KY). Polyclonal antibodies to p85, IR, IRS-1, and PTEN
phosphatase were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Phospho-specific antibodies to Akt, GSK-3
, and BAD were obtained from
Cell Signaling (Beverly, MA). Protein A-Sepharose was purchased from Amersham
Biosciences. Monoclonal antibodies to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and GSK-3
were purchased from Chemicon Corp. All other reagents
were purchased from CalBiochem or Sigma-Aldrich.
Statistical AnalysisData depicted in the graphs represent the mean ± S.D. of results. Inter-group comparisons were made with the Student's t test or analysis of variance and the Fisher least significance post hoc significance test using Number Cruncher Statistical Systems (Dr. Jerry L. Hintze, Kaysville, UT).
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RESULTS |
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Reduced PY of IR in Cerebella of Ethanol-exposed Rat
PupsPrevious in vitro studies linked ethanol-induced
neuronal loss to inhibition of insulin signaling through its receptor
(17,
47). To characterize potential
mechanisms by which chronic gestational exposure to ethanol inhibits insulin
signaling in vivo, the levels of IR
and PY-IR
were
examined in cerebellar tissue homogenates by direct Western blot analysis and
Western blot analysis of immunoprecipitates, respectively. Digital image
quantification of results obtained with 12 brains in each group demonstrated
significantly reduced levels of PY-IR
in ethanol-exposed relative to
control samples (p < 0.01; see
Fig. 3, A and
C) but similar levels of total IR
protein in
control and ethanol-exposed cerebella (Fig.
3, B and D).
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Ethanol Inhibition of Insulin-responsive Gene Expression To
examine the downstream effects of ethanol on insulin signaling, we measured
cerebellar tissue expression of GAPDH, which is an important
insulin-responsive gene product
(48). Western blot analysis
using denaturing and reducing conditions detected a single 37-kDa protein
corresponding to monomeric GAPDH (Fig.
4A). Digital image quantification of results obtained
with 12 brains in each group demonstrated significantly reduced GAPDH
expression in ethanol-exposed relative to control cereballa (p <
0.01; see Fig.
4B).
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Reduced Levels of PY-IRS-1 in Cerebella of Ethanol-exposed PupsMajor effects of insulin are mediated through IRS-1, which transmits signals downstream to regulate growth, survival, and energy metabolism. To determine whether gestational exposure to ethanol inhibits tyrosyl phosphorylation of IRS-1 in cerebellar tissue, PY-IRS-1 and IRS-1 protein levels were examined by immunoprecipitation/Western blot analysis (n = 12 per group). The studies demonstrated significantly reduced mean levels of PY-IRS-1 following gestational exposure to ethanol (p < 0.001; see Fig. 5, A and C) but similar mean levels of IRS-1 protein in ethanol-exposed and control cerebella (Fig. 5, B and D).
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Reduced PI3 Kinase Signaling in Cerebella from Ethanol-exposed PupsNeuronal survival is mediated through PI3 kinase, which can be activated through both IRS-1-dependent and IRS-1-independent mechanisms. IRS-1-associated PI3 kinase activity was assessed by measuring p85 interactions with PY-IRS-1 by Western blot analysis of IRS-1 immunoprecipitates, and total PI3 kinase activity was measured in p85 immunoprecipitates. Chronic gestational exposure to ethanol resulted in significantly reduced levels of both IRS-1-associated p85 (p < 0.001; see Fig. 6, A and C) and total PI3 kinase (p < 0.001; see Fig. 6, E and F) activities. In contrast, p85 protein levels were similar in control and ethanol-exposed cerebella (Fig. 6, B and D).
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PTEN Phosphatase ExpressionPTEN phosphatase is an important
negative regulator of PI3 kinase signaling down-stream through Akt
(27,
38). Therefore, high levels of
PTEN activity would be expected to inhibit survival signaling down-stream of
PI3 kinase. However, the effect of ethanol on PTEN expression in the brain has
not been investigated. Western blot analysis detected the expected 45-kDa
PTEN protein in all samples (Fig.
