From the Pacific Northwest Research Institute and
Department of Pharmacology, University of Washington, Seattle,
Washington 98122, the ¶ Division of Endocrinology and Metabolism,
Department of Medicine, Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, Massachusetts 02215, and the
Research Division, Joslin Diabetes Center and Program in Cell
and Developmental Biology, Harvard Medical School, Boston,
Massachusetts 02215
Received for publication, February 8, 2001
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ABSTRACT |
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It has been shown that IGF-1-induced pancreatic
The molecular defects causing obesity-linked type 2 diabetes
mellitus are not well defined. However, recently it has become clear
that key factors involved in causing type 2 diabetes are both impaired
Previous studies have shown that certain growth factors, such as growth
hormone (GH)1 and
insulin-like growth factor 1 (IGF-1), are important for stimulation of
GH signals via the janus kinase-2/signal transducer and activator of
transcription-5 pathway in INS-1 IGF-1 has been shown to activate at least two major mitogenic signaling
pathways, via PI3K and mitogen-activated protein kinase (7-10). The
mitogenic stimulation of receptors, such as the IGF-1 receptor, results
in the phosphorylation and activation of the IRS family of proteins and
the SH2-containing protein (Shc). Shc then interacts with Shc homology
2 (SH2) domain-containing proteins, such as the growth factor
receptor-bound protein-2, whereas IRS proteins interact with the
regulatory subunit of PI3K, p85, as well as growth factor
receptor-bound protein-2. The recruitment of growth factor
receptor-bound protein-2 and its association to murine sons of
sevenless-1 protein (a Ras guanine nucleotide exchange factor)
activates Ras, resulting in Raf-1 activation, which phosphorylates the
extracellular signal-regulated protein kinase (ERK) kinase, which in
turn activates ERK1/ERK2 (8). Interaction of tyrosine-phosphorylated
IRS with the p85 regulatory subunit of PI3K leads to the activation of
the catalytic subunit, p110, which in turn phosphorylates
phosphoinositides at the 3' position of the inositol ring, generating
PIP3 (4, 6). This increase in phosphorylated phosphoinositides leads to
the localization of protein kinase B (PKB) to the membrane through the
interaction of PIP3 and PIP2 with the
pleckstrin homology domain of PKB (11). Full activation of PKB
appears to be dependent on the subsequent phosphorylation of two
residues, Thr-308 in the activation loop of the kinase domain and
Ser-473 in the carboxyl-terminal tail (12). The protein kinase shown to
phosphorylate Thr-308 is 3-phosphoinositide-dependent kinase-1 (PDK1) (13, 14), whereas the kinase responsible for phosphorylation at the Ser-473 residue has not yet been identified. PKB
regulates multiple biological processes, such as cell proliferation and
apoptosis, suggesting that it may phosphorylate a number of target
proteins (reviewed in Ref 15). Glycogen synthase kinase-3 Materials--
The PDK-1 kinase assay kit and PDK-1 antibody
were from Upstate Biotechnology Inc. (Lake Placid, NY). The
phospho-GSK3 Cell Culture--
The glucose-sensitive pancreatic [3H]Thymidine
Incorporation--
Thymidine incorporation was measured essentially as
described, with minor changes. Briefly, INS-1 cells were cultured on
6-well plates to ~60% confluence and infected with adenovirus as
described below. The cells were then counted and ~1 × 104 cells were added to each well of a 96-well plate. The
cells were left to attach overnight and were then made quiescent by
incubation in starvation medium for 24 h. The INS-1 cells were
then incubated for 24 h in starvation medium with additional
glucose ± 10 nM IGF-1 as described. During the final
4 h of this incubation 5 µCi/ml [3H]thymidine was
added. The specific incorporation of [3H]thymidine into
DNA was then measured by transferring the cell lysates to UniFilter-96
GF/C filter plates using the Packard cell harvester, and the
radioactivity on the filters was counted with the Packard cell counter.
Stimulation and Lysis Conditions--
Cells were subcultured on
either 6-well or 10-cm plates to ~60% confluence. The cells were
then subjected to 24-h serum and glucose deprivation with starvation
medium (RPMI 1640 medium containing 0.1% bovine serum albumin, 0.5 mM glucose, 100 units/ml penicillin, and 100 µg/ml
streptomycin) or infected with adenovirus (see "Adenovirus Infection"), when appropriate, prior to the 24-h incubation with starvation medium. After the quiescent period the cells were
pre-treated for 15 min with or without inhibitors as indicated,
followed by incubation with fresh starvation medium with or without
inhibitors with 0.5, 3, or 15 mM glucose with or without 10 nM IGF-1 for the times indicated. The medium was removed,
the cells were washed once with ice-cold phosphate-buffered saline, and
the phosphate-buffered saline was replaced with ice-cold cell lysis
buffer consisting of 50 mM HEPES (pH 7.5), 1% (v/v)
Nonidet P-40, 2 mM activated sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 4 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
1 µg/ml leupeptin, and 1 µg/ml aprotonin. After sonication
insoluble material was removed by centrifugation, and the samples were
stored at PDK-1 and PKB Kinase Assays--
Cells were stimulated as
described (see "Stimulation and Lysis Conditions"). The PKB
immunoprecipitation and kinase assay were carried out following the
procedures described in the manual, whereas some changes were made to
the PDK-1 kinase assay. Active PDK-1 was immunoprecipitated as
described in the manual. Briefly, ~1 mg of cell lysate was
pre-cleared with 50 µl of protein G-agarose. The beads were removed,
the lysate was then added to the protein G-antibody complex, and the
samples were left rotating for 2 h at 4 °C. After two washes
with cell lysis buffer, followed by two washes with PDK-1 assay
dilution buffer, the immunoprecipitates were resuspended in 30 µl of
PDK-1 assay dilution buffer containing 10 µl (1 µg) of GSK3 fusion
protein and 10 µl of Mg/ATP mixture. The assay was started by the
addition of 10 µl (0.5 µg) of serum- and
glucocorticoid-regulated protein kinase (SGK)-inactive
recombinant protein. SGK is a member of the AGC subfamily of protein
kinases whose kinase domain is most related to that of PKB. However,
unlike PKB, SGK does not possess a pleckstrin homology domain
enabling the assay to be carried out in the absence of
PIP3. SGK is phosphorylated at the residue equivalent to
Thr-308 of PKB, resulting in activation of this kinase (21). Having
established conditions to ensure that the kinase activity was well
within the linear portion of the reaction, samples were incubated for
7.5 min at 25 °C with continuous shaking. The reaction was
terminated with 30 µl of 3× SDS gel loading buffer, and samples were
analyzed by immunoblotting (see "Protein Immunoblot Procedures")
with the phospho-GSK3 Construction of Adenoviruses--
The PI3K adenoviruses, inter
Src homology region 2 (AdV-iSH2), and the catalytic
p110 Adenovirus Infection--
The appropriate titer for each
adenovirus was determined by the addition of various dilutions of each
adenovirus to cells subcultured in 6-well plates (9.5 cm2)
to 60% confluence (~2 × 106 cells), giving a
multiplicity of infection (m.o.i.) ranging from 50 to 2000 based on
0.5-2.0 × 106 plaque forming units/ml as measured by
A260. The viral stock was replaced with
complete medium after 2 h, and the cells were incubated at
37 °C in 5% CO2 for ~16 h. The cells were then
treated as described.
