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,§1
* Institute of Pharmacological Sciences, University of Milano, Milan, Italy;
Department of Internal Medicine, Geriatrics and Gerontology Clinic,
Institute of Pharmacology University of Pavia, Pavia, Italy; and
§ IRCCS ‘Centro S. Giovanni di Dio - FBF’ Brescia, Brescia, Italy
1Correspondence: Institute of Pharmacology, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy. E-mail: racchi{at}unipv.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Key Words: signal transduction • PI3 kinase • Alzheimer’s disease • 
secretase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-secretase’, and the processing releases a large, soluble APP
fragment (sAPP) into the extracellular space (1)
.
Because cleavage occurs within the Aß domain, secretase
processing does not generate Aß (2
, 3)
. A number of
alternative metabolic pathways have also been described that have the
potential to generate intact Aß fragments (for review, see refs
4
, 5
). Because sAPP and Aß seem to be formed by two
mutually exclusive mechanisms, stimulation of secretory processing of
APP might prevent the formation of Aß and its accumulation into
amyloid plaques. Although other evidence indicates that in some cell
types the release of sAPP and Aß can proceed independently of each
other (6)
, the ability of sAPP to protect neurons
against cytotoxic insults (reviewed in ref 7
) provides an
additional basis for the hypothesis that the sustained release of
sAPP is important for neuronal viability.
Several intracellular signaling cascades are involved in the regulation
of APP processing (4
, 5)
. Protein kinase C (PKC) is
involved in the pathway that leads to the nonamyloidogenic processing
of APP; in addition, it has been suggested that other PKC-independent,
Ca2+-dependent processes are also involved in the
activation of the secretase pathway (8
, 9)
.
Neurotransmitters, hormones, or cytokines, as well as other neuroactive
compounds that activate PKC and other transduction signals, increase
secretion of sAPP via the nonamyloidogenic pathway (4
, 5)
. Many plasma membrane receptors regulate cellular processes
through protein tyrosine kinase. Ligands of receptors with intrinsic
tyrosine kinase activity, including nerve growth factor (NGF) and
epidermal growth factor (EGF), also increase sAPP secretion
(5)
. In this context, we investigated the role of insulin
in the modulation of the secretory processing of APP.
The study of the effect of insulin on APP processing is also suggested
by several other considerations detailed below. Several lines of
biochemical evidence suggest a link between glucose utilization and
energy metabolic defects to the functional alterations associated with
brain aging and with the pathogenesis of neurodegenerative disorders
such as AD. Disturbed brain energy metabolism is a prominent feature of
AD brains (10
11
12
13)
. In fact, impaired glucose metabolism
has been observed in parietal, temporal, and frontal cortex of AD
patients (14
, 15)
. Inhibition of energy metabolism can
alter APP processing and induce amyloidogenic products (16
, 17)
. Markers of altered energy metabolism in AD patients also
seem to be present peripherally (reviewed in ref 18
) and
include alteration of glucose metabolism, glutamine oxidation, and
Ca2+ homeostasis in fibroblasts (19
, 20)
as well as decreased cytochrome c oxidase
activity in platelets (21
, 22)
and fibroblasts
(23)
. Furthermore, fibroblasts from AD patients are more
sensitive to glucose deprivation and energy depletion compared to
control cells (23
, 24)
. Thus the depletion of metabolic
energy, tightly linked to glucose, may significantly alter the
metabolism of a key protein in AD.
We therefore tested the ability of insulin to regulate APP processing
in SH-SY5Y neuroblastoma, a cellular model that expresses insulin
receptors endogenously. Our findings suggest that insulin modulates APP
secretion by a tyrosine kinase-dependent and glucose-independent
mechanism. We also show that within the insulin signaling pathway, the
activity of phosphatidyl inositol 3 kinase (PI3-K) is necessary for
insulin-mediated sAPP release.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell culture and experimental treatments
SH-SY5Y neuroblastoma cells were cultured in Eagle’s minimum
essential medium (MEM) supplemented with 10% FCS,
penicillin/streptomycin, nonessential amino acids, and sodium pyruvate
(1 mM) at 37°C in 5% CO2/95% air. For the
experiments, 4 x 106 cells were seeded on
60 mm dishes and cultured for 48 h. The cells were exposed to
serum-free MEM for 24 h, then experimental treatments were
performed in serum-free MEM with incubation for 2 h at 37°C. For
the experiments without glucose or with 2-deoxy-D-glucose (2DG), cells
were incubated for 2 h at 37°C in MEM without glucose and
pyruvate with the addition of glucose or 2DG, as indicated in Results
and in the figure legends.
