Glycobiology vol. 20 no. 5 pp.

603616, 2010
doi:10.1093/glycob/cwq010
Advance Access publication on January 24, 2010
Desialylation of insulin receptors and IGF-1 receptors by
neuraminidase-1 controls the net proliferative response of L6
myoblasts to insulin
Majid Arabkhari
2
, Severa Bunda
2
, Yanting Wang
2
,
Andrew Wang
2
, Alexey V. Pshezhetsky
3
,
and Aleksander Hinek
1,2,4
2
Physiology and Experimental Medicine Program, The Hospital for Sick
Children, University of Toronto, Canada;
3
Department of Medical Genetics,
CHU Sainte Justine Research Center, Montreal, Quebec, Canada; and
4
Department of Laboratory Medicine and Pathobiology, University of Toronto,
Canada
Received on October 9, 2009; revised on December 22, 2009; accepted on
January 20, 2010
We recently established that the subunit of cell surface-
residing elastin receptor, neuraminidase-1 (Neu1), can
desialylate adjacent insulin-like growth factor 1 receptors
(IGF-1R) of arterial smooth muscle cells, thereby
quenching their proliferative response to insulin-like
growth factor II. In this study, we explored whether
Neu1 would also desialylate the insulin receptors (IR),
as well as the IGF-1R on rat skeletal L6 myoblasts, and
whether desialylation of IR and IGF-1R would affect a
net proliferative effect of insulin. First, we found that
physiological (0.51 nM) and high therapeutic (10 nM)
insulin concentrations induced a modest increase in pro-
liferation rate of cultured L6 myoblasts. While IR kinase
inhibitor could abolish the mitogenic effect of these insulin
concentrations, the observed more pronounced prolifera-
tive response to supraphysiological concentration
(100 nM) of insulin could be eliminated only by specific
inhibition of IGF-1R. Then, we found that treatment of
L6 cells with mouse-derived Neu1 or with Clostridium per-
fringens neuraminidase caused desialylation of IR, which
coincided with a significant increase of their proliferative
response to lower (0.510 nM) concentrations of insulin.
In contrast, experimental desialylation of IGF-1R coin-
cided with elimination of the heightened proliferative
response of L6 myoblasts to 100 nM insulin. Importantly,
we also found that inhibition of endogenous Neu1 abol-
ished the increase in proliferation of L6 cells induced by
1 and 10 nM of insulin, but amplified the proliferative
effect of 100 nM insulin. We therefore conclude that de-
sialylation of both IR and IGF-1R by Neu1 controls the
net proliferative response of skeletal myoblasts to insulin.
Keywords: IGF-1 receptor / insulin/ insulin receptor /
neuraminidase-1/ skeletal myoblasts proliferation
Introduction
All vertebrate skeletal muscles, except the head muscles, de-
velop from the mesodermal precursor cells originating from
the embryonic somites (Asakura and Rudnicki 2002). In this
process, mesodermal precursor cells rst dierentiate toward
the myoblasts and then majority of myoblasts proliferate and
dierentiate into the myocytes that fuse one to each other
and form multinucleated skeletal muscle bers (Parise et al.
2006). A distinct subpopulation of myoblasts does not dier-
entiate further during embryonic development, but remains in
reserve associating within the contractile muscle bers as
quiescent muscle satellite cells (Hawke and Garry 2001).
These early embryonic developmental processes leading to
the formation of two muscle-forming cell lineages are regulat-
ed by numerous positive and negative signals (Kablar et al.
1998; Palmer and Rudnicki 2001; Charge and Rudnicki
2004). During the course of further muscle elongation and
growth, satellite cells steadily exit their quiescent state and
then, after several rounds of proliferation fuse with the existing
muscle bers or form the new muscle bers. The well-devel-
oped mammalian skeletal muscles are relatively stable and
their satellite cells show little mitogenic activity and turnover
(Decary et al. 1997; Schmalbruch and Lewis 2000; Hawke and
Garry 2001). It is estimated that in a normal adult rat muscle,
no more than 12% of nuclei are replaced every week
(Schmalbruch and Lewis 2000). It has been established that
a more vigorous satellite cell activation can be triggered after
partial physical or ishemic damage of fully-grown muscles and
that their prompt proliferation, migration and nal dierentia-
tion into contractile myocytes are prerequisite steps for the
successful muscle regeneration that has to occur before forma-
tion of the intramuscular connective tissue scars (Schultz et al.
1985; Kuang and Rudnicki 2008; Le Grand and Rudnicki
2007).
The complicated process of muscle regeneration that in-
volves activation of satellite cells requires the timely,
controlled upregulation of muscle transcription factors and
muscle-specic genes. It has been established that this pro-
cess is regulated through mechanisms involving cellcell
and cellmatrix interactions, as well as through actions of
numerous secreted molecules (e.g., nitric oxide ATP) and
growth factors, including broblast growth factor, transform-
ing growth factor-, hepatocyte growth factor, tumor
necrosis factor-, insulin-like growth factors (IGF) and inter-
l euki n-6 (Grounds 1999; Hawke and Garry 2001;
Lescaudron et al. 1999; Suelves et al. 2002; Stamler and
Meissner 2001; Palsgaard et al. 2009).
The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org 603
1
To whom correspondence should be addressed: Tel: +1-416-813-6725, Fax:
+1-416-813-7480; e-mail: alek.hinek@sickkids.ca

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Conventionally, both insulin and IGF-1 are believed to in-
duce a metabolic and mitogenic response of myoblats.
However, the roles of insulin and IGF-1 may not be that easy
to separate in vivo. While one recent study indicated that IGF-
1 is a more potent regulator of gene expression than insulin in
primary human myoblasts and myotubes (Palsgaard et al.
2009), the other suggests that the immature muscle has a
heightened capacity to activate signaling cascades that promote
translation initiation in response to the postprandial rise in in-
sulin, thereby enabling their ecient utilization for muscle
growth. This further coincides with enhanced satellite cell pro-
liferation (Davis and Fiorotto 2009), but how these two
processes are linked is not entirely clear.
Thus, our present studies were aimed at exploration whether
insulin, a hormone structurally similar to IGF-1, would also
contribute to the mechanisms that modulate proliferation of
myoblasts. Our experimental model constituted of L6 rat skel-
etal myoblasts that can dierentiate spontaneously from
myoblasts to multinucleated myotubes (Tsakiridis et al.