7A). Digital image analysis of the Western blot signals
demonstrated significantly higher mean levels of PTEN in ethanol-exposed
relative to control cerebella (p < 0.001, n = 12 per
group; see Fig. 7B).
Correspondingly, the mean level of PTEN phosphatase activity was significantly
higher in cerebellar tissue from ethanol exposed relative to control pups
(p < 0.001) (Fig.
7C).
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Real-time quantitative RT-PCR was used to determine whether PTEN mRNA levels were increased in the ethanol-exposed relative to control brains. PTEN and 18 S RNA transcripts were quantified in parallel reactions using RT products generated from 20 ng of total RNA. Serial dilutions of plasmid DNA (0.002 to 20 ng) containing PTEN or 18 S cDNA target sequences were used as standards in all experiments. Graphs relating the threshold cycle values and input target DNA amounts (ng) resulted in standard curves that were linear over the 5 orders of magnitude tested and had correlation coefficients (r2) of 0.99 for 18 S and 0.97 for PTEN. Equations generated from the standard curves were used to calculate 18 S and PTEN mRNA abundance in the samples. No template and total RNA controls were included in all experiments. In addition, studies showed that rat genomic DNA could not be amplified using the conditions employed for real-time RT-PCR. All reactions were performed in triplicate, and all assays were repeated at least three times.
The mean threshold cycle value for PTEN was 25.4 ± 1.43 for the control group and 26.2 ± 1.2 in the ethanol-exposed group. The mean threshold cycle value for 18 S was 12.8 ± 3.2 in the control group and 12.1 ± 1.5 in the ethanol-exposed group. The PTEN mRNA/18 S RNA ratios were calculated for each sample to normalize for small difference in RNA content in the initial reactions. Inter-group comparisons made using Student's t test analysis demonstrated that the mean percentages of PTEN/18 S for control (1.35 ± 0.06) and ethanol-exposed (1.55 ± 0.1) brains were not statistically significant (p = 0.183; see Fig. 7D).
To more directly evaluate the roles of ethanol and impaired insulin/IGF-1 signaling in relation to PTEN expression and function, experiments were conducted using primary cerebellar neuron cultures. Insulin was found to be more effective than IGF-1 in stimulating Ser phosphorylation of PTEN (Fig. 8, A and B) and suppressing PTEN phosphatase activity (data not shown). In contrast, PTEN protein expression was not modulated by short term insulin or IGF-1 stimulation (Fig. 8A). In the in vitro ethanol-treated cultures, insulin-stimulated levels of phospho-PTEN were reduced by 7080% (p < 0.001), and IGF-1-stimulated levels of phospho-PTEN were reduced by 1520% (p < 0.01) relative to control (Fig. 8, C and D). Correspondingly, in vitro ethanol treatment was associated with reduced insulin and IGF-1 suppression of PTEN phosphatase activity, with greater impairment of insulin compared with IGF-1-mediated responses (Fig. 8, E and F).
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Ethanol Inhibits Survival Signaling Downstream of PI3
KinasePI3 kinase mediates survival by phosphorylating Akt and
GSK-3, which results in activation of Akt kinase
(28,
49) and inhibition of
GSK-3
activity (50). Akt
kinase also phosphorylates and inactivates GSK-3
, as well as BAD
(29,
31,
32), which are both
pro-apoptotic. To determine whether the hypoplasia and increased apoptosis
observed in cerebella from ethanol-exposed pups were associated with impaired
signaling downstream of PI3 kinase, we examined Akt, phospho (p)-Akt, GSK,
pGSK, BAD, and pBAD protein levels by Western blot analysis and measured Akt
and GSK-3
kinase activities in corresponding immunoprecipitates. Chronic
gestational exposure to ethanol resulted in significantly reduced levels of
pAkt, pGSK-3
, pBAD, and Akt kinase activity and increased levels of
total BAD protein and GSK-3
kinase activity (p < 0.05 or
p < 0.001; see Fig.
9). In contrast, the levels of Akt and GSK-3
proteins were
similar in control and ethanol-exposed cerebella.