Harvesting the cells and preparation of conditioned medium
Proteins in conditioned medium were quantitatively precipitated
by the deoxycholate/trichloroacetic acid procedure as described
previously (25)
. Cell monolayers were washed twice with
ice-cold phosphate-buffered saline and lysed on the tissue culture dish
by addition of ice-cold lysis buffer (50 mM Tris/HCl, pH=7.5, 150 mM
NaCl, 5 mM EDTA, and 1% Triton X-100). An aliquot of the cell lysate
was used for protein analysis with the Bio-Rad Bradford kit for protein
quantification.
Immunodetection of sAPP
Normalization of protein loading on each blot was obtained by
loading a volume of sample of conditioned medium standardized to the
protein concentration in the total cell lysate. Proteins were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and
transferred onto PVDF membrane (DuPont NEN, Boston, Mass.). The
membrane was blocked for 1 h with 10% non-fat dry milk in
Tris-buffered saline containing 1% Tween 20. Membranes were
immunoblotted with the antibodies 22C11 (Boehringer Mannheim, Mannheim,
Germany) or 6E10 (Senetek, St. Louis Mo.). The detection was carried
out by incubation with alkaline phosphatase-conjugated goat anti-mouse
immunoclobulin G (IgG) (Kirkegaard and Perry Laboratories,
Gaithersburg, Md.) for 1 h. The blots were then washed extensively
and sAPP was visualized using an enhanced chemiluminescent method
(DuPont NEN, Boston, Mass.).
Quantitative densitometry and statistics
Quantitative analysis of Western blots was performed by
calculating the relative density of the immunoreactive bands after
acquisition of the blot image with a Nikon CCD video camera module and
analysis with the Image 1.47 program (Wayne Rasband, NIH, Research
service Branch, NIMH, Bethesda, Md.). Statistical analyses were done by
one-way ANOVA, followed by a two-tailed student’s t test or
multiple comparison test where appropriate; a value of
P<0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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release release into the
conditioned media of the cells. The maximal effect was obtained at the
concentration of 1 µM insulin (Fig. 1A
), which resulted in an ~2.5-fold increase of sAPP over
the basal level (Fig. 1B
). Western blots show that sAPP
released into the conditioned medium migrates as a doublet with an
apparent molecular mass of 100–120 kDa (Fig. 1A
). This
protein pattern is consistent with previous reports using the same
cellular model (26
, 27)
and likely reflects the expression
of the major isoforms of APP (M. Racchi, unpublished data). Since the
same immunoreactive band was detected by the antibodies 22C11 and 6E10
and the latter antibody recognizes epitopes in the first 16 amino acids
of Aß (which also constitutes the carboxyl terminus of APP cleaved by
-secretase), the identified bands can be assumed to be authentic
sAPP.
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Insulin-induced sAPP release is independent of glucose
The effect of insulin on sAPP release is independent of the
presence of glucose. As previously shown in COS cells and fibroblasts
from AD patients (17
, 24)
, basal sAPP release is
decreased in conditions of glucose deprivation (~30% below control
basal levels) and even more so in the presence of 2DG (~50%).
Treatment of SH-SY5Y cells with 1 µM insulin in medium without
glucose showed an increase of sAPP release relative to basal levels,
similar to that obtained in glucose-containing (50 mM) medium
(Fig. 2
). The presence of 2-DG similarly did not block the relative ability of
insulin to stimulate sAPP release (Fig. 2)
.
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Tyrosine kinase activity is necessary for insulin-induced sAPP
secretion
Cells were treated for 2 h with 1 µM insulin in the
presence or absence of 20 µM genistein. Western blot analysis showed
that genistein completely blocked the effect of insulin, returning the
level of sAPP release to basal levels, whereas the constitutive
sAPP release of SH-SY5Y cells was not affected by the inhibitor
(Fig. 3
).