1995). Cultured L6 cells respond well to insulin and have been
used in numerous studies aimed at metabolic eect of this hor-
mone (Samokhvalov et al. 2009; Klip et al. 2009).
Since we recently established that enzymatic desialylation
of plasmalemma-residing sialo-glycoprotein, insulin-like
growth factor 1 receptors (IGF-1R), by endogenous neuramin-
idase-1 (Neu1) or Clostridium perfringens neuraminidase
(cPNase) inhibited propagation of mitogenic signals induced
by insulin-like growth factor II (IGF-II) (Hinek et al. 2008),
we decided to test now whether experimental desialylation
of both insulin-binding receptors [insulin receptors (IR) and
IGF-IR] would modulate a net proliferative response of skele-
tal myoblasts to insulin. We were particularly interested
whether such process would be also modulated by endogenous
Neu1, a predominantly lysosomal enzyme that is also targeted
to the cell surface, as a subunit of elastin receptor (Hinek et al.
1993; Privitera et al. 1998), and is specically active toward
numerous sialylated glycoproteins, including cell surface
IGF-1R and platelet-derived growth factor receptors (PDGFR)
(Hinek et al. 2008).
The presented data indicate that the proliferative response of
cultured skeletal L6 myoblasts to physiological and therapeu-
tical doses of insulin can be enhanced after IR desialylation by
both endogenous Neu1 and exogenous neuraminidases (mouse
kidney-derived Neu1 and cPNase). In contrast, a more potent
proliferative response to supraphysiological concentrations of
insulin that engage IGF-1R signaling could be eliminated fol-
lowing desialylation of IGF-1R.
Results
Results of our initial experiments measuring [
3
H]-thymidine
incorporation, DNA levels and the number of cells expressing
ki67 in 5-day-old L6 myoblast cultures consistently demon-
strated that daily treatment with broad spectrum doses of
insulin (0.5100 nM) induced a dose-dependent increase in
their proliferation rate. While the physiological concentrations
of insulin (0.51 nM) produced only small (515%) increase
in L6 cells proliferation rate, the 10 and 100 nM insulin doses
induced a signicant 3544 and 7586% increases in their
proliferation rate, respectively (Figure 1A).
We then demonstrated that the signicant proliferative ef-
fects induced by 1 and 10 nM insulin could be completely
repressed in cultures pretreated with the specic IR kinase
inhibitor, hydroxy-2-naphthalenylmethylphosphonic acid tris-
acetoxy-methyl ester (HNMPA-(AM)3), but not in cultures
incubated with a noncompetitive IGF-1 receptor inhibitor,
picropodophylin (PPP).
In contrast, the most potent proliferative response of L6 cells
to 100 nM insulin was not aected by pretreatment with IR
inhibitor, but signicantly reduced by pretreatment with IGF-
1R inhibitor or with anti-IGF-1R antibody (Figure 2). These
data suggested that while the proliferative response of cultured
L6 cells to lower (0.510 nM) doses of insulin is mostly trans-
duced through IR, the more potent proliferative response to
supraphysiological concentration (100 nM) of insulin could
be also transduced through the IGF-1R.
Results of the next series of our experiments provided im-
portant novel observation that pretreatment of cultured L6
myoblasts with exogenous neuraminidases, Neu1 isolated from
mouse kidneys (MNeu1) or cPNase, induced desialylation of
both their IR and IGF-1R. This conclusion was based on two
crucial observations depicted in Figure 3. First, western blot
analysis with antibodies recognizing the subunits of respec-
tive IR or IGF-1R showed that lysates of L6 cells that were
incubated for 30 min in the presence of 1 mU/mL of MNeu1
or 25 mU/mL of cPNase caused a down-shift in the molecular
weight of these immunodetectable components of IR and the
IGF-IR. Then, we found that digoxigenin-labeled Maackia
amurensis agglutinin lectin (Dig-MAA) that recognized the
23-linked sialic acids residues of immunoprecipitated IR
and IGF-1R (higher molecular weight) from extracts of untreat-
ed control cultures failed to react with immunoprecipitated IR
and IGF-1R (smaller molecular weight receptors) from Neu1-
treated cultures. In contrast, these smaller molecular weight re-
ceptors demonstrated reactivity with peanut agglutinin (PNA)
that specically recognizes Gal13GalNac residues. This ob-
servation conrmed that the used preparations of exogenous
neuraminidases did not contain any admixture of active galac-
tosidases. Since used preparations of both exogenous
neuraminidases did not demonstrate any proteolytic activities
and lysis of L6 cells was performed in the radio immunopre-
cipitation assay (RIPA) buer containing cocktail of class-
specic protease inhibitors, we conclude that the observed
down-shift in the molecular weights of both IR and the
IGF-1R was solely caused by their desialylation. A similar
neuraminidase-induced decrease in the molecular mass of
both receptors has been previously described in adipocytes
(Heidenreich and Brandenburg 1986) and in neuroblastoma
cells (Neilsen et al. 2004).
We next showed that these desialylated IR were more
sensitive to insulin (1 or 10 nM) as the phosphorylation
status of IR in cells cotreated with insulin and cPNase
was markedly higher than in those treated with insulin
alone (Figure 4A). Furthermore, we showed that treatment
of L6 cells with 10 nM insulin also led to the activation
of the phosphatidylinositol-3 kinase (PI3K)/Akt-involving
signaling pathway (phosphorylation of Akt protein) that
could be further increased in cells cotreated with insulin
and exogenous Neu1 (Figure 4B). Since these experiments
were performed in the presence of IGF-1R inhibitor, PPP,
604
M Arabkhari et al.

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we conclude that even high therapeutic concentration of in-
sulin (10 nM) initiated its intracellular signaling via IR
activation (Figure 4B).
We also documented that in contrast to smaller doses of
insulin that transduce their eect via the IR, the supraphysio-
logical dose (100 nM) of this hormone induced IGF-1R
phosphorylation in cultured L6 cells. Meaningfully, this dose
of insulin did not cause phosphorylation of the desialylated
smaller molecular weight IGF-1R subunit detected in cul-
tures pretreated with MNeu1 or cPNase (Figure 4C).