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Chronic Gestational Exposure to Ethanol Impairs Insulin-stimulated Survival Signaling in the BrainIn vitro studies were used to help validate the in vivo observations and demonstrate that chronic gestational exposure to ethanol inhibits insulin-stimulated survival mechanisms in CNS neurons. Cultures generated from ethanol-exposed pups had significantly reduced levels of insulin-stimulated neuronal viability (p < 0.005), IRS-1-associated PI3 kinase, total PI3 kinase, and Akt kinase (all p < 0.001) activities and increased levels of GSK-3 activity (p < 0.001) relative to neuronal cultures generated from control pups (Fig. 10, AC, E, and F). In addition, insulin significantly suppressed PTEN phosphatase activity in control cultures (p < 0.01), whereas in cultures generated from ethanol-exposed pups, the basal levels of PTEN were significantly higher than control (p < 0.001), and insulin failed to suppress the phosphatase activity (Fig. 10D).
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DISCUSSION |
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The finding that chronic gestational exposure to ethanol resulted in
reduced levels of PY-IR yet had no significant effect on IR
protein expression corresponds with previous in vitro results
(47,
52) and indicates that the
inhibitory effects of ethanol on insulin signaling in the developing brain
begin at the level of IR
function rather than IR
expression. These
results are consistent with findings in a previous report
(53) demonstrating that
chronic gestational exposure to ethanol impaired IGF-1 receptor function but
not the receptor expression. Potential consequences of impaired insulin
signaling include inhibition of insulin-responsive gene expression and
cellular functions such as proliferation, neurite outgrowth, glucose
utilization, phospholipid metabolism, and amino acid transport. In this
regard, we demonstrated that ethanol-exposed cerebella had significantly
reduced levels of an important insulin-responsive gene, GAPDH
(48), suggesting that CNS
glucose utilization and energy metabolism may have been impaired. In addition
to regulating glucose utilization, insulin stimulates glucose uptake. Glucose
uptake is mediated by glucose transporter molecules termed GLUTs
(54), and previous studies
(5557)
demonstrated that chronic gestational exposure to ethanol inhibits expression
and function of glucose transporter molecules GLUT1 and GLUT3 in the brain.
Together, these observations potentially link ethanol-impaired insulin
signaling and neuronal loss to deficiencies in uptake and utilization of
glucose in the developing brain.
Our finding of reduced PY-IRS-1 levels in cerebellar tissue from ethanol-exposed pups indicates that chronic gestational exposure to ethanol impairs IRS-1 signaling, which could lead to reduced activation of growth and survival mechanisms. In this regard, we also detected reduced binding of the p85 subunit of PI3 kinase to IRS-1 in cerebellar tissue and reduced levels of insulin-stimulated IRS-1-associated PI3 kinase activity in cultured cerebellar neurons from ethanol-exposed pups. These results indicate that IRS-1-mediated survival mechanisms, including those stimulated by insulin, were significantly impaired by ethanol, consistent with previous results obtained using in vitro ethanol exposure models (9, 17, 42, 47, 58, 59). However, further studies demonstrated that ethanol caused even greater reductions in total PI3 kinase activity, both in vivo and following insulin stimulation in vitro. Therefore, PI3 kinase was likely inhibited through both IRS-dependent and IRS-independent, e.g. PTEN, pathways. Indeed, impaired signaling through IRS-2 is another candidate for mediating the IRS-dependent effects, because insulin-stimulated tyrosyl phosphorylation of IRS-2 can activate PI3 kinase (60), IRS-2 is expressed in the brain and functionally active in CNS neurons (61), and ethanol inhibits growth factor signaling through IRS-2 (62). The results from both the in vivo and in vitro experiments showed that ethanol inhibited PI3 kinase activity and suggest that inhibition of insulin signaling through IRS-dependent and IRS-independent pathways contributed to the observed impairments in survival signaling and development of cerebellar hypoplasia.
