From the Departments of Internal Medicine and Pharmacology, Gifford
Laboratories for Diabetes Research, University of Texas Southwestern
Medical Center, Dallas, Texas 75235-8854 and the
Research
Division, Joslin Diabetes Center, Harvard Medical School,
Boston, Massachusetts 02215
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INTRODUCTION |
Adult pancreatic
-cells are relatively well differentiated and
consequently have a low mitotic index (1, 2). Under normal
circumstances the proportion of
-cells undergoing mitosis is about
0.5% of the population of
-cells in a pancreatic islet (1).
Nonetheless,
-cell proliferation can be increased by several
nutrient factors, such as glucose and amino acids (2). Glucose
increases mitogenesis in islet
-cells so that around 5% of the
-cell population are undergoing DNA synthesis (2-4), for which
glucose metabolism is required (3). However, the intracellular
signaling pathways responsible for glucose-mediated
-cell
proliferation, beyond a requirement for glucose metabolism, are not
particularly well understood. Nonetheless, it has been postulated that
this may be via elevation of intracellular cAMP (2, 5),
glucose-mediated activated protein kinase C (6), and/or a
Ca2+-dependent activation of the
mitogen-activated protein kinase (MAPK)1 (7, 8).
Pancreatic
-cell proliferation can also be stimulated by several
growth factors (2, 9). In particular, somatolactogenic hormones
(prolactin and growth hormone (GH)) and insulin-like growth factor I
(IGF-I) have been shown to increase the number of replicating
-cells
in rodent islets by up to 6% of the islet cell population (2, 10, 11).
A prolactin-induced increase in
-cell proliferation is likely
associated with an increase in
-cell mass observed during pregnancy
(12). GH is perhaps the most potent of peptide growth factors to induce
proliferation of differentiated
-cells (10, 13). It has been
postulated that GH mediates
-cell growth via local IGF-I production
(14), however, it is more likely that GH mediates a direct effect on
-cell proliferation (15) probably via a JAK2/STAT5 signal
transduction pathway (16). Likewise, IGF-I stimulates
-cell
proliferation independently of GH or prolactin (17), most probably via
the IGF-I receptor and subsequent protein tyrosine phosphorylation signal transduction pathway found in other mammalian cell types (17-20). IGF-I binds to the IGF-I-receptor resulting in activation of
its intrinsic tyrosine kinase activity that in turn tyrosine phosphorylates members of the insulin receptor substrate (IRS) family
(18-20). Tyrosine-phosphorylated IRS is then able to recruit the
85-kDa regulatory subunit of phosphatidylinositol 3'-kinase (PI
3'-kinase) via its SH2 domain, leading to activation of the enzyme
(17-19). This then leads to PI 3'-kinase-dependent
downstream activation of the 70-kDa S6 kinase (p70S6K)
(21). IGF-I-mediated tyrosine phosphorylation of IRS also engages the
bridging molecule growth factor receptor-bound protein-2 (Grb-2) via
its SH2 domain to phosphotyrosine site on IRS (17-19). This results in
an increased binding of IRS-docked Grb-2 to the murine Son-of-Sevenless
1 protein (mSOS), a guanine nucleotide exchange factor which converts
inactive Ras-GDP into active Ras-GTP (17-19). Activated GTP-bound Ras
then recruits the Raf serine kinase that phosphorylates MAP kinase
kinase (MEK), resulting in MEK-mediated phosphorylation activation of
the MAPK (erk-1 and -2 isoforms) (18, 19, 22). Furthermore, IGF can
also activate the Ras/MAPK branch of the pathway independently of IRS,
via IGF-I receptor kinase tyrosine phosphorylation of the
SH2-containing protein (Shc) which then directly binds Grb-2/mSOS
resulting activation of Ras/MAPK (17-20). Notwithstanding, by which
ever signal transduction pathway it is mediated, activation of MAPK and
p70S6K are known to be a requirement for induction of a
mitogenesis in most mammalian cell types (17-20).
In the pancreatic
-cell, although several peptide growth factors and
nutrients have been shown to increase
-cell proliferation (2), the
intracellular signal transduction pathway(s) involved in induction of
-cell mitogenesis have not been well defined. Moreover, the
regulation of
-cell growth may actually be uniquely different from
other cell types, since
-cell function is exquisitely related to its
metabolic state (23). In these studies we have used the
-cell line
INS-1 as a model to better characterize glucose and IGF-I-mediated
signal transduction pathways that specially influence
-cell
proliferation. Compared with other
-cell lines, despite a higher
mitotic index than primary
-cells, INS-1 cells are relatively well
differentiated and respond to glucose in terms of insulin secretion in
a physiologically relevant glucose concentration range (24).
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EXPERIMENTAL PROCEDURES |
Materials--
The [methyl-3H]thymidine
(20 Ci/mmol) was from NEN Life Science Products Inc. (Boston, MA).
Anti-"active-MAPK" antiserum was purchased from Promega Corp.