Most importantly, we have established that desialylation of
both structurally similar receptors (IR and IGF-1R) by exoge-
nous sialidases modulated proliferative response of skeletal
myoblasts to insulin in the opposite ways. Results of this series
605
Fig. 1. Results of assays measuring [
3
H]-thymidine incorporation, DNA levels and the frequency of cells expressing proliferative ki67 antigen in 5-day-old cultures
of L6 cells consistently demonstrate that treatment with broad spectrum of insulin (0.5 to 100 nM) induced a dose-dependent increase in their proliferation.
Desialylation of insulin-responsive receptors on myoblasts

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606
Fig. 2. Results of assays measuring [
3
H]-thymidine incorporation, DNA levels and the frequency of cells expressing proliferative ki67 antigen in 5-day-old cultures
of L6 cells treated with 1100 nM of insulin in the presence and absence of specic IR kinase inhibitor (HNMPA-(AM)3) or IGF-1R inhibitor (PPP). Results from
these assays demonstrate that signicant proliferative eects induced by 1 and 10 nM insulin were completely repressed in cultures pretreated with HNMPA-(AM)3,
but not diminished in cultures incubated with IGF-1R inhibitor (PPP) that eliminated the proliferative response in cultures treated with 100 nM of insulin.
M Arabkhari et al.

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of experiments clearly demonstrated that pretreatment of L6
cells with cPNase signicantly enhanced their proliferative re-
sponse to physiological doses of insulin (0.5 and 1 nM) and
that this eect was eliminated in cultures pretreated with IR
inhibitor (HNMPA-(AM)3), but not with IGF-1R inhibitor
(PPP) (Figure 5A).
We also found that pretreatment of L6 cells with 1 mU/mL
of mouse-derived Neu1 signicantly enhanced the proliferative
response of L6 cells to 10 nM insulin. While culture of L6 cells
treated with 10 nM of insulin alone demonstrated an average
24% increase in their basal proliferation rate, cells pretreated
with 1 mU/mL of MNeu1 showed an average 79% increase
in their proliferation rate after treatment with 10 nM insulin.
The magnitude of an enhancement of insulin-induced prolifer-
ative eect, caused by treatment with 1 mU/mL of MNeu1,
was higher than the eect of comparable concentration of
cPNase that produced an average 43% increase in L6 cells pro-
liferation. The application of higher concentration of cPNase
(25 mU/mL) produced only slightly higher enhancement of
the proliferative response to 10 nM of insulin (Figure 5B). In
contrast, pretreatment of parallel cultures with the same dose of
cPNase practically eliminated the proliferative response of L6
cells to high (100 nM) dose of insulin (Figure 5B). This obser-
vation, endorsed the notion that the proliferative eect of the
extremely high dose of insulin, transduced through IGF-1R,
could be inhibited after desialylation of this receptor.
Finally, we demonstrated that preincubation of L6 cells
with 500 nM of 2,3-dehydro-2-deoxy-N-acetylneuraminic ac-
id (ddNANA) [the competitive inhibitor of endogenous
mammalian neuraminidases that most potently aects Neu1
(Hinek et al. 2006, 2008)] or with blocking anti-Neu1 anti-
body (Hinek et al. 2008) eliminated the enhancement of
proliferative response of myoblasts to 1 and 10 nM of insu-
lin. Meaningfully, similar inhibition of endogenous Neu1
actually potentiated the proliferative eect of L6 cells to
100 nM insulin (Figure 6A and B). Moreover, L6 myoblasts
transfected with Neu1-specic siRNA revealed a remarkable
decrease in level of Neu1 protein and in sialidase activity
(Figure 6C, left panel). These Neu1-decient cells incorpo-
rated signicantly less [
3
H]-thymidine than their counterparts
transfected with nontargeting siRNA, when treated with
1 nM insulin or 10 nM insulin. In contrast, Neu1 siRNA-
transfected cells demonstrated heightened proliferative re-
sponse to 100 nM insulin (Figure 6C, right panel).
Thus, our data clearly indicate that the activity of endoge-
nous Neu1 can modulate the net proliferative response of L6
cells to low and high doses of insulin in the opposite manner.
Discussion
The sialidases (neuraminidases) (EC 3.2.1.18; N-acylneurami-
nosyl glycohydrolase) are a family of exoglycosidases that
catalyze the hydrolytic cleavage of nonreducing sialic acid re-
sidues ketositically linked to mono- or oligosaccharide chains
of glycoconjugates. They have been identied in numerous vi-
ral, bacterial, fungal, protozoan, avian and mammalian species
(Monti et al. 2002; Saito and Yu 1995).
In mammalian cells, four genetically distinct neuraminidases,
diering in their tissue distribution, subcellular localization
and substrate specicity, have been characterized. They have
been localized to lysosomes: Neu1 (Igdoura et al. 1998;
Pshezhetsky and Potier 1996; Pshezhetsky et al. 1997); to cy-
tosol: neuraminidase-2 (Neu2) (Miyagi et al. 1993; Monti,
Preti, Nesti et al. 1999; Monti, Preti, Rossi et al. 1999,
2000; Saito and Yu 1995; Wada et al. 1999); to the plasma
membrane: neuraminidase-3 (Neu3, also known as ganglio-
607
Fig. 3. Pretreatment of L6 cells with exogenous neuraminidases, MNeu1 or
cPNase induced desialylation of both insulin-(IR) and IGF-1 receptors (IGF-1R)
residing on their surface. (Upper panels) Representative western blots with
antibodies recognizing the subunits of IR (A) and IGF-1R (B) indicate that
lysates of L6 cells that were incubated for 30 min in the presence of 1 mU/mL of
MNeu1 or 25 mU/mL of cPNase contained the immunoreactive protein bands of
slightly smaller molecular weight than their counterparts in lysates of untreated
cells. (Lower panels) These lower molecular weight forms of both receptors
subunits did not react with the Maackia amurensis agglutinin lectin (Dig-MAA)
that recognized the 23-linked sialic acids on intact subunits of both IR and
IGF-1R present in extracts of control cells. In contrast, these smaller molecular
weight receptors reacted with peanut agglutinin (PNA) that recognized their
intact Gal13GalNac residues.
Desialylation of insulin-responsive receptors on myoblasts

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side sialidase) (Miyagi et al. 1999; Monti et al. 2000, 2004;
Yamaguchi et al. 2005); and to mitochondria and lysosomes:
neuraminidase-4 (Neu4) (d'Azzo et al. 2001; Pattison et al.