PI3 kinase promotes survival by phosphorylating Akt and activating its
kinase (27,
28), which then phosphorylates
Ser and Thr residues on target pro-apoptosis molecules, including GSK-3
(50), BAD
(31), and caspase-9
(63,
64), and rendering them
inactive. Our experiments demonstrated that ethanol impaired survival
signaling downstream of PI3 kinase, as was manifested by the reduced levels of
phospho-Akt, phospho-BAD, phospho-GSK-3, and Akt kinase activity, and
increased levels of activated BAD and GSK-3
activity. Further in
vitro studies showed that insulin activation of Akt kinase and inhibition
of GSK-3
were impaired by ethanol. Therefore, these studies linked
ethanol impairment of insulin-stimulated survival signaling downstream of PI3
kinase to the increased cell loss, apoptosis, and cerebellar hypoplasia
observed in vivo.
Further studies revealed that PTEN phosphatase activation represents an
additional non-IRS-1 mechanism by which survival signaling downstream of PI3
kinase could be inhibited by ethanol in CNS neurons. PTEN dephosphorylates and
reverses the activation of PI3 kinase
(38,
65), whereas inactivation of
PTEN promotes membrane recruitment of Akt, leading to increased Akt
phosphorylation and kinase activity
(38,
65). PTEN expression and
phosphatase activity are negatively regulated by phosphorylation of PTEN
protein (66); however, the
effects of ethanol exposure and growth factor stimulation on PTEN expression
and function were previously unknown. Our in vivo studies
demonstrated significantly higher levels of PTEN protein and phosphatase
activity, without correspondingly increased PTEN mRNA expression in
ethanol-exposed cerebella, indicating post-transcriptional modulation of PTEN
by ethanol. In addition, the magnitude of increased PTEN expression was
similar to that of GSK-3 activity, corresponding with the expected
inhibitory effects of PTEN on survival signaling downstream of PI3 kinase.
In vitro studies were used to characterize the effects of ethanol on the levels of PTEN protein, phosphorylation, and phosphatase activity and to determine whether the adverse effects of ethanol on PTEN were linked to impaired insulin signaling. Insulin, and to a lesser extent IGF-1, stimulated PTEN phosphorylation and correspondingly inhibited PTEN phosphatase activity in control cerebellar neuron cultures. Chronic gestational exposure to ethanol impaired the insulin-stimulated phosphorylation of PTEN and suppression of PTEN phosphatase activity. Therefore, the adverse effect of ethanol on insulin signaling to inhibit the function of PTEN in CNS neurons probably represents an important mechanism of impaired survival and increased apoptosis.
The results from our in vivo and in vitro experiments
linked chronic gestational exposure to ethanol to aberrantly increased
expression and enzymatic activity of PTEN, which has a pivotal role in
regulating PI3 kinase-activated survival signaling. Although PTEN has been
shown to inhibit insulin-stimulated Akt phosphorylation without affecting
IR activation of IRS-1
(67), other more recent data
(68) suggest that PTEN can
also inhibit insulin-stimulated IRS-1 phosphorylation and the attendant
downstream signaling. Therefore, in the ethanol-exposed cerebella, the
impaired survival signaling may have been mediated by the combined effects of
the following: 1) ethanol inhibition of IR
phosphorylation and function,
2) PTEN inhibition of IRS-1 phosphorylation, and 3) PTEN inhibition of Akt
kinase. These studies suggest important mechanisms by which ethanol-impaired
insulin signaling could promote the development of cerebellar hypoplasia in
fetal alcohol syndrome.
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FOOTNOTES |
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To whom correspondence should be addressed: Pierre Galletti Research Bldg.,
Rhode Island Hospital, 55 Claverick St., Rm. 419, Providence, RI 02903. Tel.:
401-444-7364; Fax: 401-444-2939; E-mail:
Suzanne_DeLaMonte_MD{at}Brown.edu.
1 The abbreviations used are: IGF-1, insulin-like growth factor type 1;
IRS-1, insulin receptor substrate-1; PY, tyrosyl-phosphorylated; PI3 kinase,
phosphatidylinositol 3-kinase; GSK-3, glycogen synthase kinase-3; P, postnatal
day; TUNEL, terminal transferase dUTP endlabeling; BCIP/NBT,
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; MOPS,
4-morpholinepropanesulfonic acid; RT, reversetranscribed; IR, insulin
receptor
; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p,
phospho.
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REFERENCES |
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