(Madison, WI), the "total MAPK" antiserum (erk1/erk2) was a gift
from Dr. M. Cobb (University of Texas Southwestern Medical Center,
Dallas, TX), and the IRS-1 and IRS-2 antisera were generated as
described previously (20). All other antisera were purchased from
Upstate Biotechnology (Lake Placid, NY). Transblot nitrocellulose
membrane (0.45 µm pore size) was from Bio-Rad, immunoblot
chemiluminescence detection kit from NEN Life Science Products. IGF-I
and protein kinase/phosphatase inhibitors were purchased from
Calbiochem-Novabiochem (La Jolla, CA). The
(Rp)-2'-O-monobutyryl-cAMP and
(Sp)-2'-O-monobutyryl-cAMP were from Biolog Life
Sciences Institute (La Jolla, CA). All the other biochemicals were
purchased from either Sigma or Fisher Scientific (Pittsburgh, PA) and
were of the highest purity available.
Cell Culture--
The glucose-sensitive pancreatic
-cell
line, INS-1 (24), was used in the experiments. INS-1 cells were
maintained in RPMI 1640 medium containing 2 mM
L-glutamine, 1 mM sodium pyruvate, 50 µM
-mercaptoethanol, 100 units/ml penicillin, 100 µg/ml streptomycin, 10% fetal calf serum, and 11.2 mM
glucose, and incubated at 37 °C, 5% CO2 as described
(24). Cells were subcultured at 80% confluence.
[3H]Thymidine Incorporation--
Incorporation of
[3H]thymidine was used as an indicator of DNA synthesis
and INS-1 cell proliferation (7, 25). INS-1 cells were cultured on
96-well plates (105 cells/well) and incubated for 2 days at
37 °C in INS-1 medium. The medium was removed and the cells made
quiescent by serum and glucose deprivation for 24 h in RPMI 1640 containing 0.1% BSA instead of serum and no glucose. The INS-1 cells
were then incubated for a further 24 h in RPMI 1640, 0.1% BSA at
different glucose concentrations (0-24 mM glucose) with or
without IGF-I (0.1-100 nM), ± various inhibitors. The
last 4 h of this latter incubation period was carried out in the
additional presence of 5 µCi/ml [3H]thymidine to
monitor the degree of DNA synthesis and gain an assessment of the
-cell proliferation rate. After this final incubation period, the
cells were collected and lysed using a semi-automatic cell harvester
(Cambridge Technology Inc.) and the cell lysates transferred to
Whatmann glass fiber micropore filters. The [3H]thymidine
specifically incorporated into the INS-1 cell DNA trapped on glass
fiber filters was counted by liquid scintillation counting.
Protein Immunoblot and Co-Immunoprecipitation
Analysis--
INS-1 cells were subcultured on 10-cm plates to about
50% confluence as described previously (24). The cells were then
subjected to a 24-h period of quiescence by serum and glucose
deprivation in RPMI 1640 medium containing 0.1% BSA instead of serum
and no glucose. After the quiescent period, INS-1 cells were then
incubated in fresh RPMI 1640 medium containing 0, 3, 6, 9, or 18 mM glucose ± 10 nM IGF-I for between 5 and 60 min as indicated. The cells were then lysed in 0.5 ml of
ice-cold lysis buffer consisting of 50 mM Hepes (pH 7.5),
1% (v/v) Nonidet P-40, 2 mM sodium vanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate,
4 mM EDTA, 10 µM leupeptin, 10 µg/ml
aprotonin, and 100 µM phenylmethylsulfonyl fluoride.
Immunoblot analysis of mitogenic signal transduction protein expression
and protein tyrosine phosphorylation was as described previously, using
horseradish peroxidase based chemiluminescence reaction as a secondary
detection method (21, 26). Examination of stimulated protein-protein
interactions between mitogenic signal transduction pathway proteins was
by co-immunoprecipitation analysis as described previously (21, 26).
For immunoblot analysis, 50-75 µg of INS-1 cell total protein lysate
was used, and for immunoprecipitation 750 µg of INS-1 cell total
protein.
Other Procedures--
Protein assay was by the bicinchoninic
acid method (Pierce, Rockford, IL). Data are presented as a mean ± S.E. Statistically significant differences between groups were
analyzed using Student's t test, where p < 0.05 was considered statistically significant.
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RESULTS |
IGF-I Only Stimulates INS-1 Cell Proliferation in the Presence of
Physiological Glucose Concentrations--
The effect of glucose
(0.1-24 mM) ± IGF-I (10 nM) on INS-1 cell DNA
synthesis was determined by [3H]thymidine incorporation
as an index of
-cell proliferation. Glucose independently increased
[3H]thymidine incorporation into INS-1 cells (Fig.