2004). The quantitative real time RT-PCR assessments indica-
ted that in human tissues Neu1 generally shows 1020 times
higher expression than those of Neu3 and Neu4, while
Neu2 expression is only four- to ten thousandth of the
Neu1 value (Yamaguchi et al. 2005; Miyagi 2008).
Previous studies from our laboratory indicated that lyso-
somal Neu1 can be also targeted to the cell surface, as a
subunit of elastin receptor (Hinek et al. 1993; Privitera et al.
1998). It is important to mention that the cell surface-residing
Neu1 remains specically active toward adjacent sialylated
glycoproteins (Hinek et al. 2006, 2008). Thus, it functionally
diers from the other cell membrane-residing sialidase, Neu3
that strictly modulates gangliosides (Monti et al. 2000; Sato
and Miyagi 1996; Anastasia et al. 2008).
We have to mention that Neu1 residing on the cell surface of
human aortic smooth muscle cells (SMC) and dermal bro-
blasts, as well as cPNase, sharing a partial homology and
substrate specicity with mammalian Neu1, can desialylate
elastic ber-associated microbrillar glycoproteins and bro-
nectin (Hinek et al. 2006). We also provided experimental
evidence that both endogenous Neu1 and exogenous cPNase
can remove terminal sialic acids from IGF-1R and PDGFR re-
siding on the surface of human arterial SMC, thereby
abolishing the proliferative response of these cells to their re-
spective mitogenic ligands IGF-II and PDGF BB (Hinek et al.
2008). Other investigators also shown that the Neu1 causes de-
sialylation of Toll-like receptor 4 (Amith et al. 2010), Fc
receptors for immunoglobulin G, Fc gammaR (Seyrantepe et
al. 2009) and 4 integrin (Uemura et al. 2009) and consequent-
ly modulates the respective signals transduced through these
receptors. However, we do not consider that desialylation of
the above-mentioned proteins and receptors would be poten-
tially involved in the modulation of the described mitogenic
response of L6 cells to dierent doses of insulin. In fact, our
data demonstrating that treatment of L6 cells with cPNase or
MNeu1 alone did not aect the basic rate of L6 cells prolifer-
ation (Figure 5) endorse this notion.
In this report, we rst demonstrated that physiological (0.5
1 nM) and therapeutic doses (10 nM) of insulin induced pro-
liferation of cultured L6 cells via activation of their IR and
consecutive triggering of the downstream PI3K/Akt signaling
pathway. Then, we found that the proliferative eect of such
insulin concentrations on L6 cells could be further enhanced
Fig. 4. (A) (Upper panel) Representative western blots with antibody
recognizing the subunits of IR show that lysates of cultured L6 cells that
were rst incubated for 30 min in the presence of 25 mU/mL cPNase contain
the immunoreactive protein bands of slightly smaller molecular weight than
untreated cells. (Lower panel) Representative western blots with antibody
recognizing the phosphorylated form of subunits of IR demonstrate that
10-min treatment of L6 cells with 1 or 10 nM of insulin induced higher
phosphorylation of 95 kDa subunits of IR than treatment with 1 nM insulin.
Also, slightly smaller (desialylated) form of these subunits present in extracts
of cells pretreated for 30 min with cPNase are stronger phosphorylated in
response to both doses of insulin. (B) Representative western blots and results
of densitometric evaluations of four western blots with anti-Phospho-Akt and
anti-Akt antibodies demonstrating that extracts of L6 cells pretreated with
1 mU/mL of mouse-derived Neu1 show signicantly higher phosphorylation of
Akt in response to similar doses of insulin than their counterparts that were not
exposed to MNeu1. The phosphorylation of Akt (induced by both does of
insulin) was not aected by the presence of IGF-1R inhibitor, PPP. (C) (Upper
panel) Representative western blots with antibody recognizing the subunits
of IGF-1R show that lysates of cultured L6 cells that were rst incubated for
30 min in the presence of 1 mU MNeu1 or with 25 mU/mL cPNase contain the
immunoreactive protein bands of slightly smaller molecular weight than
untreated cells. (Lower panel) Representative western blots of parallel extracts
of cultured L6 cells with anti-Phospho-Tyr (PY-20) antibody demonstrate that
10-min treatment with 100 nM insulin, but not with 10 nM insulin, induced
phosphorylation of 110 kDa subunits of their IGF-1 receptors. One hundred
nanomolar insulin could not induce phosphorylation of smaller (desialylated)
form of these subunits present in extracts of cells pretreated for 30 min with
both neuraminidases.
608
M Arabkhari et al.

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609
Fig. 5. (A) Results of [
3
H]-thymidine incorporation assay performed on 5-day-old cultures of L6 cells indicate that treatment with 25 mU/mL of cPNase
signicantly enhanced their proliferative response of L6 cells to physiological concentrations (0.5 and 1 nM) of insulin. The enhancement of the proliferative eect
induced by 1 nM insulin in cultures incubated with cPNase was eliminated in cultures treated with the inhibitor of insulin receptor (HNMPA-(AM)3), but not
aected in cultures pretreated with IGF-1 receptor inhibitor (PPP). (B) Results of [
3
H]-thymidine incorporation also demonstrate that pretreatment of L6 cells with
1 mU/mL of mouse-derived Neu1 signicantly enhanced the proliferative response of L6 cells to 10 nM insulin. The magnitude of an enhancement of
insulin-induced proliferative eect, caused by treatment with 1 mU/mL of MNeu1, was higher than the eect of comparable concentration of cPNase. The
application of higher concentration of cPNase (25 mU/mL) produced only slightly higher enhancement of the proliferative response to 10 nM of insulin. In contrast,
pretreatment of parallel cultures with 25 mU cPNase practically eliminated the proliferative response of L6 cells to high (100 nM) dose of insulin.