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Insulin-induced sAPP release is independent from PKC activation
We tested the possibility that insulin regulation of the release
of sAPP was secondary to activation of PKC by using the selective
PKC inhibitor GF-109203X (28)
. The cells were incubated
for 2 h with 200 nM PdBu in the presence or absence of 2.5 µM
GF-109203X. Treatment of SH-SY5Y cells with PdBu elicited an increase
of sAPP release of approximately twofold over basal levels. As
expected, simultaneous treatment with GF-109203X abolished the response
to phorbol ester (Fig. 4
). As previously shown, 1 µM insulin increased the release of sAPP
whereas addition of GF-109203X did not inhibit the effect of insulin.
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Insulin-induced sAPP release is independent of MAP kinase
activation
Activation of tyrosine kinase receptors can activate MAP Kinase
through a Ras/Raf/MAP-K kinase (ERK) pathway. Cells were treated with 1
µM insulin as indicated before, but with the simultaneous addition of
30 µM PD98059, a MEK inhibitor. This concentration was chosen on the
basis of previously published data (29
30
31)
. As shown in
Fig. 5
, inhibition of MEK by PD-98059 did not block the secretory effect of
insulin, suggesting that the MAP-K pathway was not involved. Treatment
of the cells with PD98059 in the absence of insulin did not inhibit
constitutive release of sAPP (Fig. 5)
.
|
Insulin-mediated sAPP release is dependent on PI3-K activation
Insulin receptor phosphorylation results in the activation of
PI3-K. Figure 6
shows that treatment of the cells with 1 µM insulin and 10 µM
wortmannin, a specific inhibitor of PI3-K (32)
, blocked
the insulin-induced increase of sAPP release. To confirm the result,
we also used the compound LY-294002, another selective inhibitor of
PI3-K (33)
. We observed that 10 µM LY-294002 blocked the
release of sAPP induced by insulin (Fig. 6)
. Neither wortmannin nor
LY-294002 was able to affect constitutive release of sAPP (data not
shown).
|
Insulin-mediated sAPP release is independent from
p70s6K activity
PI3-K and subsequent activation of the PKB/PDK system are involved
in the activation of p70S6K. There is evidence
that the phosphorylation of this kinase plays a role in the regulation
of protein synthesis (34
, 35)
. We tested whether this
branch of the insulin signaling pathway was involved in the regulation
of sAPP release. Cells were incubated with 1 µM insulin and in the
absence or presence of 20 ng/ml rapamycin, a macrolide that inhibits
the activation pathway of p70s6K downstream of
PI3-K. After treatment, Western blot and densitometric analysis
demonstrated that rapamycin did not interfere with insulin-induced
sAPP release, thus ruling out the involvement of
p70s6K (Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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release from SH-SY5Y cells in a concentration- and
tyrosine kinase-dependent manner.
It has been extensively shown that secretase cleavage of APP is
increased by the activation of a variety of signal transduction
pathways (for review, see refs 4
, 5
), including tyrosine
kinase receptors. Among other ligands, insulin acts through a receptor
with intrinsic protein tyrosine kinase activity. Genistein has been
reported to exhibit specific inhibitory activity against tyrosine
kinases. As reported here, 20 µM and 40 µM genistein (the latter
concentration not shown) blocked sAPP release induced by insulin,
thus showing that a tyrosine kinase dependent mechanism was necessary
for the effect of insulin on APP metabolism, a mechanism similar to
that described for EGF (36)
.
Deprivation of glucose from the culture medium in our experiments
decreased the basal release of sAPP, although insulin was still able
to induce a relative increase of sAPP secretion. Reduction of basal
release by glucose deprivation clearly indicates that glucose is
necessary in a key step of APP metabolism. Nevertheless, the finding
that glucose deprivation did not interfere with insulin-mediated
increase of sAPP secretion suggests the possibility that insulin can
act as a growth factor to induce sAPP release through a mechanism
similar to EGF and other tyrosine kinase receptor ligands.
The activation of the secretory metabolism of APP by EGF is mediated at
least partially by PKC, because treatment of the EGF-stimulated cells
with the specific PKC inhibitor GF-109203X decreases the response by
~35% (36)
. In our experiments, the simultaneous
treatment of neuroblastoma cells with GF-109203X at the same
concentration used in the former study completely blocked the effect of
phorbol esters, but did not block the activation of sAPP release by
insulin. This result suggests that PKC is not involved in
insulin-mediated release of sAPP.