1). In the absence of IGF-I, no change in
[3H]thymidine incorporation in INS-1 cells was observed
between 0 and 0.5 mM glucose (Fig. 1), but at 1 mM glucose a modest increase in INS-1 cell proliferation
was observed (1.4-fold increase compared with no glucose,
p < 0.001; Fig. 1). However, the most effective glucose concentration range on INS-1 cell proliferation occurred in the
physiologically relevant range between 6 and 18 mM glucose (4-19-fold above that in the absence of glucose; p < 0.001; Fig. 1). Maximum [3H]thymidine incorporation into
INS-1 cells above "zero glucose" occurred at 18 mM
glucose (19-fold increase, p < 0.0001; Fig. 1), and
declined above this glucose concentration (Fig. 1). Notwithstanding, a
14-fold higher INS-1 cell proliferation rate at 24 mM
glucose was significantly higher than that observed in the absence of glucose (p < 0.001; Fig. 1).

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Fig. 1.
[3H]Thymidine incorporation in
INS-1 cells with different glucose concentrations. Approximately
105 quiescent INS-1 cells/well were incubated for 24 h
in RPMI 1640 medium containing 0.1% BSA, 0-24 mM
glucose ± 10 nM IGF-I, then assessed for
proliferation rate by [3H]thymidine incorporation as
described under "Experimental Procedures." All experiments were
done in triplicate on at least eight independent occasions. The data
are expressed as a fold increase above the control observation in the
absence of glucose and IGF-I (i.e. 500-1200
cpm/105 cells), and depicted as a mean ± S.E.
(n 8).
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It was apparent that IGF-I only exerted a notable effect on
[3H]thymidine incorporation into INS-1 cells when glucose
was present at physiologically relevant concentrations (6-18
mM glucose; Fig. 1). Below 1 mM glucose, IGF-I
modestly increased [3H]thymidine incorporation into INS-1
cells (1.9-fold increase above zero glucose in the absence of IGF-I,
p < 0.001; Fig. 1), however, this effect was rather
small compared with the synergistic effects of IGF-I and glucose in the
physiologically relevant range (6-18 mM; Fig. 1). At 6 mM glucose, IGF-I instigated a 10-fold increase in
[3H]thymidine incorporation above that in the absence of
glucose and IGF-I (p < 0.001; Fig. 1), which was
2.5-fold higher than the rate of INS-1 cell proliferation at 6 mM glucose alone (p < 0.01; Fig. 1). IGF-I
instigated a maximum increase in INS-1 cell [3H]thymidine
incorporation at 15 mM glucose (52-fold above that in the
absence of glucose and IGF-I glucose, p < 0.001; Fig.
1), which was 4.2-fold higher than that at 15 mM glucose
alone (p < 0.01; Fig. 1). Above 18 mM
glucose the synergistic effect of IGF-I and glucose on
[3H]thymidine incorporation into INS-1 cells was
significantly diminished (p < 0.001 above 15 mM glucose + 10 nM IGF-I; Fig. 1), so that at
24 mM glucose addition of IGF-I only surpassed the glucose effect by 1.4-fold (Fig. 1). Notwithstanding, this was 21-fold higher
than the proliferation rate in the absence of glucose and IGF-I
(p < 0.001; Fig. 1).
Both IGF-I and glucose-stimulated INS-1 cell proliferation required
glucose metabolism (Fig. 2).
Mannoheptulose (15 mM), a competitive inhibitor of
glycolysis (27), completely inhibited 15 mM
glucose-stimulated INS-1 cell [3H]thymidine incorporation
in the presence or absence of IGF-I (p < 0.001; Fig.
2). Not surprisingly, mannoheptulose (3 or 15 mM) had no
independent effect on INS-1 cell proliferation whether IGF-I was
present or not (Fig. 2). Likewise, the non-metabolizable glucose
analogues, 2-deoxyglucose or 3-O-methyl glucose (at
concentrations of 3 or 15 mM), had no effect on
[3H]thymidine incorporation into INS-1 cells (Fig. 2),
and could not provide a suitable platform for IGF-I instigated INS-1
cell proliferation (Fig. 2). Fructose (15 mM), which is not
efficiently metabolized in pancreatic
-cells (27), did not
significantly increase INS-1 cell proliferation in the presence or
absence of IGF-I (Fig. 2). These observations further emphasize the
requirement of physiologically relevant concentrations of glucose for
IGF-I to stimulate INS-1 cell proliferation.

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Fig. 2.
[3H]Thymidine incorporation in
INS-1 cells with different monosaccharides. Approximately
105 quiescent INS-1 cells/well were incubated for 24 h
in RPMI 1640 medium containing 0.1% BSA, 3 or 15 mM
glucose, 3 or 15 mM 2-deoxyglucose, 3 or 15 mM
3-O-methylglucose, 3 or 15 mM mannoheptulose,
mannoheptulose ± 3 or 15 mM glucose or 15 mM fructose, ± 10 nM IGF-I, then assessed for
proliferation rate by [3H]thymidine incorporation as
described under "Experimental Procedures." All experiments were
done in triplicate on three independent occasions. The data are
expressed as a fold increase above the control observation in the
absence of glucose and IGF-I (i.e. 500-1200
cpm/105 cells), and depicted as a mean ± S.E.
(n = 3).
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The effect of increasing IGF-I concentrations on
[3H]thymidine incorporation in INS-1 cells at various
glucose concentrations was examined (Fig.