Desialylation of insulin-responsive receptors on myoblasts

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Fig. 6. (A) Results of [
3
H]-thymidine incorporation performing in 5-day-old cultures of L6 cells showing that inhibition of endogenous neuraminidase with
500 M ddNANA or with 2 g/mL of polyclonal anti-Neu1 antibody eliminated the proliferative response of L6 cells to 1 and 10 nM of insulin. (B) The similar
inhibition of endogenous Neu1 actually potentiated the proliferative response of parallel cultures to 100 nM of insulin. (C) Representative western blots and results
of densitometric evaluations show that extracts of L6 myoblasts that were transfected for 36 h with the Neu1-specic siRNA demonstrate 6-fold decrease in the
level of immunodetected Neu1 protein, as compared to the counterparts transfected with the nontargeting siRNA. The media collected from the parallel cultures of
Neu1 siRNA-transfected cells also demonstrated 10-fold decrease in sialidase activity, assessed by spectroscopic estimation of the uorescent product released
from the synthetic substrate, 4MU-NANA (left panel). The Neu1 decient cells (transfected with the Neu1-specic siRNA) incorporated signicantly
less [
3
H]-thymidine than their counterparts transfected with nontargeting siRNA, when treated for 48 h with 1 nM insulin or 10 nM insulin. In contrast,
Neu1 siRNA-transfected cells demonstrated heightened proliferative response to 100 nM insulin (right panel).
610
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following experimental desialylation of their IR, induced by
exogenous Neu1 or cPNase. Importantly, we also found that
inhibition of endogenous Neu1 with a competitive inhibitor
ddNANA or with anti-Neu1 antibody abolished the prolifera-
tive response of L6 cells to 1 and 10 nM of insulin. A similar
trend was observed in cultures in which expression of endog-
enous Neu1 has been inhibited by the siRNA method. This
particular data suggested that activity of endogenous Neu1
would cause desialylation of the adjacent cell surface IR,
which somehow enhances proliferative signals induced by
physiological and therapeutic doses of insulin.
While one would expect that the proposed exclusive in-
volvement of Neu1 might be additionally endorsed in cells
overexpressing this enzyme, we did not attempt to overexpress
Neu1 in L6 myoblasts, because the transduction of cells with
the sole Neu1 cDNA does not lead to a consequent increase in
the activity of this enzyme (Igdoura et al. 1998; Pattison et al.
2004; Vinogradova et al. 1998). In order to obtain heightened
Neu1 activity, cells have to be transduced with the double con-
struct encoding Neu1 and its partner, protective protein/
cathepsin A (PPCA). Since increased proteolytic activity of
cell surface-delivered PPCA in transfected cells could poten-
t i al l y i nact i vat e ot her mi t ogeni c pol ypept i des (e. g. ,
endothelin-1 or angiotensin II) (Bonten et al. 1995; Bonten
and d'Azzo, 2000; Seyrantepe et al. 2008), results obtained
from such an experimental model might not be conclusive.
However, our major claim that endogenous Neu1 may be a po-
tent modulator of cellular response to insulin was recently
endorsed by results of other study from our laboratory indicat-
ing that dermal broblasts obtained from sialidosis patients
(OMIM 256550), decient in Neu1, but expressing normal le-
vels of Neu2, Neu3 and Neu4, demonstrate signicantly lower
metabolic response to physiological doses of insulin, as com-
pared with response of normal broblasts. We found that
sialidosis broblasts demonstrated much lower levels of Akt
phosphorylation in response to the same low dose of insulin
than normal broblasts. Meaningfully, preincubation of siali-
dosis broblasts with exogenous Neu1 and cPNase restores
normal levels of Akt phosphorylation in response to insulin
(A. Hinek, unpublished observation).
It is also important to mention that the described mitogenic re-
sponse of L6 myoblasts to insulin could not be observed in
cultures of human arterial SMC. We have established that cul-
tured SMC could not be stimulated to proliferate following
treatment with 1100 nM of insulin, even after exposure to ex-
ogenous neuraminidase (data not shown). Thus, the observed
selectivity in the proliferative responsiveness of skeletal myo-
blasts to insulin might indicate for a unique setting of IR on L6
myoblasts. This assumption seems to be consistent with previ-
ously published data indicating that vascular SMC are
insensitive to insulin, and that this is due to a preponderance of
IGF-IR and the presence of insulin/IGF-1 hybrid receptors with
high anity to IGF-1 and extremely low anity to insulin
(Arnqvist, 2008). In context of this information, our data may
further point out that the proliferative response of cultured L6
myoblasts to physiological and low therapeutic concentrations
of insulin (0.510 nM) is propagated solely through the IR and
not involve hybrid receptors that preferably interact with IGF-1.
On the other hand, we also demonstrated that supraphysio-
logical concentration (100 nM) of insulin induced the most
potent proliferative response of L6 myoblasts that was eec-
tively transduced through their IGF-1R. While the activation
of IGF-1R by such a high dose of insulin has been previously
documented in other cell types (Chisalita and Arnqvist 2005;
Johansson and Arnqvist 2006), we now demonstrated that the
desialylation of IGF-1R following exogenous neuraminidases
actually inhibited the heightened proliferative response of skel-
etal L6 myoblasts to 100 nM of insulin. Meaningfully, we also
documented that proliferative response of tested myoblasts to
100 nM of insulin could be additionally enhanced in cultures
treated with ddNANA, with anti-Neu1 antibody or in cultures
in which expression of endogenous Neu1 has been inhibited by
siRNA method. These data endorse the notion that the enzy-
matic activity of Neu1 residing on the cell surface could
structurally alter and functionally inactivate IGF-1R, thereby
decreasing the proliferative responsiveness of skeletal myo-
blasts to the extremely high doses of insulin. This also
prompted the hypothesis that desialylation of IGF-1R could al-
so serve as a negative modulator that prevents the overzealous
response of myoblasts to the extremely high dose of insulin.
Further studies are needed to elucidate the exact molecular
mechanism, in which enzymatic removal of sialic acid residues
from both IR and IGF-1R dierently aects the proliferative re-
sponse of skeletal myoblasts to insulin. Now, we can only
speculate that desialylation of both insulin sensitive receptors
would change their three-dimensional organization in such a
way that either upregulate (IR) or decrease (IGF-1R) their
insulin-induced activation. We particularly suggest that
changes in three-dimensional folding of these two receptors,
induced by desialylation, would dierently aect their auto-
phosphorylation prerequisite for the subsequent downstream
signals (Amoui et al. 2001; Arnqvist 2008). This speculation
is endorsed by our data demonstrating that the phosphorylation
status of IR in L6 cells cotreated with 1 or 10 nM of insulin and
exogenous neuraminidases was markedly higher and coincided
with a stronger activation of the PI3K/Akt-involving signaling
than in cells treated with insulin alone (Figure 4A and B). In
contrast, pretreatment of L6 myoblasts with MNeu1 or cPNase
abolished IGF-1R phosphorylation induced by 100 nM insu-
lin (Figure 4B). Our explanation suggesting that desialylation
of IR would expose their kinase domains correlates well
with the results of older studies demonstrating that neur-
aminidase treatment of triton-solubilized IR from rat fat cell
membranes released sialic acid and changed their isoelectric
point from 4.7 to 5.6 and mobility on the sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDSPAGE).