Protein Immunoblot Procedures--
Cell lysates were normalized
for total protein after levels were determined using the bicinchoninic
acid protein assay kit. For immunoblot analysis 25-50 µg of protein
was separated by SDS-polyacrylamide gel electrophoresis. After transfer
to nitrocellulose, membranes were immersed in blocking buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.1% Tween,
and 5 or 10% (w/v) nonfat dry milk) and incubated with gentle
agitation for 1 h. This was followed by incubation overnight at
4 °C with the appropriate primary antibody diluted in primary
antibody dilution buffer (10 mM Tris/HCl, pH 8.0, 150 mM NaCl, 0.1% Tween, and 5% bovine serum albumin or 10%
(w/v) nonfat dry milk). After a series of washes with blocking buffer without milk, the membranes were incubated for 1 h with gentle agitation with horseradish peroxidase-conjugated secondary antibody diluted in blocking buffer. Washes were repeated as before, and the
positive signals were visualized with chemiluminescence reagent and
x-ray film.
Constitutive Activation of PI3K in INS-1 Cells Only Increases
IGF-1-induced Glucose and/or IGF-1 Does Not Affect PDK-1 Kinase Activity in INS-1
Cells--
PDK-1 has been suggested to lie downstream of PI3K in
signal transduction pathways (15). It was determined whether PDK-1 plays a role in the mitogenic signal transduction in INS-1 cells in
response to glucose ± IGF-1. PDK-1 activity was assessed in INS-1
cells incubated with 0.5, 3.0, or 15 mM glucose ± 10 nm IGF-1 for 2, 10, and 40 min. PDK-1 was found to be active at 0.5 mM glucose, but neither an increase in glucose nor the
presence of IGF-1 had any significant effect on further increasing
PDK-1 activity (Fig. 2B).
Total levels of PDK-1 in each of the samples did not vary, indicating
that equivalent amounts of PDK-1 were immunoprecipitated (Fig.
2A). Thus, it would appear that PDK-1 is present in INS-1
cells and is constitutively active.
PKB Kinase Is Stimulated by IGF-1 but Not by Glucose in INS-1
Cells--
Activation of PI3K leads to downstream activation of PKB
(25). It has been proposed that PKB translocation to the plasma membrane via its pleckstrin homology domain is increased via
interaction with the PIP2 and PIP3 products
synthesized by increased PI3K activity at the cell plasma membrane
(11). PKB membrane localization appears to be sufficient to allow
PDK-1-mediated phosphorylation of PKB at Thr-308 and partial
activation, although full activation requires additional
phosphorylation at Ser-473 by an as yet unidentified kinase (13,
14).
PKB Thr-308 and Ser-473 phosphorylation and consequential regulation of
PKB activity was examined in INS-1 cells incubated at 0.5, 3.0, or 15 mM glucose ± 10 nM IGF-1 for 2, 5, 10, 20, and 40 min. PKB activity did not change significantly when cells were incubated at basal 3 mM glucose or stimulatory 15 mM glucose (Fig.
3A). In contrast, IGF-1
rapidly increased PKB activity, which reached a maximum after 5 min of
IGF-1 treatment and was maintained over the 40-min period at a similar
level whether at a basal 3 mM or stimulatory 15 mM glucose level (Fig. 3A). As such,
IGF-1-induced activation of PKB activity did not appear to be
glucose-dependent. The PKB phosphorylation state was
determined in parallel, and negligible phosphorylation at Thr-308 or
Ser-473 was observed in INS-1 cells incubated at 3 or 15 mM
glucose, correlating with a lack of glucose-induced PKB activity (Fig.
3B). In contrast, IGF-1 induced a rapid and marked increase
in PKB Thr-308 phosphorylation, which was maximal at 2 min and then
trailed off but was still significantly above that at time zero by 40 min (Fig. 3B). IGF-1 also markedly increased phosphorylation
of PKB at Ser-473 within 2 min, reaching a maximum between 10 and 20 min, which was maintained at 40 min (Fig. 3B). In general,
the Thr-308/Ser-473 phosphorylation of PKB by IGF-1 correlated with
IGF-1-induced PKB activity (Fig. 3A). Neither 3 or 15 mM glucose affected IGF-1-induced PKB Thr-308 or Ser-473
phosphorylation, also correlating with a lack of influence of glucose
on IGF-1-induced PKB activity. The total amount of PKB did not
significantly alter with glucose/IGF-1 treatment (Fig. 3B).
GSK3 Time-dependent Phosphorylation of p70S6K by
Glucose and IGF-1--
Phosphorylation and activation of
p70S6K has also been postulated to be downstream of PKB
activation via activation of mTOR by PKB (17, 18). However, mTOR
activation can also be mediated by nutrients such as branch chain amino
acids (27-30). It was investigated whether p70S6K
activation paralleled that of PKB in INS-1 cells. The activation of
p70S6K protein is accompanied by phosphorylation at
multiple Ser/Thr residues that retards its migration during
SDS-polyacrylamide gel electrophoresis (4, 5, 31). The
time-dependent activation of p70S6K was
investigated in INS-1 cells incubated at 3 or 15 mM
glucose ± 10 nM IGF-1 for 2, 5, 10, 20, and 40 min.
The cell lysates were then subjected to immunoblot analysis with the
p70S6K antibody. The results show that the phosphorylation
of p70S6K was apparent after 10 min but only in the
presence of 15 mM glucose + 10 nM IGF-1.
Phosphorylation of p70S6K was not observed until after 20 min at 3 mM glucose + 10 nM IGF-1 or 15 mM glucose alone (Fig. 5).
There was no apparent change in p70S6K phosphorylation at 3 mM glucose over the 40-min time period (Fig. 5). The
greatest level of activation was observed after 40 min of stimulation
in the presence of both 15 mM glucose and 10 nM IGF-1 (Fig. 5). It should be noted that although glucose activated p70S6K, it had no apparent effect on PKB activity (Fig. 5
versus Fig. 3). Moreover, IGF-1-induced activation of
p70S6K was relatively slow compared with that of
IGF-1-induced PKB activation and was enhanced by stimulatory glucose
concentrations, unlike PKB activity, which was not influenced by
glucose (Fig. 5 versus Fig. 3).