3). In the absence of IGF-I there was an
incremental increase in [3H]thymidine incorporation with
increasing glucose between 3 and 18 mM as observed
previously (Fig. 1). IGF-I above 1 nM increased [3H]thymidine incorporation >2-fold compared with cells
treated with glucose alone, with a maximal increase at 10 nM IGF-I, 18 mM glucose (49-fold above zero
glucose, p < 0.001; Fig. 3). This effect was not
exceeded above a concentration of 10 nM IGF-I (Fig. 3), and
subsequently 10 nM IGF-I was used as an optimal IGF-I concentration in following experiments.

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Fig. 3.
[3H]Thymidine
incorporation in INS-1 cells with different IGF-I
concentrations. Approximately 105 quiescent
INS-1 cells/well were incubated for 24 h in RPMI 1640 medium
containing 0.1% BSA, 0-100 nM IGF, 0-18 mM
glucose, then assessed for proliferation rate by
[3H]thymidine incorporation as described under
"Experimental Procedures." All experiments were done in
triplicate on six independent occasions. The data are expressed
as a fold increase above the control observation in the absence of
glucose and IGF-I (i.e. 500-1200 cpm/105
cells), and depicted as a mean ± S.E. (n = 6).
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The effect of insulin to compete for IGF-I (10 nM)-stimulated [3H]thymidine incorporation
into INS-1 cells was also examined at various glucose concentrations
(Fig. 4). Only at very high insulin concentrations (>10 µM) was any significant competition
for IGF-I-stimulated INS-1 cell proliferation observed, which then
rendered only a 20-30% inhibition (p < 0.02; Fig.
4). In the absence of IGF-I, only high concentrations of insulin (>10
µM) could instigate a modest increase in
[3H]thymidine incorporation into INS-1 cells at 15 mM glucose (1.3-fold above 15 mM glucose
without IGF-I, p < 0.05; Fig. 4). Thus, the observations found in this study were predominately attributable to
IGF-I working through the IGF-I receptor.

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Fig. 4.
The effect of exogenous insulin on
glucose/IGF-I-stimulated [3H]thymidine incorporation in
INS-1 cells. Approximately 105 quiescent INS-1
cells/well were incubated for 24 h in RPMI 1640 medium containing
0.1% BSA, 0, 3, or 15 mM glucose, or 15 mM
glucose + 10 5-10 9 M bovine
insulin, then assessed for proliferation rate by
[3H]thymidine incorporation as described under
"Experimental Procedures." All experiments were done in triplicate
on three independent occasions. The data are expressed as a fold
increase above the control observation in the absence of glucose,
IGF-I, and insulin (i.e. 500-1200 cpm/105
cells), and depicted as a mean ± S.E. (n = 3).
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The Effect of Various Protein Phosphorylation Inhibitors on Glucose
and IGF-I-stimulated INS-1 Cell Proliferation--
The effect of
specific protein kinase and phosphatase inhibitors on stimulation of
[3H]thymidine incorporation in INS-1 cells by 15 mM glucose ± 10 nM IGF-I was examined
(Table I). In these series experiments 15 mM glucose instigated a 20-fold increase in
[3H]thymidine incorporation above that in the absence of
glucose (p < 0.001), and the combination of 15 mM glucose and 10 nM IGF-I gave a 52-fold
increase above that in the absence of glucose and IGF-I
(p < 0.001) similar to that observed previously (Fig.
1). Addition of (Sp)-2'-O-monobutyryl-cAMP (5 µM), a cell permeable PKA agonist (28), did not show any
significant changes in the rate of INS-1 cell
[3H]thymidine incorporation at either 15 mM
glucose alone or in the additional presence of IGF-I (Table I).
However, in the presence of
(Rp)-O2'-monobutyryl-cAMP (5 µM), a cell permeable cAMP analogue which inhibits
protein kinase-A (PKA) activity (28), 15 mM glucose-induced [3H]thymidine incorporation in INS-1 cells was inhibited
by 87% (p < 0.001; Table I), whereas it was not
significantly affected in the additional presence of IGF-I (Table I).
This suggested a possible role for PKA in glucose-induced INS-1 cell
mitogenesis (29), but this was overcome by addition of IGF-I.
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Table I
The effect of protein phosphorylation inhibitors on glucose/IGF-I
stimulated [3H]thymidine incorporation in INS-1 cells
Approximately 105 quiescent INS-1 cells/well were incubated for
24 h in RPM1 1640 medium containing 0.1% BSA, 15 mM
glucose ± 10 nM IGF-I, ± various inhibitors of
protein kinases, phosphoprotein phosphatases, or tyrosine kinase
signaling cascades as indicated, then assessed for proliferation rate
by [3H]thymidine incorporation as outlined under
"Experimental Procedures." The data are presented as a percentage
of either the control [3H]thymidine incorporation at 15 mM glucose in the absence of IGF-1, or the control
[3H]thymidine incorporation at 15 mM glucose + 10 nM IGF-1, as appropriate. A mean ± S.E. are
depicted of at least five experiments done in triplicate.