These papers also documented that desialylation of IR subu-
nits did not change their insulin-binding capabilities (Kasuga
et al. 1980), but resulted in enhanced IR kinase activities
(Fujita-Yamaguchi et al. 1985). We would also speculate that
desialylation of both IR and IGF-1R may in turn aect the
tyrosine phosphorylation of the insulin receptor substrates
(IRS 14) that act as a multisite docking proteins by binding
to downstream signal-transducing molecules. The dierential
site-specic phosphorylation of IRS-1 has been shown as an
important step explaining the dierences in the downstream
signaling pathways transduced through IGF-1R (that couples
IRS-1 preferentially to Grb2) and through IR that preferentially
connects IRS-1 to the p85 subunit of PI3K and to Nck (Giraud
et al. 2004, 2007; Luo et al. 2007).
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Results of our studies suggesting a crucial role of Neu1 in
skeletal muscle growth and regeneration correlate well with
the fact that progressive muscular atrophy and skeletal defor-
mities are present in children with sialidosis, disease
characterized with the exclusive deciency of active Neu1
(de Geest et al. 2002). Our data also comply with recent re-
sults from Dr. d'Azzo group demonstrating that mice
nullzygous at the Neu1 locus that develop clinical abnormal-
ities reminiscent of early-onset sialidosis in children also
develop muscular atrophy (Zanoteli et al. 2007). Our results
also endorse previous claim from Dr. Igdoura's group demon-
strating that the expression of Neu1 is upregulated by MyoD
on early stage of myogenesis and that experimental overex-
pression of Neu1 coincides with heightened proliferation of
myoblastic cells and inhibition of their dierentiation cascade
(Champigny et al. 2005).
Our novel claim that the activity of Neu1, but not other
sialidases, can induce enhancement of mitogenic response
of L6 myoblasts to low dose of insulin is also endorsed by
the fact that two other sialidases, cytosolic sialidase (Neu2)
and cell membrane-bound sialidase (Neu3), have been previ-
ously implicated as inducers of myoblast dierentiation, but
not proliferation, through the strict modulation of the GM3
ganglioside content in myoblasts and subsequent protection
against the apoptotic stimuli (Sato and Miyagi 1996; Fanzani
et al. 2003; Anastasia et al. 2008). Moreover, it has been re-
ported that a transient upregulation of Neu3 expression in L6
myocytes caused a signicant decrease in IR signaling, via
modulation of plasma membrane gangliosides and interaction
with Grb2 (Sasaki et al. 2003).
In summary, results of our study strongly indicate that insu-
lin, similar to IGF-I and -II, contributes to a process of
muscular development and repair by stimulating myoblast pro-
liferation. They may also encourage further studies aimed at
the possible use of low (not hypoglycemic) doses of insulin
for stimulation of skeletal muscle regeneration and for the
treatment of selected musculopathies. Most importantly, for
the rst time we demonstrated that Neu1-induced desialylation
of IR residing on skeletal myoblasts sensitizes them to a more
potent proliferative response to physiological and low thera-
peutic doses of insulin.
Materials and methods
Materials
Culture media (Dulbecco's modied eagle's medium, DMEM),
fetal bovine serum (FBS), 0.2% trypsin0.02% ethylenediami-
netetraacetic acid (EDTA) and other tissue culture reagents were
obtained from Invitrogen (Burlington, Ontario, Canada). The
ddNANA that inhibits mammalian Neu1 (Hinek et al. 2006,
2008) was purchased from Sigma (St. Louis, MO). Rabbit poly-
clonal antibody specic to human Neu1 and not cross-reacting
with other human sialidases was also used (Vinogradova et al.
1998). Insulin was obtained from Eli Lilly Canada, Inc. (Toron-
to, Ontario). A cell permeable IR kinase inhibitor, HNMPA-
(AM)3, was purchased from Biomol (Philadelphia, PA), and
PPP, a cell permeable, noncompetitive IGF-1 receptor inhib-
itor, was from Calbiochem/EMD Chemicals Inc., (Gibbstown,
NJ). The monoclonal anti-phospho-Tyr (PY-20) antibody and
puried rabbit polyclonal antibodies to subunits of IR and
IGF-1 receptor were obtained from Santa Cruz Biotechnolo-
gy, Inc. (Santa Cruz, CA). Polyclonal antibody recognizing
phosphorylated IR was from Cell Applications (San Diego,
CA) (CB4386). Polyclonal anti-phospho-Akt (Ser473) and
anti-Akt antibodies were purchased from Cell Signaling Tech-
nology (Beverly, MA), and monoclonal anti--actin from
ABCam (Cambridge, MA). The DNA isolation kit was pur-
chased from Qiagen, Inc. (Mississauga, Ontario, Canada).
Rabbit Anti-ki67 polyclonal antibody was purchased from
Chemicon (Temecula, CA), and the immunostaining kit,
DakoCytomation, was obtained from Dako North America,
Inc. (Carpinteria, CA). The enhanced chemiluminescence west-
ern blotting detection kit and [
3
H]-thymidine were purchased
from Amersham Bioscience Canada, Ltd. (Oakville, Ontario,
Canada). The digoxigenin glycan dierentiation kit (used to
identify sialylated glycoproteins) was purchased from Roche
(Mannheim, Germany). The uorescein-labeled F(ab)2 frag-
ments of goat anti-rabbit secondary immunoglobulin, nuclear
stains, propidium iodide and 4,6-diamidino-2-phenylindole
were purchased from Sigma (St Louis, MO). The Cytomation
LSAB2 System (horseradish peroxidase liquid diaminobenzi-
dine kit and hematoxylin counterstain) was supplied by
DakoCytomation (Carpinteria, CA).
Exogenous neuraminidases
In all described experiments we used two exogenous
neuraminidases.