Glucose-stimulated p70S6K Phosphorylation Is Partially
PI3K-independent--
It was investigated whether p70S6K
phosphorylation activation by glucose and IGF-1 was dependent on
upstream PI3K and/or mTOR activation by the use of two specific
inhibitors, wortmannin and rapamycin. Wortmannin specifically inhibits
PI3K (32), and rapamycin blocks mTOR activation by forming a complex
with the immunophilin FKBP12 to generate a potent inhibitor of mTOR
(33). INS-1 cells were pre-treated with or without inhibitors and
stimulated with 3 or 15 mM glucose ± 10 nM IGF-1 for 40 min with or without inhibitor. To confirm
that the wortmannin and rapamycin were specifically inhibiting their
target, we analyzed their effects on phosphorylation of PKB (as a
positive control) and ERK1/ERK2 (as a negative control) in the same
INS-1 cells. In the absence of inhibitor (control) PKB was shown to be
phosphorylated on Thr-308 and Ser-473 by IGF-1 but not glucose (Fig.
6A), as previously
demonstrated (Fig. 3). The IGF-1-induced phosphorylation of PKB was
completely inhibited by wortmannin (100 nM) but unaffected
by rapamycin (25 mM), as compared with that of the control
(Fig. 6A). PKB levels were equivalent in each sample, as
indicated by total PKB immunoblot analysis (Fig. 6A).
ERK1/ERK2 phosphorylation activation in INS-1 cells was detected by
incubation with stimulatory 15 mM glucose and was increased
further with additional IGF-1 (Fig. 6B), as previously observed (4, 5, 34). Phosphorylation of ERK1/ERK2 in response to 15 mM glucose ± 10 nM IGF-1 was not
significantly affected by either wortmannin or rapamycin. ERK1/ERK2
levels were equivalent in each sample, as indicated by immunoblot
analysis using a total ERK1/ERK2 antibody (Fig. 6B). In
control cells, the phosphorylation activation of p70S6K was
stimulated in samples treated with 15 mM glucose and by 10 nM IGF-1 at both 3 and 15 mM glucose (Fig.
6C). The greatest degree of phosphorylation was observed in
cells treated with 15 mM glucose + 10 nM IGF-1
(Fig. 6C). In the presence of 100 nM wortmannin, the phosphorylation of p70S6K in cells treated with 15 mM glucose or 15 mM glucose + 10 nM IGF-1 was reduced but not completely blocked, whereas phosphorylation appeared to be completely inhibited in cells stimulated with 3 mM glucose + 10 nM IGF-1 (Fig.
6C). It appeared that IGF-1 no longer potentiated 15 mM glucose-induced p70S6K phosphorylation,
because p70S6K phosphorylation by 15 mM
glucose + 10 nM IGF-1 in the presence of wortmannin
was equivalent to that by 15 mM glucose alone (Fig. 6C). In contrast, in the presence of rapamycin, the
phosphorylation activation of p70S6K by glucose and IGF-1
was completely inhibited (Fig. 6C).
Effect of PKB Overexpression on Mitogenesis in INS-1 Cells--
It
was investigated whether increased PKB expression would influence
glucose/IGF-1-induced
PKB overexpression was analyzed by titration of adenovirus infection in
INS-1 cells with subsequent PKB immunoblot analysis (Fig.
7). A GFP-expressing adenovirus (AdV-GFP)
was used as a control and illustrated the endogenous levels of PKB
(Fig. 7). Immunoblot analysis of INS-1 cells infected with AdV-PKB-WT,
AdV-PKB-CA, or AdV-PKB-KD indicated a higher molecular weight PKB
protein, in addition to the endogenous PKB (Fig. 7). This was due to
the additional Myc/His tags on the adenovirally introduced PKB and was
confirmed by immunoblotting with a c-Myc antibody (data not shown). The highest levels of expression (that did not adversely affect
INS-1 cells) were obtained at a m.o.i. of 10 × 102,
and this was the amount of adenovirus used for all subsequent experiments.
INS-1 cells were infected with AdV-PKB-WT, AdV-PKB-CA, AdV-PKB-KD, or a
control adenovirus expressing luciferase (AdV-Luc) and were assayed for
glucose/IGF-1-induced [3H]thymidine incorporation in
comparison with uninfected INS-1 cells (Fig.
8). The uninfected INS-1 cells showed
3.9 ± 0.1-fold (n = 3) higher levels of
incorporation at 3 mM glucose compared with 12.6 ± 2.1-fold (n = 3) higher levels at 3 mM
glucose + 10 nM IGF-1 above 0.5 mM glucose
(p Effect of PKB Overexpression on Glucose/IGF-1 Activation of PKB,
GSK3
GSK3
ERK1/ERK2 phosphorylation activation was investigated in
PKB-overexpressing INS-1 cells (Fig. 9C). In
AdV-GFP-infected INS-1 cells, ERK1/ERK2 phosphorylation was increased
by 15 mM glucose, compared with basal 3 mM
glucose and further enhanced on addition of 10 nM IGF-1
(Fig. 9C). Surprisingly, 15 mM glucose ± 10 nM IGF-1-induced ERK1/ERK2 phosphorylation was partly
inhibited in AdV-PKB-WT-infected INS-1 cells and abolished in
AdV-PKB-CA-infected
The phosphorylation state of p70S6K in response to 40 min
of incubation with glucose and IGF-1 was examined in adenoviral
mediated PKB-overexpressing INS-1 cells (Fig. 9D).
Phosphorylation of p70S6K was not detected in the
AdV-GFP-infected control cells at basal 3 mM glucose,
slightly increased at 3 mM glucose + 10 nM
IGF-1 or 15 mM glucose alone, and further enhanced after
treatment with 15 mM glucose + 10 nM IGF-1
(Fig. 9D), similar to observations in uninfected INS-1 cells
(Fig. 6C). Similar patterns of p70S6K
phosphorylation were observed in AdV-PKB-WT- and AdV-PKB-KD-infected INS-1 cells (Fig. 9D). In AdV-PKB-CA-infected INS-1 cells,
phosphorylation of p70S6K at basal 3 mM glucose
was increased, but at stimulatory 15 mM glucose or in the
added presence of IGF-1, p70S6K phosphorylation was not
significantly different from that in AdV-GFP-infected control Previous studies have not only shown that PI3K is important for
glucose-dependent Two such components downstream of PI3K are PDK-1 and PKB (15). The
proposed model for PKB activation describes a complex process with a
number of steps, one of which involves the protein PDK-1 (15). The
first step is translocation of PKB from the cytosol to the plasma
membrane, which is induced by increased levels of PIP3,
synthesized at the plasma membrane by PI3K. The interaction of
PIP3/PIP2 with the pleckstrin homology
domain of PKB causes a conformational change allowing phosphorylation
at Thr-308 and Ser-473, which appear to be important steps for full activation of PKB. PDK-1 has been shown to phosphorylate PKB at Thr-308; however, the kinase that phosphorylates PKB at Ser-473 has not
yet been identified. We first analyzed the activity of PDK-1 in
response to glucose and IGF-1. PDK-1 activity did not respond to
glucose or IGF-1 in INS-1 cells (Fig. 2B). Hence, it appeared that PDK-1 exists in an active state under basal conditions in
pancreatic Similar studies were carried out to determine whether PKB activity was
induced by glucose. However, only IGF-1 stimulated the activation of
PKB, whereas glucose had no significant effect (Fig. 3). This lack of
response to glucose is consistent with the data obtained from INS-1
cells infected with the constitutively active PI3K in which mitogenesis
was only enhanced by IGF-1 and not glucose alone (Fig. 1B).