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In the presence of sphingosine (10 µM), a selective
inhibitor of protein kinase C (30), both 15 mM
glucose-stimulated and IGF-I + 15 mM glucose-stimulated
[3H]thymidine incorporation in INS-1 cells were
significantly inhibited (50-60% inhibition; p < 0.02; Table I). This suggested that certain protein kinase C isoform(s)
could be involved in regulating glucose and IGF-I-mediated INS-1 cell
proliferation (29). Staurosporine (20 nM) an inhibitor of
protein kinase C, PKA, and protein kinase G (30), inhibited 15 mM glucose-stimulated [3H]thymidine
incorporation into INS-1 cells (50% inhibition, p < 0.001; Table I), but not that in the additional presence of IGF-I
(Table I). This was similar to the effect of
(Rp)-O2'-monobutyryl-cAMP on INS-1
cell proliferation (Fig. 5), and
suggested that staurosporine may be mediating its effect via inhibition of PKA.

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Fig. 5.
IGF-I and glucose both increase protein
phosphotyrosine phosphorylation of IRS-2 and pp60, resulting in
increased association of PI 3'-kinase and mSOS. INS-1 cells (50%
confluent on a 15-cm diameter dish) were stimulated with 3 or 15 mM glucose ± 10 nM IGF-I for 10 min, and
cell lysates generated as described under "Experimental
Procedures." INS-1 cell lysates were then subjected to
immunoprecipitation (IP) with antiserum against the p85
regulatory subunit of PI 3'-kinase (panels A-D) or
anti-phosphotyrosine (PY, panels E and
F) antibody. Immunoprecipitates were then subjected to
immunoblot (IB) analysis with IRS-2 (panel A),
mSOS (panel B), anti-phosphotyrosine (panels C
and E), and p85 PI 3'-kinase (panels D and
F) antibodies, as described under "Experimental
Procedures." An example blot for such co-immunoprecipitation analysis
is shown.
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It has been implicated that intracellular [Ca2+] might be
involved in signaling pathways that lead to glucose-induced
-cell mitogenesis (7, 8), that may be mediated by activation of Ca2+/calmodulin-dependent proteins.
Calmidazolium (50 nM), a calmodulin antagonist,
significantly reduced 15 mM glucose-stimulated
[3H]thymidine incorporation into INS-1 cells by 86%
(p < 0.002; Table I), but did not inhibit
IGF-I-stimulated INS-1 cell proliferation at 15 mM glucose
(Table I). KN-93 (1 µM), an inhibitor of calmodulin kinase II activity in
-cells (31), showed no significant decrease in
15 mM glucose-stimulated INS-1 cell proliferation whether
IGF-I was present or not (Table I). These data suggested a role for Ca2+/calmodulin (but not necessarily calmodulin kinase II)
for glucose-induced INS-1 cell proliferation (29), however, this was
averted by the addition of IGF-I.
The possible role of phosphoprotein phosphatase activities on
glucose/IGF-I-induced INS-1 cell proliferation was investigated. Okadaic acid (50 nM), an inhibitor of phosphoprotein
phosphatase 1 and phosphoprotein phosphatase 2A, and cyclosporin A
(5 µM), an inhibitor of phosphoprotein phosphatase 2B,
had no significant effect on 15 mM glucose or glucose + IGF-I- stimulated INS-1 cells proliferation (Table I). However, the
protein tyrosine phosphatase inhibitor, orthovanadate (0.5 mM), markedly inhibited 15 mM glucose and IGF-I + 15 mM glucose-stimulated [3H]thymidine
incorporation into INS-1 cells by >95% (p < 0.001; Table I). This was indicative of protein tyrosine phosphorylation as an
important aspect of glucose/IGF-I mitogenic signal transduction pathway(s) in INS-1 cells.
Protein tyrosine phosphorylation cascades have been strongly implicated
in mitogenic signal transduction pathways (19, 20). Genistein (25 µM), an inhibitor of protein tyrosine kinase activity, markedly inhibited both 15 mM glucose and IGF-I-stimulated
[3H]thymidine incorporation into INS-1 cells by >95%
(p < 0.001; Table I). Specific inhibitors of PI
3'-kinase activity (wortmannin (10 nM) and LY294002 (5 µM)) also markedly inhibited by both 15 mM
glucose and IGF-I-induced INS-1 cell proliferation by >90% (p < 0.001; Table I). Activation of p70S6K
lies downstream of PI 3'-kinase activation in mitogenic signaling pathways, and correspondingly specific inhibition of p70S6K
by rapamycin (10 nM) resulted in a significant 50%
inhibition of both 15 mM glucose (p < 0.02) and IGF-I (p < 0.001)-induced [3H]thymidine incorporation into INS-1 cells incubated
(Table I). The MEK inhibitor, PD98059 (50 µM), inhibited
INS-1 cell proliferation as stimulated by the combination of 10 nM IGF-I + 15 mM glucose by >80% compared
with the equivalent control (p < 0.001; Table I). In
contrast, PD98059 had no significant effect on
[3H]thymidine incorporation into INS-1 cells incubated
with 15 mM glucose in the absence of IGF-I (Table I). These
data implicate that the PI 3'-kinase branch of mitogenic signal
transduction pathways was important for both glucose and
IGF-I-stimulated INS-1 cell proliferation, whereas the MAPK branch
might only be relevant for IGF-I-mediated INS-1 cell mitogenesis.