1. The cPNase Type V (Sigma N2133) displays a similar sub-
strate specicity toward sialylated glycoproteins as mammalian
Neu1. The cPNase was puried by anity chromatography
using p-aminophenyl oxamic acid agarose. This preparation
was additionally subjected to an ion-exchange chromatogra-
phy that has been proven for removing protease activity
from neuraminidase preparations (Hatton and Regoeczi
1973). The nal cPNase preparation liberated 1 mmole of
N-acetylneuraminic acid per minute at pH 5.0 at 37C from
N-acetylneuraminyl-lactose substrate but not demonstrated
detectable proteolytic activity against casein substrate.
This cPNase preparation did not reveal any -galactosidase
activity against the synthetic when tested against 4-methyl-
umbelliferyl-D-galactopyranoside substrate (D'Agrosa et al.
1992).
2. The exogenous Neu1 that was initially isolated from
mouse kidney tissue as a part of the multienzyme complex
by the anity chromatography on a concanavalin A-Sepharose
column. Further purication involved anity chromatography
on a p-aminophenyl -D-thiogalactopyranoside-agarose col-
umn and fast protein liquid chromatography gel-ltration on
Superose 6 column, as previously described (Pshezhetsky
and Potier 1996). This preparation did not display any endo-
peptidase activity (Vinogradova et al, 1998). Since this
preparation could possible contain the admixture of protective
protein with cathepsin A activity (PPCA), it has been kept in
the presence of phenylmethylsulphonyl uoride (PMSF) which
is an ecient PPCA inhibitor (Itoh et al. 1993).
Cells
All experiments aimed at proliferative eects of insulin, and
desialylation of the IGF-1R and IR was performed on cultures
of L6 rat skeletal myoblasts purchased from ATCC (Manassas,
VA). These myoblasts can dierentiate spontaneously from
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myoblasts to multinucleated myotubes that express muscle-
specic proteins (Tsakiridis et al. 1995), and have been pre-
viously recognized as a stable and reproducible model for
studying the hormonal and metabolic regulation of the glu-
cose transporting system and other cellular eects of insulin
(Antonescu et al. 2008; Samokhvalov et al. 2009).
Analysis of IGF-1R and IR desialylation
To provide direct evidence that the IRand IGF-1Rpresent on the
surface of L6 cells are sialylated glycoproteins and that exposure
of these cells to exogenous neuraminidases would cause their
desialylation, the following procedures were performed. The
conuent cultures of L6 cells were transferred for 6 h to se-
rum-free DMEM. The media were then changed, and the
cultures were maintained for 30 min in the presence or absence
of 1 mU/mL of mouse Neu1 or 25 mU/mL of cPNase. At the end
of this incubation period, cultures were washed in phosphate
buered saline (PBS) and then lysed in RIPA buer containing
cocktail of class-specic protease inhibitors in the following -
nal concentrations: 2 mM benzamidine, 2 mM -Aminocaproic
acid (EACA), 1 mM EDTA, 1 mM Ethylene glycol-bis(2-ami-
noethylether)-N,N,N,N-tetraacetic acid (EGTA), 1 mg/mL
Trasylol and 2 mM PMSF. The aliquots of cell lysates (con-
taining equal 400 g protein concentrations) were directly
resolved by 10% SDSPAGE and then analyzed by western
blotting using antibodies raised to the subunits of the IGF-
1R and IR, respectively (Hinek et al. 2008). The parallel ali-
quots of the same lysates were rst immunoprecipitated for
1 h at 4C (in buer containing cocktail of proteases inhibi-
tors) with antibodies recognizing subunits of respective
IGF-1R and IR, and then the immunoprecipitation products
bound to 4% Protein A-beaded agarose were centrifuged and
washed four times with PBS and then resuspended in sample
buer with 2--mercaptoethanol, resolved by 10% SDS
PAGE, and transferred onto a nitrocellulose membrane.
To visualize the sialylated glycoprotein bands, the mem-
branes were then probed with the Dig-MAA, recognizing
23-linked sialic acids from the Roche digoxigenin glycan
dierentiation kit, in accordance with the manufacturer's proto-
col. To further test whether preparations of exogenous
neuraminidases, cPNase and MNeu1, would potentially contain
admixture of other active glycosidases, e.g. galactosidases, the
proteins immunoprecipitated with antibodies recognizing
subunits IR and IGF-1R were additionally probed with PNA
that specically recognizes Gal13GalNac residues (Knibbs
et al. 1993; Gillespie et al. 1993).
Testing whether desialylation of IR and IGF-1R would affect
their insulin-induced phosphorylation
L6 were initially plated at a density of 500,000 cells/culture and
grown in medium with 10% FBS for 24 h. The subconuent
cultures were then serum starved for 24 h. Cultures kept in se-
rum-free medium were then preincubated for 30 min with
25 mU of cPNase before they were exposed for 10 min to 1
or 10 nM of human recombinant insulin. At the end of this in-
cubation period, cultures were washed in PBS and then lysed in
RIPA buer containing a proteinase inhibitor cocktail and
1 mM of the endogenous phosphatase inhibitor sodium ortho-
vanadate. The aliquots of cell lysates (containing equal protein
concentration) were resuspended in sample buer with 2--
mercaptoethanol, resolved by 10% SDSPAGE, and trans-
ferred onto a nitrocellulose membranes and immunoblotted
with anti-IR- subunit antibody and with anti-phospho-IR-
subunit antibody, as described in the gure legend.
The parallel group of cultures were preincubated for
30 min either with 1 mU of mouse-derived Neu1 or with
25 mU of cPNase before they were exposed for 10 min to
100 nM of human recombinant insulin. The aliquots of these
culture lysates (containing equal protein concentration) were
then resolved by 10% SDSPAGE, transferred to the nitrocel-
lulose membranes and immunoblotted with anti-IGF-1R-
subunit antibody or with anti-phosphotyrosine (PY-20) anti-
body, as described in gure legend.
Analysis of Akt phosphorylation in response to insulin
L6 were cultured as described above, preincubated for 30 min
with 1 mU of mouse-derived Neu1 before they were exposed
for 10 min to 1 or 10 nM of human recombinant insulin in the
presence or absence of 100 nM of the specic IGF-R inhibitor,
PPP. At the end of this incubation period, cultures were lysed
and aliquots of each cell lysate (containing equal protein
concentration) were then resolved by 10% SDSPAGE,
transferred to the nitrocellulose membranes and immunoblotted
rst with anti-Phospho-Akt antibody and then re-probed with
anti-Akt antibody. Loading of equivalent amounts of protein
was additionally conrmed by stripping the membranes and
reprobing with monoclonal antibody to -actin.