Interestingly, the results from the time course of IGF-1-enhanced PKB
phosphorylation show that phosphorylation of PKB at Ser-473 increases
at a slower rate compared with PKB Thr-308 phosphorylation (Fig.
3B). In addition, the time course of PKB phosphorylated at
Thr-308 shows a mobility shift to a slower migrating species that
mirrors the increase in PKB phosphorylated at Ser-473 (Fig.
3B). Hence, the slower migrating species could be PKB
phosphorylated at both Thr-308 and Ser-473. As such, these results
suggested that phosphorylation of PKB at Thr-308 was first, followed by
phosphorylation at Ser-473. Intriguingly, the PKB kinase-dead variant,
although inactive (as confirmed by lack of GSK3 A known substrate of PKB is GSK3 Additional proteins that are important in mitogenic signaling are mTOR
and p70S6K. These enzymes are activated, in part, by a
PI3K-dependent pathway and are critical for cell
proliferation (18, 41-43). Although a role for mTOR in mitogenesis has
been implicated, little is known about the molecular mechanisms
involved in regulating the activation of this large molecular mass
protein. The mitogenic activation of p70S6K is also complex
due to a requirement for hierarchical phosphorylation at multiple sites
(31, 44). However, there is substantial evidence that mTOR mediates the
phosphorylation of p70S6K, because rapamycin, which
interacts with and inhibits mTOR, blocks the activation of
p70S6K in response to mitogenic stimuli (45). A number of
reports exist that strongly suggest that p70S6K is
downstream of PKB via the activation of mTOR (17, 18). However, the
phosphorylation activation of p70S6K in pancreatic
It was not too surprising that the kinase-dead form of PKB, which does
not appear to act as a dominant negative protein, had little effect on
-cell proliferation is glucose-dependent; however, the
mechanisms responsible for this glucose dependence are not known.
Adenoviral mediated expression of constitutively active
phosphatidylinositol 3-kinase (PI3K) in the pancreatic
-cells,
INS-1, suggested that PI3K was not necessary for glucose-induced
-cell proliferation but was required for IGF-1-induced mitogenesis.
Examination of the signaling components downstream of PI3K,
3-phosphoinositide-dependent kinase 1, protein
kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70S6K), suggested that a major part of
glucose-dependent
-cell proliferation requires
activation of mammalian target of rapamycin/p70S6K,
independent of phosphoinositide-dependent kinase 1/PKB
activation. Adenoviral expression of the kinase-dead form of PKB in
INS-1 cells decreased IGF-1-induced
-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type
and constitutively active PKB-infected cells. Upon analysis of
extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an
increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1
was observed in kinase-dead PKB-infected cells, but this
phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of
rapamycin/p70S6K signal transduction pathways for a full
commitment to glucose-induced pancreatic
-cell mitogenesis. However,
for IGF-1-induced activation, these pathways must be carefully
balanced, because chronic activation of one (PI3K/PKB) can lead to
dampening of the other (ERK1/2), reducing the mitogenic response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell function (1) and a failure to increase
-cell mass to
compensate for peripheral insulin resistance (2, 3). It is therefore
critical that strategies be developed to replenish the loss of
-cells and/or to expand existing
-cell mass to compensate for
insulin resistance. Such an approach relies on defining the mechanisms
involved in regulating
-cell mitogenic signaling pathways in
response to growth factors and nutrients.
-cell proliferation (4, 5). In addition, these studies showed that
GH and IGF-1 are dependent on glucose at concentrations in the
physiologically relevant range of 6-18 mM to increase
-cell mitogenesis. However, the basis of this glucose dependence of
-cell proliferation remains unknown.
-cells. Although glucose has no
independent effect on the activation of this pathway, it is nonetheless
required for GH mitogenic action (4, 6). There appears to be no
cross-talk of janus kinase-2 activation to insulin receptor substrate
(IRS)-mediated signaling by GH. However, activation of
phosphatidylinositol 3-kinase (PI3K) mediated by glucose is at least
partially required for full GH-induced
-cell proliferation.
and
(GSK3
/
) have been shown to be two such targets, and their
phosphorylation on Ser-21 and Ser-9, respectively, negatively regulates
GSK3
/
activity (16). PKB has also been implicated in catalyzing
phosphorylation activation of mammalian target of rapamycin (mTOR),
which then mediates the phosphorylation of p70-kDa-S6-kinase (p70S6K) (17, 18). In
-cells, glucose and IGF-1
activation of p70S6K via the IRS-mediated signal
transduction pathway is dependent on PI3K. However, the important
signaling components involved in the regulation of mitogenesis that are
downstream of PI3K, such as PDK-1, PKB, mTOR, and GSK3
/
, have not
been characterized in
-cells, even though it has been shown that
p70S6K is activated by glucose and IGF-1 (4, 5). Therefore,
in this study we set out to characterize the signaling elements
downstream of PI3K that are regulated by glucose and IGF-1 and found
that PKB and p70S6K are differentially activated. These
data suggested that a major part of the glucose dependence of
-cell
proliferation is via a direct activation of mTOR/p70S6K,
independent of PDK-1/PKB activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
(Ser-21/9), total PKB, phosphoThr-308-PKB,
phosphoSer-473-PKB antibodies, the GSK3 fusion protein, and the PKB
kinase assay kit were from New England Biolabs Inc. (Beverly, MA).
Anti-phospho-ERK1/ERK2 was obtained from Promega Corporation (Madison,
WI), and the total ERK1/ERK2 antiserum was a gift from Dr. M. Cobb
(University of Texas Southwestern Medical Center, Dallas, TX). The
p70S6K antisera were generated as described (19).
Monoclonal anti-c-Myc clone 9E10 was obtained from
Sigma. The total GSK3
/
antibody was from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Anti-rabbit and anti-sheep IgG
horseradish peroxidase conjugates were from JacksonImmuno Research
(West Grove, PA), and the anti-mouse IgG horseradish peroxidase
conjugate was from Upstate Biotechnology Inc. Wortmannin and rapamycin
were from Calbiochem-Novabiochem. IGF-1 was purchased from Gro Pep Pty
Ltd. (Adelaide, Australia). The pUSEamp expression vectors containing
Myc-His-tagged mouse PKB
(wild type), myr-PKB
(constitutively
active), and PKB
-K179M (kinase-dead) were from Upstate Biotechnology
Inc. DNA purification kits and Superfect transfection reagents were
purchased from Qiagen (Valencia, CA). Restriction enzymes were from New
England Biolabs Inc. The bicinchoninic acid protein assay kit was
purchased from Pierce. The
[methyl-3H]thymidine and chemiluminescence
reagent was from PerkinElmer Life Sciences. All other reagents
were of analytical grade from either Sigma or Fisher.