IGF-I and Glucose Activate IRS-mediated Mitogenic Signal
Transduction Pathways in INS-1 Cells--
Protein phosphorylation
activation of mitogenic signal transduction pathways by 15 mM glucose ± 10 nM IGF-I in INS-1 cells was investigated using co-immunoprecipitation and immunoblot analysis. Immunoprecipitation of the 85-kDa regulatory subunit of PI 3'-kinase followed by immunoblot analysis with IRS-2 antiserum, revealed a
specific increased association of PI 3'-kinase and IRS-2 instigated by
both 15 mM glucose and IGF-I. At a basal 3 mM
glucose, 10 nM IGF-I increased the amount of IRS-2
associated with PI 3'-kinase within 10 min (Fig. 5A).
Increasing the glucose concentration to 15 mM further
increased IRS-2/PI 3'-kinase association, which was not particularly
affected by the additional presence of IGF-I (Fig. 5A). The
specific nature of this glucose/IGF-I-induced IRS-2/PI 3'-kinase
interaction was illustrated in that PI 3'-kinase immunoblot analysis of
PI 3'-kinase immunoprecipitates revealed that an equivalent amount of
PI 3'-kinase present in each sample (Fig. 5D). In contrast to the PI 3'-kinase/IRS-2 interaction in INS-1 cells, no detectable difference in the PI 3'-kinase/IRS-1 interaction could be found at 15 mM glucose ± IGF-I in INS-1 cells (data not shown).
However, immunoblotting of the PI 3'-kinase immunoprecipitates with
antiserum recognizing the C-terminal region of mSOS (Fig.
5B) indicated an increased association of mSOS within 10 min
in INS-1 cells stimulated with IGF-I at 3 mM glucose
(presumably via increased Grb2 association with tyrosine-phosphorylated
IRS (19, 20)). The PI 3'-kinase/mSOS association in INS-1 cells was
further increased by 15 mM glucose alone, but this was not
substantially increased by the additional presence of IGF-I at 15 mM glucose (Fig. 5B).
Immunoprecipitation of the p85 PI 3'-kinase regulatory subunit followed
by anti-phosphotyrosine (PY) immunoblot analysis revealed an increase
in the presence of tyrosine-phosphorylated 95-kDa
-subunit of
the IGF-I receptor only in the presence of IGF-I (Fig. 5C).
In the same analysis a prominent 60-kDa tyrosine-phosphorylated protein
(pp60) showed increased association to the PI 3'-kinase immunoprecipitate after a 10-min exposure to 15 mM glucose
compared with that at a basal 3 mM glucose (Fig.
5C). The association of tyrosine-phosphorylated pp60 with PI
3'-kinase was further increased by IGF-I at both 3 and 15 mM glucose within 10 min (Fig. 5C). Correspondingly, immunoprecipitation with an anti-phosphotyrosine antibody followed by anti-phosphotyrosine immunoblot analysis indicated
an increase in the tyrosine phosphorylated state of pp60 by IGF-I at a
basal 3 mM glucose, and at 15 mM glucose ± IGF-I (Fig. 5E). Immunoblot analysis of
anti-phosphotyrosine immunoprecipitates with p85 PI 3'-kinase antisera
revealed an increased association of PI 3'-kinase with
tyrosine-phosphorylated proteins instigated by IGF-I at a basal 3 mM glucose (Fig. 5F). The p85 PI 3'-kinase association with phosphotyrosine proteins was increased at 15 mM glucose, compared with that at 3 mM
glucose ± IGF-I, and further enhanced by the addition of IGF-I
(Fig. 5F).
IGF-I was also able to activate the Ras/Raf/MEK/MAPK mitogenic signal
transduction pathway independent of IRS by IGF-I receptor tyrosine
kinase-mediated phosphorylation of Shc (18-20). Immunoprecipitation of
Shc followed by immunoblot analysis of mSOS (Fig.
6A) and Grb2 (Fig.
6B) from INS-1 cells incubated for 10 min at 3 or 15 mM glucose ± IGF-I, indicated that IGF-I at a basal 3 mM glucose could promote the association of mSOS/Grb2 to
Shc (Fig. 6, A and B). Furthermore, 15 mM glucose alone was found to promote the association of
Grb2/mSOS with Shc, that was further enhanced by the additional
presence of IGF-I (Fig. 6, A and B). The specific nature of glucose/IGF-I-induced Grb2-mSOS interaction with Shc in INS-1
cells was indicated in that an equivalent amount of Shc was detected by
immunoblot analysis of Shc immunoprecipitates (Fig. 6C).

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Fig. 6.