Inhibition of endogenous Neu1 expression by siRNA method
The predesigned ON-TARGETplus SMARTpools of rat Neu1-
specic siRNA and control nontargeting siRNAs were pur-
chased from Thermo Scientic, Dharmacon, Inc. (Chicago,
IL). The following sequences were used:
Rat Neu1 pool: GGAGUAAGGAUGACGGCGU,
GGGCUGUGGUGAACGACGU,
GGUAUUGGAGGCAGGUACU,
CGGCUUUCAUUGUAGACGA,
Nontargeting pool: AAACUGAGCUGGAAUCUAA,
GUAACUAAGUGUCCCAACA,
GUAUCAAGCUCUACUUUAC,
GUCCAUGGGUUUAUAUAUG.
L6WT cells were seeded in six-well tissue culture dishes at
2 10
5
per well. Twenty-four hours later, the cultures were di-
vided into two experimental groups and then incubated, for the
next 36 h either with 160 pmol of rat Neu1-specic siRNA or
with nontargeting siRNA, in the presence of 10 L of X-treme-
GENE siRNA transfection reagent (Cat. # 04476093001,
Roche, (Laval, Quebec, Canada) Diagnostics GmbH), accord-
ing to the manufacturer's instructions. The ecacy of Neu1
siRNA transfection was veried in extracts of L6 (incubated
for 36 h with transfecting reagents) when the protein levels of
Neu1 were estimated by western blotting with anti-Neu1 anti-
body and by spectroscopic estimation of the uorescent
product (detected at 445 nM) released from the synthetic sub-
strate, 4-methylumbelliferyl-N-acetyl--D-N-acetylneuraminic
acid (4MU-NANA) that was added to the culture media, as pre-
viously described (Duca et al. 2007). All transfected cultures
were then maintained in media containing 2% FBS and 1 Ci/
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mL of
3
H-thymidine was added, and then the parallel quadrupli-
cate cultures from both experimental groups transfected with
Neu1-specic siRNAor with control nontargeting siRNAs were
exposed either to 1, 10 or 100 nM insulin. The nal rates of cel-
lular proliferation were estimated, 48 h later by the
3
H-
thymidine incorporation method, as described in Quantitative
assays of cellular proliferation section.
Quantitative assays of cellular proliferation
[
3
H]-Thymidine Incorporation Assay. L6 myoblasts were
plated in six-well culture dishes (5 10
5
/dish) and maintained
in medium supplemented with 10% FBS for 24 h to achieve 70
to 80% conuency. Cells were then made quiescent by
incubation with serum-free medium for 24 h. Subsequently,
quadruplicate cultures of each experimental group were
maintained for the next 72 h in fresh medium supplemented
with 2% FBS in the presence or absence of dierent
concentrations of insulin (0.5100 nM) and other reagents
(blockers and inhibitors as described in gure legends), and
[
3
H]-thymidine (2 Ci/mL). At the end of each experiment,
cultures were washed with PBS. DNA was extracted with 5%
trichloroacetic acid at 4C and then solubilized in 0.3 M
sodium hydroxide. Finally, 200 L aliquots from each culture
were analyzed by scintillation counting as previously described
(Hinek et al. 2008).
DNA Assay. The DNeasy Tissue System (Qiagen) was used in
accordance with the manufacturer's protocol to assess the total
DNA content of quadruplicate cultures subjected to dierent
treatments in the individual experiments, as previously
described (Hinek et al. 2008).
Detection of Proliferative Antigen, ki67. All cultures of L6
myoblasts (initially plated at cover slips and subjected to
dierent treatments) were xed in 100% methanol for 30 min
at 20C and in 0.03% hydrogen peroxide for 10 min at room
temperature. Then, the slides were washed with PBS and
exposed to 1% normal goat serum blocking solution for half
an hour. Subsequently, the slides were immunostained using
the DakoCytomation kit (Dako North America, Inc.),
according to the manufacturer's instructions, counterstained
with hematoxylin, dehydrated and mounted in Elvanol.
Twenty pictures were randomly taken of each culture, and the
number of ki67-positive cells, as well as the total number of
cells, was counted. The nal results were expressed as the %
of positive cells (mean +/ SD).
Statistical analysis
The raw data (means and standard deviations) obtained from
quadruplicate cultures belonging to each experimental group
were collected and then results from three separate experiments
were pooled together, expressed as the % change of control and
statistically evaluated by analysis of variance, followed by a
Tukey post test to establish which groups were dierent.
Funding
This work was supported by the Canadian Institute of Health
Research through grant PG 13920 and by the Heart and Stroke
Foundation of Ontario through grants NA 5435 and NA 4381
to A.H., as well as by the operating grant from the Canadian
Diabetes Foundation to A.V.P.
Conflict of interest statement
None declared.
Abbreviations
cPNase, Clostridium perfringens neuraminidase; ddNANA,
2,3-dehydro-2-deoxy-N-acetylneuraminic acid; Dig-MAA,
digoxigenin-labeled Maackia amurensis agglutinin lectin;
DMEM, Dulbecco's modied eagle's medium; EDTA, ethy-
lenediaminetetraacetic acid; FBS, fetal bovine serum; HNMPA-
(AM)3, hydroxy-2-naphthalenylmethylphosphonic acid
tris-acetoxy-methyl ester; IGF, insulin-like growth factors;
IGF-1, insulin-like growth factor 1; IGF-II, insulin-like
growth factor II; IGF-1R, insulin-like growth factor 1 recep-
tors; IR, insulin receptors; IRS, insulin receptor substrates;
Neu1, neuraminidase-1; Neu2, neuraminidase-2; Neu3,
neuraminidase-3; Neu4, neuraminidase-4; PBS, phosphate
buered saline; PDGFR, platelet-derived growth factor recep-
tors; PI3K, phosphatidylinositol-3 kinase; PMSF, phenyl-
methylsulphonyl uoride; PNA, peanut agglutinin; PPCA,
protective protein/cathepsin A; PPP, picropodophylin; RIPA,
radio immunoprecipitation assay; SDSPAGE, sodium dode-
cyl sulfatepolyacrylamide gel electrophoresis; SMC, smooth
muscle cells.
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