-cell line
INS-1 (20) was maintained in the complete medium RPMI 1640 (11.2 mM glucose) containing 10% (v/v) fetal calf serum, 50 µm
-mercaptoethanol, 10 mM HEPES, 2 mM
glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin,
100 µg/ml streptomycin and incubated at 37 °C in 5% CO2 as described (20).
80 °C.
/
(Ser-21/9) antibody.
subunit (AdV-p110) were constructed as described (22). The
pUSEamp expression vectors containing Myc-His-tagged mouse PKB
(wild
type), myr-PKB
(constitutively active), and PKB
-K179M
(kinase-dead) were digested with the restriction enzymes
HindIII and Pme1. Each of the cDNAs was
inserted between the HindIII and SmaI sites of
pBluescript, providing the necessary restriction sites for insertion
into pAdTrack-CMV between KpnI and NotI. The PKB
adenoviruses were generated and purified as described (23, 24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cell Proliferation--
It has previously been shown
that activation of PI3K is required for glucose and IGF-1-induced
-cell proliferation (4, 5). As such, it was examined whether
bypassing the requirement for PI3K activation by an adenoviral mediated
constitutively active form of PI3K would unveil the extent of the
requirement for this enzyme activity on glucose and IGF-1-induced
-cell proliferation. Previous studies have shown that adenoviral
expression of the iSH2 of the p85
subunit with the
catalytic p110
subunit (p110) results in constitutive activation of
PI3K (22). Because of the presence of a Myc tag on each of the
subunits, INS-1 cells infected with recombinant adenovirus to express
iSH2 (AdV-iSH2), p110 (AdV-p110), or
iSH2 + p110 (AdV-p110 + AdV-iSH2) could be confirmed by subjecting the lysates to immunoblot analysis with the
anti-Myc antibody (Fig. 1A).
Bands at 35 and 115 kDa correspond to the predicted molecular
mass of Myc-tagged iSH2 and p110, respectively, whereas the band at 40 kDa is nonspecific (as indicated by its presence
in uninfected and AdV-
-Gal-infected control cells). The
-cell
mitogenesis was determined in INS-1 cells infected with the control
-Gal (AdV-
Gal), AdV-iSH2, AdV-p110, or AdV-p110 + AdV-iSH2. The uninfected and AdV-
Gal controls showed
similar increases in [3H]thymidine incorporation in
response to 3 or 15 mM glucose ± 10 nM
IGF-1 (Fig. 1B), as observed previously (4, 5). For uninfected INS-1 cells at 15 mM glucose,
-cell
mitogenesis was increased 19-fold (p < 0.01), and in
the additional presence of 10 nM IGF-1 was 48-fold
(p < 0.005) above the 0.5 mM glucose
control (Fig. 1B). In AdV-p110-infected INS-1 cells
-cell
mitogenesis in response to glucose and IGF-1 was not significantly
different from the uninfected control. In addition, in
AdV-iSH2-infected INS-1 cells glucose-induced
-cell
mitogenesis was similar to the uninfected control; however, that at 15 mM glucose + 10 nM IGF-1 was elevated to
68.2 ± 8.4-fold (n = 3) above 0.5 mM
glucose (Fig. 1B; p < 0.02). In
AdV-p110/AdV-iSH2-coinfected cells glucose-induced
-cell
mitogenesis was not significantly altered compared with that in
uninfected or AdV-
Gal controls in INS-1 cells (Fig. 1B). However, IGF-1-induced
-cell mitogenesis in
AdV-p110/AdV-iSH2 coinfected INS-1 cells was significantly
increased 22.2 ± 0.5-fold (n = 3;
p < 0.01) at basal 3 mM glucose and
126.5 ± 12.9-fold (n = 3; p < 0.01) at a stimulatory 15 mM glucose above 0.5 mM glucose control (Fig. 1B).
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Fig. 1.
[3H]Thymidine incorporation
assays in INS-1 cells overexpressing PI3K. INS-1 cells were
cultured on 6-well plates and infected with adenoviruses
AdV- -Gal, AdV-p110, AdV-iSH2, AdV-p110 + AdV-iSH2 (using an equivalent m.o.i. in each well) or
uninfected. A, cell lysates were prepared as described under
"Experimental Procedures" and subjected to immunoblot
(IB) analysis with the c-Myc antibody. B,
[3H]thymidine incorporation assays were carried out as
described under "Experimental Procedures" with 3 or 15 mM glucose ± 10 nM IGF-1 and 0.5 mM glucose as control.
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Fig. 2.
Analysis of PDK-1 kinase activity in INS-1
cells stimulated with glucose and/or IGF-1. Cell lysates were
prepared from INS-1 cells subjected to 24-h serum and glucose
deprivation (see "Experimental Procedures") prior to stimulation
with 3 or 15 mM glucose ± 10 nM IGF-1 for
the times indicated. PDK-1 was immunoprecipitated and assayed for
kinase activity as described under "Experimental Procedures." As an
indication of PDK-1 kinase activity, the phosphorylation of GSK3 /
was measured by immunoblot (IB) using phospho-GSK3
/
(Ser-21/9) antibody (A). For the controls, cells were
stimulated with 15 mM glucose + 10 nM IGF-1,
and either SGK was omitted from the assay or sheep IgG was used in
place of the anti-PDK-1 kinase sheep polyclonal antibody. The
total cell lysates were immunoblotted for PDK-1 (B). The
results shown are representative of three independent
experiments.
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Fig. 3.
The effects of glucose and IGF-1 on PKB
activity in INS-1 cells. Cell lysates were prepared from INS-1
cells subjected to 24-h serum and glucose deprivation (see
"Experimental Procedures") prior to stimulation with 3 or 15 mM glucose ± 10 nM IGF-1 for the times
indicated and 0.5 mM glucose without IGF-1 as control. The
level of GSK3 /
phosphorylation was used to measure the PKB
activity of each sample (as described under "Experimental
Procedures"), and an immunoblot (IB) using
phospho-GSK3
/
(Ser-21/9) antibody is shown in A. Cell
lysates were subjected to immunoblot analysis as described under
"Experimental Procedures" using phospho-Thr-308-PKB,
phospho-Ser-473-PKB, and total PKB antibodies (B). The
results shown are representative of three independent
experiments.
/
has been shown to be a phosphorylation substrate of PKB,
where phosphorylation of GSK3
/
by PKB inhibits GSK3
/
activity (26). It was determined whether glucose and/or IGF-1 could
influence endogenous GSK3
/
phosphorylation in
-cells (Fig.