IGF-I and glucose both increase the
association of Grb2 and mSOS with Shc. INS-1 cells (50% confluent
on a 15-cm diameter dish) were stimulated with 3 or 15 mM
glucose ± 10 nM IGF-I for 10 min, and cell lysates
generated as described under "Experimental Procedures." INS-1 cell
lysates were then subjected to immunoprecipitation (IP) with
antiserum Shc. The Shc immunoprecipitates were then subjected to
immunoblot (IB) analysis with mSOS (panel A),
Grb2 (panel B), and Shc (panel C) antibodies, as
described under "Experimental Procedures." An example blot for such
co-immunoprecipitation analysis is shown.
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The mSOS activation, by its Grb2-mediated association with
tyrosine-phosphorylated IRS and/or Shc, results in downstream
activation of Ras which in turn stimulates Raf-1 serine kinase activity
(18-20). Raf-1 then activates MEK which in turn activates MAPK (erk-1
and -2 isoforms) by serine phosphorylation (7, 8, 18-20). Activated MAPK can be detected with specific antiserum that only recognizes the
phosphorylation activated forms of erk-1 and -2 (7, 8). Immunoblot
analysis of glucose ± IGF-I-stimulated INS-1 cells with
"activated phospho-MAPK" antiserum showed little activated MAPK in
the absence of glucose or IGF-I (Fig. 7).
However, an increase in active phospho-MAPK after 10 min exposure to 3 mM glucose was detected which reached a maximum at 18 mM glucose independently of IGF-I (Fig. 7). IGF-I treatment
of INS-1 cells for 10 min in the absence of glucose activated MAPK
(Fig. 7). In the additional presence of glucose (3-9 mM),
IGF-I instigated a further increase in activation of MAPK in INS-1
cells above that of glucose alone (Fig. 7). The IGF-I activation of
MAPK reached a maximum at 6 mM glucose, but thereafter
decreased so that at 18 mM glucose IGF-I could instigate an
activation of MAPK above that of 18 mM glucose alone (Fig.
7). The amount of total MAPK in INS-1 cells, as ascertained by
immunoblot analysis with antisera recognizing both active and inactive
forms of MAPK, was not significantly altered by glucose and IGF-I
treatment (Fig. 7). Similarly immunoblot analysis of total MEK and Shc
in INS-1 cells treated with various glucose concentrations ± IGF-I indicated that levels of these proteins did not noticeably alter
(Fig. 7).

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Fig. 7.
IGF-I and glucose both stimulated
phosphorylation activation of MAPK (erk-1/erk-2 isoforms) in INS-1
cells. INS-1 cells (50% confluent on a 10-cm diameter dish) were
stimulated with 3, 6, 9, or 18 mM glucose ± 10 nM IGF-I for 10 min, and cell lysates generated as
described under "Experimental Procedures." Specific immunoblot
analysis for phosphorylation activated MAPK, total MAPK, MEK, and Shc
was examined in the INS-1 cell lysates as described under
"Experimental Procedures." A representative immunoblot
(IB) analysis of "activated" MAPK, total MAPK, MEK, and
Shc is shown.
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In the IGF-I mediated signal transduction pathway, p70S6K
is activated downstream of PI 3'-kinase activation (18).
Phosphorylation activation of p70S6K occurs on multiple
sites so that p70S6K phosphorylation can be detected on
immunoblot analysis by an apparent electrophoresis mobility retardation
(26). INS-1 cells were incubated for 5-60 min in the presence of 3-18
mM glucose ± 10 nM IGF-I. Maximal
p70S6K phosphorylation was observed at 30 min (data not
shown). Immunoblot analysis with p70S6K-specific antiserum
indicated phosphorylation activation of p70S6K in response
to both glucose and IGF-I (Fig. 8). In the absence of IGF-I, glucose
p70S6K phosphorylation above 6 mM glucose
reached a maximum at 18 mM glucose (Fig.
8). In the added presence of IGF-I no
activation of p70S6K was observed below 3 mM
glucose, however, above 3 mM glucose IGF-I increased
p70S6K activation by glucose alone, reaching a maximum
potentiating effect of IGF-I at 18 mM glucose (Fig. 8). A
p85 PI 3'-kinase immunoblot analysis of the same INS-1 cell lysates
used for p70S6K analysis indicated that there was little
change in the total amount of PI 3'-kinase protein per sample, that
emphasized a specific activation of p70S6K instigated by
glucose and IGF-I (Fig. 8).

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Fig. 8.
IGF-I and glucose both stimulated
phosphorylation activation of p70S6K in INS-1 cells.
INS-1 cells (50% confluent on a 15-cm diameter dish) were stimulated
with 3, 6, 9, or 18 mM glucose ± 10 nM
IGF-I for 30 min, and cell lysates generated as described under
"Experimental Procedures." Specific phosphorylation activation of
p70S6K was examined in the INS-1 cell lysates by immunoblot
(IB) analysis as described under "Experimental
Procedures." A representative immunoblot for p70S6K
is shown. Phosphorylated forms of p70S6K become retarded on
SDS-polyacrylamide gel electrophoresis analysis, and these
multi-phosphorylated p70S6K forms are indicated by the
arrows.