Insulin receptor kinase activity results in the tyrosine
phosphorylation of substrates like the insulin receptor substrate 1
(IRS-1). Phosphorylated IRS-1 interacts with many other proteins that
bind phosphotyrosine with their SH2 domains. GRB2 is one such protein,
and its function involves the role of an ‘adapter molecule’ that
links the guanine nucleotide exchange factor for
p21ras (named mSOS) to phosphorylated IRS-1. This
complex may then activate Ras and the MAP-K cascade (37)
.
Recent reports showed that MAP-K (ERK) is involved in NGF, phorbol
esters, and carbachol-stimulated sAPP secretion; it is notable that
a reduction of sAPP secretion via inhibition of MAP-K was reported
for several cell lines (29
30
31)
. Our experiments
demonstrate that the effect of insulin signaling on sAPP release was
not inhibited by the addition of PD-98059, a selective MEK inhibitor
ruling out the involvement of the MAP-K pathway in the effect of
insulin on APP metabolism.
Phosphorylated IRS-1 can bind and activate PI3-K, and it is now
suggested that such a pathway may be one of the key signaling events in
the insulin effect on end point responses (38)
. PI3-K
plays a critical role in growth factor signaling to cell growth and
proliferation, differentiation, apoptosis, glycogen synthesis, protein
synthesis, vesicle traffic, and glucose transporter translocation.
PI3-K appears to be an upstream activator of uptake of amino acids,
gene transcription, and mRNA translation. Acute cellular responses to
insulin include the activation of protein synthesis (39)
through a pathway involving PI3-K, PKB and p70s6K
(40)
. The latter kinase is activated through a pathway
sensitive to the macrolide rapamycin, which can block the pathway of
activation of p70s6K downstream of PI3-K. We show
here that the compound does not inhibit insulin-mediated sAPP
release, suggesting that the effect is not mediated by an increase in
protein translation.
The activation of sAPP release by insulin is inhibited by wortmannin
and LY-294002. The former is a cell-permeable fungal metabolite that
inhibits PI3-K by covalent modification of the catalytic subunit of
PI3-K. The compound LY-294002, another specific inhibitor of PI3-K, is
mechanistically different from wortmannin since it directly competes
for the ATP binding sites of the catalytic subunit of PI3-K. The kinase
targeted to phosphoinositides is important for one of the basic
physiological roles of insulin: translocation of the Glut4 glucose
transporter to the cell surface to increase the rate of glucose uptake
into target cells. Wortmannin blocks the rate of insulin-stimulated
exocytosis of Glut4 glucose transporters, with little effect on
endocytosis (41
, 42)
. Similarly, PI3-K inhibitors block
insulin-stimulated trafficking of transferrin receptors
(43)
. Since transferrin receptors are not present on
Glut4-containing vesicles, this suggests that PI3-K is involved in the
regulation of a step in exocytosis that is not restricted to movement
of Glut4-containing vesicles. Moreover, phosphoinositides could serve
as specific membrane targets that bind proteins required for the
formation of transport vesicles. For example, the targets could be the
polypeptides of the adaptor complex that link clathrin to the
cytoplasmic tail of certain transmembrane receptor proteins
(44)
. Thus, all these observations lead us to the
conclusion that the effect of insulin on APP metabolism may be mediated
by a PI3-K-dependent modulation of vesicular trafficking. This would
mean either an increase of the transport of APP into secretory
compartments or trafficking of ‘secretase’ containing vesicles toward
APP-containing membrane domains. These hypotheses are based on
experimental evidence that ruled out all alternatives within insulin
signaling pathway and describe a novel intracellular pathway involved
in the regulation of APP metabolism.
Finally, we can speculate that the mechanism described here
may also serve a function of neuroprotection from the toxic
activity of Aß itself. It has been demonstrated that insulin and
insulin-like growth factors can protect neurons in culture from Aß
toxicity (45
, 46)
. Thus, sAPP derived from
insulin-mediated metabolism of APP can also serve as a neuroprotective
molecule (see ref. 7
) and work synergistically with insulin in a
putative system that can both prevent and offer rescue from toxic
events.
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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