4). INS-1 cells were treated at basal 3 mM glucose or stimulatory 15 mM glucose ± 10 nM IGF-1 for 2, 5, 10, 20, and 40 min and then subjected
to immunoblot analysis with the phospho-GSK3
/
antibody or
total-GSK3
/
antibody (Fig. 4). Neither 3 or 15 mM
glucose had any significant effect on GSK3
/
phosphorylation over
the 40-min time course (Fig. 4), correlating with a lack of effect of
glucose on PKB activity (Fig. 3A). In contrast, IGF-1
rapidly and markedly increased GSK3
/
phosphorylation within 2 min
that was sustained throughout the 40-min time course and was
independent of the glucose activation (Fig. 4). This GSK3
/
phosphorylation pattern correlated with IGF-1-induced PKB activity and
phosphorylation (Fig. 3, A and B). The total
amount of GSK3
/
did not alter with glucose/IGF-1 treatment (Fig.
4).
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Fig. 4.
The effects of glucose and IGF-1 on
GSK3 /
phosphorylation
in INS-1 cells. Cell lysates were prepared from INS-1 cells
subjected to 24-h serum and glucose deprivation (see "Experimental
Procedures") prior to stimulation with 3 or 15 mM
glucose ± 10 nM IGF-1 for the times indicated and 0.5 mM glucose without IGF-1 as control. The level of
GSK3
/
phosphorylation was measured by subjecting cell lysates to
immunoblot (IB) analysis as described under "Experimental
Procedures" using phospho-GSK3
/
(Ser-21/9) antibody
(A). The total levels of GSK3
/
were measured by
immunoblot with the total GSK3
/
antibody (B). The
results shown are representative of three independent
experiments.
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Fig. 5.
The time-dependent
phosphorylation of p70s6K in INS-1 cells by glucose and
IGF-1. Cell lysates were prepared from INS-1 cells subjected to
24-h serum and glucose deprivation (see "Experimental Procedures")
prior to stimulation with 3 or 15 mM glucose ± 10 nM IGF-1 for the times indicated and 0.5 mM
glucose without IGF-1 as control. The cell lysates were immunoblotted
(IB) with p70s6K antibody as described under
"Experimental Procedures" with 10% milk in place of 5% milk and
5% bovine serum albumin for blocking and antibody dilution buffers.
The results shown are representative of three independent
experiments.
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Fig. 6.
The effect of PI3K and mTOR inhibition on
various components of the signaling pathway in INS-1 cells. INS-1
cells subjected to 24-h serum and glucose deprivation were pre-treated
for 15 min with 100 nM wortmannin or 25 nM
rapamycin or without inhibitor. This was followed by stimulation with 3 or 15 mM glucose ± 10 nM IGF-1 in the
presence of 100 nM wortmannin or 25 nM
rapamycin or without inhibitor for 40 min and 0.5 mM
glucose without IGF-1 or inhibitor as control. Cell lysates were
subjected to immunoblot (IB) analysis as described under
"Experimental Procedures" using phospho-Thr-308-PKB,
phospho-Ser-473-PKB, and total-PKB antibodies (A);
phospho-ERK 1/ERK2 and total ERK 1/ERK2 antibodies (B); and
p70s6K antibody (C). The results shown are
representative of three independent experiments.
-cell mitogenesis. Recombinant adenoviruses
were generated to express three Myc/His-tagged constructs of PKB,
consisting of a wild type (AdV-PKB-WT), a constitutively active
(AdV-PKB-CA), and a kinase-dead form (AdV-PKB-KD), in INS-1 cells. The
constitutively active PKB was created by fusing c-Src-derived residues, required for myristoylation, to PKB. This additional sequence
directly targets PKB to the plasma membrane, leading to its
constitutive activation (35). The kinase-dead form has a point
mutation, K179M, removing the ATP-binding site, which results in a loss
of kinase activity (36).
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Fig. 7.
Overexpression of AdV-PKB wild type,
activated, and kinase-dead forms in INS-1
cells. INS-1 cells were infected with the following adenoviruses:
AdV-PKB wild type (AdV-WT), AdV-PKB constitutively active (AdV-CA),
AdV-PKB kinase-dead (AdV-KD), and the control adenovirus-green
fluorescent protein (AdV-GFP). A multiplicity of infection
(MOI) from 0.5 to 20 × 102 was
used as described under "Experimental Procedures." After the 16-h
incubation in complete medium, cell lysates were subjected to
immunoblot (IB) analysis as described under "Experimental
Procedures" using the total PKB antibody.
0.05; Fig. 8). The 15 mM
glucose-induced increase in INS-1 cell mitogenesis was 24.4 ± 1.3-fold (n = 3) above the 0.5 mM glucose
background and 52.2 ± 6.4-fold (n = 3) higher in
the added presence of IGF-1 (p
0.05; Fig. 8). These results are consistent with previous findings (4, 5). Control AdV-Luc-infected INS-1 cells were comparable with the uninfected cells
in their proliferative response to glucose/IGF-1, indicating no adverse
effects of the adenovirus vector per se (Fig. 8).
AdV-PKB-WT, AdV-PKB-CA, and AdV-PKB-KD all increased basal 3 mM glucose 2-3-fold above uninfected control, but there
was no significant effect with 10 nM IGF-1 at 3 mM glucose. In addition, although the PKB-AdVs did not
significantly alter
-cell mitogenesis at 15 mM glucose compared with the uninfected control, at 15 mM
glucose + 10 nM IGF-1, the mitogenic response significantly
decreased by 37.0, 53.8, and 41.4% for AdV-PKB-WT, AdV-PKB-CA, and
AdV-PKB-KD respectively, compared with the uninfected control
(p
0.05; Fig. 8).
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Fig. 8.
[3H]Thymidine incorporation
assays in INS-1 cells overexpressing PKB wild type, activated, and
kinase-dead forms. INS-1 cells were cultured on 6-well
plates and infected with adenovirus-luciferase (AdV-Luc), AdV-PKB-WT,
AdV-PKB-CA, or AdV-PKB-KD or uninfected, and
[3H]thymidine incorporation assays were carried out as
described under "Experimental Procedures" with 3 or 15 mM glucose ± 10 nM IGF-1 and 0.5 mM glucose as control.
/
, ERK1/ERK2, and p70S6K--
Our data indicated
that PKB activation was involved in IGF-1-induced
-cell
proliferation but not necessarily that by glucose (Figs. 3-6). As
such, one might have predicted that overexpression of AdV-PKB-KD would
inhibit IGF-1-induced
-cell mitogenesis at 15 mM glucose
(as observed, Fig. 8), but it was surprising that overexpression of
AdV-PKB-WT and especially AdV-PKB-CA also inhibited IGF-1-induced INS-1
cell proliferation at 15 mM glucose (Fig. 8). Hence, we
investigated the activation of elements in the IGF-1 signal
transduction pathway in PKB-overexpressing cells (Fig. 9). INS-1 cells were infected with
AdV-PKB-WT, AdV-PKB-CA, AdV-PKB-KD, or AdV-GFP as a control adenovirus,
made quiescent, and then incubated with 3 or 15 mM
glucose ± 10 nM IGF-1 for 40 min. Immunoblot analysis with total PKB showed similar levels of adenoviral expressed PKB protein, which was much higher than the endogenous expressed PKB (Fig.
9A). In the AdV-GFP-infected cells, PKB phosphorylation was
only detected on Thr-308 and Ser-473 residues when stimulated with 10 nM IGF-1 at 3 mM glucose or stimulatory
15 mM glucose (Fig. 9A), as previously seen
(Fig. 3). In AdV-PKB-WT-infected cells, the level of Thr-308 and
Ser-473 phosphorylation, although slightly lower for the endogenous PKB
compared with the AdV-GFP-infected control cells, was much greater for
the adenoviral wild type-expressed PKB (Fig. 9A). In the
AdV-PKB-WT-infected cells there was increased basal phosphorylation,
which was induced by IGF-1 on both Thr-308 and Ser-473 residues of PKB,
independently of glucose, and this was in parallel with the endogenous
PKB (Fig. 9A). In AdV-PKB-CA-infected INS-1 cells the level
of phosphorylated adenovirally expressed PKB on both Thr-308 and
Ser-473 residues was maximal under all conditions tested, as expected
for the constitutively active variant of PKB, compared with wild type
or kinase-dead PKB variants, and dampened the phosphorylation
activation of endogenous PKB by IGF-1 (Fig. 9A). In
AdV-PKB-KD-infected INS-1 cells, IGF-1-induced phosphorylation of
Thr-308 and Ser-473 residues of endogenous PKB was similar to control
AdV-GFP-infected
-cells. However, the levels of
phosphorylation on Ser-473 of the adenovirally expressed kinase-dead
PKB were markedly reduced under all conditions tested (Fig.
9A). In contrast, Thr-308 phosphorylation of the adenoviral
expressed kinase-dead PKB was increased under basal conditions and
stimulated by IGF-1 in a glucose-independent manner (Fig.
9A).
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Fig. 9.
Effect of PKB overexpression of
glucose/IGF-1-induced activation of PKB,
GSK3 /
,
mitogen-activated protein kinase, and p70S6K. INS-1
cells were infected (as described under "Experimental Procedures")
at an m.o.i. of 10 × 102 with the following
adenoviruses: AdV-PKB-WT, AdV-PKB-CA, AdV-PKB-KD, and the control
AdV-GFP. Following the 24-h serum and glucose deprivation described
under "Experimental Procedures," the cells were stimulated with 3 or 15 mM glucose ± 10 nM IGF-1 for 40 min
and 0.5 mM glucose without IGF-1 as control. Cell lysates
were subjected to immunoblot (IB) analysis as described
under "Experimental Procedures" using phospho-Thr-308-PKB,
phospho-Ser-473-PKB, and total PKB antibodies (A),
phospho-GSK3
/
(Ser-21/9) and total GSK3
/
antibodies
(B), phospho-ERK 1/2 and total ERK 1/2 antibodies
(C), or p70s6K antibody (D). The
results shown are representative of three independent
experiments.
/
is a downstream phosphorylation substrate of PKB, and as
such its phosphorylation should parallel that of PKB Thr-308/Ser-473 phosphorylation activation (26). In AdV-GFP-infected control INS-1
cells endogenous GSK3
/
phosphorylation was increased by IGF-1 in
a glucose-independent manner (Fig. 9B) that paralleled PKB
Thr-308/Ser-473 phosphorylation activation (Fig. 9A). In
AdV-PKB-WT-infected cells basal phosphorylation of GSK3
/
was
raised and further increased in response to IGF-1 (Fig. 9B),
in parallel with PKB phosphorylation (Fig. 9A). In
AdV-PKB-CA-infected INS-1 cells GSK3
/
phosphorylation was
maximal, irrespective of the glucose/IGF-1 conditions (Fig.
9B), correlating with constitutive activation of PKB in
these cells (Fig. 9A). In AdV-PKB-KD-infected INS-1 cells, phosphorylation of GSK3
/
was comparable with that in AdV-GFP-infected cells and as such was likely to be due to endogenous PKB activity. This is consistent with previous results, which showed
that the kinase-dead variant of PKB is not a dominant negative protein
(15). Total levels of GSK3
/
were equivalent in all samples
analyzed (Fig. 9B).
-cells. In contrast, ERK1/ERK2 phosphorylation
activation by 15 mM glucose ± IGF-1 was not inhibited
in AdV-PKB-KD-infected
-cells but, if anything, was enhanced
(Fig. 9C). Total ERK1/ERK2 levels were equivalent in all
samples examined (Fig. 9C). The inhibition of glucose ± IGF-1 ERK1/ERK2 phosphorylation in the presence of constitutively
active PKB strongly suggests that there is cross-talk between the
PI3K/PKB and ERK1/ERK2 signaling pathways.
-cells
(Fig. 9D). These results suggested that constitutive
activation of PKB did not overly affect glucose/IGF-1-induced phosphorylation activation of p70S6K.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell mitogenesis (4, 5) but that glucose induces the activity of PI3K (37). Hence, to gain more insight
into the mechanisms involved in glucose-dependent IGF-1
-cell mitogenesis, we studied the effects of overexpressing
constitutively active PI3K by infecting INS-1 cells with AdV-p110 and
AdV-iSH2. However, because AdV-p110- and
AdV-iSH2-infected INS-1 cells only showed IGF-1-enhanced
-cell mitogenesis (Fig. 1B), it suggested that the
glucose-dependent signaling events must lie elsewhere. This
led us to focus on events downstream of PI3K.
-cells and does not appear to be stimulated in response
to glucose or IGF-1. This is consistent with reports in other cells
that showed that PDK-1 is constitutively active in vivo (38,
39). In addition, the expression of AdV-PKB-CA, which is constitutively
active because of a membrane-targeting motif attached to the
amino terminus, in INS-1 cells is sufficient to
localize PKB to the membrane, resulting in phosphorylation of the
Thr-308 and Ser-473 residues in unstimulated cells (Fig. 9A). These results suggest that there is a significant
amount of active PDK-1 present at the membrane of unstimulated cells. Therefore, although PDK-1 is an important component of the PI3K pathway, it does not appear to be a key regulatory component of the
glucose or IGF-1 mitogenic response in pancreatic
-cells.
/
phosphorylation), was still phosphorylated at Thr-308 in a manner
regulated by IGF-1. However, lack of phosphorylation at Ser-473 of the
kinase-dead PKB suggested that Ser-473 phosphorylation of PKB might
well be an autophosphorylation event. If so, the slight amount of
IGF-1-induced kinase-dead PKB Ser-473 phosphorylation observed was
probably due to endogenous PKB activity (Fig. 9A). These
data are consistent with a recent report suggesting that Thr-308 is
phosphorylated first, followed by autophosphorylation of the Ser-473
residue of PKB (40).
/
, a protein kinase implicated in
several biological and metabolic processes (26). GSK3
/
phosphorylation by PKB leads to down-regulation of its activity. However, only IGF-1 stimulated GSK3
/
phosphorylation, whereas glucose had no effect (Figs. 4 and 9B). This lack of
response to glucose and the stimulation of GSK3
/
phosphorylation
by IGF-1 correlated with the activation of PKB (Fig. 4). Hence, these
results suggest that GSK3
/
is activated by IGF-1 in a
glucose-independent manner. However, it is not clear what lies
downstream of IGF-1-mediated GSK3
/
inhibition in
-cells and if
it relates to the control of
-cell mitogenesis. Hence the potential
biological role of GSK3
/
in
-cells awaits future experimental investigation.
-cells did not correlate with the activation of PKB. In this regard,
the activation of p70S6K was much slower compared with PKB
activation (Fig. 5 versus Fig. 3), and whereas glucose
stimulated p70S6K activation (Figs. 5 and 9), glucose had
no effect on upstream control of PKB activity (Fig. 3). In addition,
p70S6K phosphorylation activation by glucose was not
inhibited by the PI3K-specific inhibitor, wortmannin, in contrast to an
apparent complete inhibition of IGF-1-induced p70S6K and
PKB phosphorylation activation by wortmannin (Fig. 6). In addition,
overexpression of AdV-PKB-KD did not appear to change the activation of
p70S6K in response to IGF-1 and/or glucose compared with
the AdV-PKB-WT (Fig. 9D), even though complete
phosphorylation activation of AdV-PKB-KD did not occur (Fig.
9A). However, the mTOR-specific inhibitor, rapamycin,
completely abolished p70S6K activation phosphorylation by
IGF-1 and glucose (Fig. 6D). Hence, taken together, these
results strongly support a model in which p70S6K activation
by glucose is via mTOR, bypassing a requirement for PKB activation in
-cells. This is consistent with other reports that have shown
p70S6K activation via mTOR, independent of PKB, by other
nutrient sources such as branched chain amino acids (27, 28, 30). In
addition, studies in yeast suggest that mTOR is involved in regulating
cellular responses to nutrient availability (46). In contrast, IGF-1 activated the PI3K/PKB/mTOR signaling pathway. Therefore the mitogenic stimulation by glucose is, at least in part, PKB-independent, and mTOR
may mediate the glucose-dependent aspect of the stimulated phosphorylation of p70S6K. Hence, mTOR may be one major
component regulating glucose-dependent
-cell mitogenesis
in addition to the glucose activation of ERK1/ERK2 (34). Although it is
clear that glucose metabolism is required, the secondary signals
that activate kinases to induce the activation of signaling proteins,
such as mTOR and ERK1/ERK2, have yet to be identified. One possible
candidate is Ca2+, because the inhibition of
glucose-induced Ca2+ levels in
-cells prevents the
phosphorylation of ERK1/ERK2, suggesting that ERK1/ERK2 phosphorylation
is in part regulated by a Ca2+-dependent
protein kinase (34).
-cell mitogenesis, due to compensation by endogenous PKB (Fig. 8).
However, it was unexpected that overexpression of the wild type or
constitutively active forms of PKB did not increase
-cell
mitogenesis. The phosphorylation activation of p70S6K by
glucose and IGF-1 was not enhanced by overexpression of the wild type
or constitutively active forms of PKB (Fig. 9D), reaffirming the importance of glucose activation bypassing PKB and acting directly
through mTOR activation (as discussed above). Surprisingly, the
constitutively active PKB inhibited the IGF-1 and/or glucose phosphorylation activation of ERK1/ERK2 (Fig. 9C). One
possible mechanism by which this may occur is through inhibition of
Raf1 activity, because studies have shown that this enzyme is
negatively regulated by PKB phosphorylation (47, 48). Hence, these PKB inhibitory effects on the ERK1/ERK2 pathway probably contribute to the
decrease in
-cell mitogenesis observed in IGF-1-treated cells
expressing the constitutively active or wild type forms of PKB.
Interestingly, the ERK1/ERK2 phosphorylation levels in IGF-1- and/or
glucose-stimulated INS-1 cells expressing the kinase-dead form of PKB
were slightly higher compared with those in control-infected cells
(Fig. 9C). Therefore, although PKB activity is diminished in
AdV-PKB-KD-expressing INS-1 cells, the ERK1/ERK2 signaling pathway
appears to be more active. These observations of adenovirus-mediated PKB overexpression in
-cells strongly suggest that there is a requirement for both the PI3K and ERK1/ERK2 signaling pathways for
glucose-dependent IGF-1-induced
-cell mitogenesis.
Moreover, the data imply that there is a tightly controlled
balance between PKB and ERK1/ERK2 signaling pathway elements, both
kinetically and of their expression levels; otherwise, negative
cross-talk between the pathways would likely dampen signal
transduction. It should also be considered that other targets
downstream of PKB, such as BAD, caspase-9, and forkhead transcription
factors (reviewed in Ref. 49), which affect the mitogenesis and/or
survival of
-cells, may contribute to control of
-cell mass and
warrant further investigation. These elements downstream of PKB may be important factors that play a major role in the pathogenesis of type 2 diabetes if adversely regulated by prolonged hyperglycemia and/or
hyperlipidemia, which in turn could contribute to the failure of
-cell expansion. Alternatively, if PKB downstream elements can be
carefully controlled pharmacologically, this may provide a means of
preserving or even enhancing
-cell mass as a potential therapy for
both type 1 and 2 diabetes (2).
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Cynthia Jacobs in the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK 55267 (to C. J. R.) and RO1 43051 (to B. B. K.) and by the Juvenile Diabetes Foundation International (to C. J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Medizinische Klinik I, Universität Regensburg, F. J. Strauss-Allee, 93042 Regensburg, Germany.
** To whom correspondence and reprint requests should be addressed: Pacific Northwest Research Inst., 720 Broadway, Seattle, WA 98122. Tel.: 206-860-6777; Fax: 206-726-1202; E-mail: cjr@pnri.org.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M101257200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GH, growth hormone;
IGF-1, insulin-like growth factor 1;
IRS, insulin
receptor substrate;
PI3K, phosphatidylinositol 3-kinase;
SH2, Shc
homology 2;
ERK, extracellular signal-regulated protein kinase;
PIP3, phosphatidylinositol 3,4,5-trisphosphate;
PKB, protein kinase B;
PIP2, phosphatidylinositol
3,4-diphosphate;
PDK-1, 3-phosphoinositide-dependent
kinase 1;
GSK3, glycogen synthase kinase-3;
mTOR, mammalian target of
rapamycin;
p70S6K, p70-kDa-S6-kinase;
SGK, serum- and
glucorticoid-regulated protein kinase;
iSH2, inter SH2 of
p85;
m.o.i., multiplicity of infection;
AdV, adenovirus;
WT, wild
type;
CA, constitutively active;
KD, kinase-dead;
GFP, green
fluorescent protein;
Luc, luciferase.
![]() |
REFERENCES |
---|
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