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DISCUSSION |
Although adult pancreatic
-cells have a relatively low mitotic
index (1), both IGF-I and glucose have been shown to stimulate pancreatic
-cell proliferation (2, 15, 32). However, little has been
revealed about the mitogenic signal transduction pathways in
-cells
that are activated by glucose and certain growth factors, which
irreversibly leads to committing a
-cell into a growth phase of the
cell cycle (33). Moreover, the regulation of mitogenesis in pancreatic
-cells is likely to be more complicated than in other eukaryotic
cells due to the unique characteristic of the
-cell's
stimulus-coupling mechanisms being tightly linked to its metabolic
state (23, 34). Indeed, glucose has been shown to induce adult
-cell
replication both in vitro and in vivo (2, 3),
although the concentration dependence has not been previously established. In this study glucose-induced
-cell proliferation has
been examined in the relatively well differentiated INS-1 cell line.
INS-1 cells respond to glucose in terms of insulin secretion in the
physiologically relevant range (5-20 mM) (24). Likewise,
it was found that glucose-induced [3H]thymidine
incorporation into INS-1 cells was only apparent at glucose
concentrations between 6 and 24 mM. Similar to
glucose-induced insulin secretion (24), the threshold glucose
concentration to instigate an increase in INS-1 cell proliferation was
between 3 and 6 mM that reached a maximum at 18 mM glucose. Glucose metabolism was required to provoke a
mitogenic response in INS-1 cells, as previously established in adult
and fetal pancreatic
-cells (2), however, it remains unclear what
the appropriate signaling elements are immediately downstream of
glucose metabolism required for a glucose-induced mitogenic response
(2).
IGF-I has been shown to be an effective stimulus for inducing
differentiated pancreatic
-cell growth (2). Furthermore, IGF-I has
been implicated to play a prominent role for increasing the population
of pancreatic islets in the regenerating pancreas (3). In this study,
it was found that IGF-I (1-10 nM) could markedly increase
INS-1 cell proliferation, but only in the physiologically relevant
glucose concentration range between 6 and 18 mM (Figs. 1
and 3). Thus, unlike the mitogenic effect of IGF-I on other eukaryotic
cells (17), in pancreatic
-cells IGF-I was dependent on glucose
being present to provoke a mitogenic response. The additional presence
of IGF-I at 6-18 mM glucose increased INS-1 cell
proliferation 2-3-fold above that at glucose alone. Thus, there was a
degree of synergy between IGF-I and glucose (6-18 mM) to
increase the pancreatic
-cell proliferation rate. A further indication of the glucose-dependent nature of IGF-I-induced
INS-1 cell replication was that metabolism of glucose was required for the IGF-I mitogenic effect (Fig. 2). However, at higher glucose concentrations (24 mM), the effect of IGF-I on INS-1 cell
[3H]thymidine incorporation was reduced, perhaps
indicative of the adverse effects of elevated glucose concentrations on
-cell function (35). Nonetheless, at 15 mM glucose, it
was apparent that it was a specific effect of IGF-I working through
IGF-I receptors (previously shown to be present on pancreatic
-cells
(36)), and not a secondary effect of insulin secreted from INS-1 cells working via insulin and/or IGF-I receptors or IGF-I operating via
insulin receptors (36, 37). Insulin could only slightly inhibit 10 nM IGF-I-induced
-cell proliferation at very high concentrations (>10 µM insulin; Fig. 4). Likewise, in
the absence of IGF-I, insulin only modestly potentiated glucose-induced
INS-1 cell growth at very high unphysiological concentrations (>10
µM insulin; Fig. 4).
A degree of insight for signaling requirements of glucose-induced and
glucose-dependent IGF-I-stimulated
-cell proliferation were gained from inhibitor studies (Table I). Glucose-induced INS-1
cell proliferation appeared to require both PKA and
Ca2+/calmodulin as previously suggested (2, 38), but this
requirement was vanquished in the additional presence of IGF-I.
Notably, inhibition of protein phosphotyrosine phosphatases or tyrosine
protein kinases resulted in complete inhibition of glucose and
IGF-I-induced INS-1 cell replication, implicating certain protein
tyrosine phosphorylation/dephosphorylation signaling cascades were an
important ingredient in INS-1 cell mitogenic signaling. Activation of
PI 3'-kinase occurs along IRS-mediated tyrosine phosphorylation
signaling pathways (19, 20), and PI 3'-kinase activity appeared
essential for both glucose or IGF-I to provoke a mitogenic response.
This was substantiated by the finding that rapamycin, an inhibitor of
p70S6K activation that occurs downstream of PI 3'-kinase
activation (19), also inhibited glucose and IGF-I-induced INS-1 cell
proliferation. However, it should be noted that rapamycin only partly
inhibited the glucose/IGF-I-induced mitogenic response in INS-1 cells,
perhaps suggesting that alternative factors downstream of PI 3'-kinase activation (e.g. other protein substrates of
phosphatidylinositide 3,4,5-triphosphate-activated protein kinase B
(39)), ought to be considered. Intriguingly, inhibition of MEK and
consequential MAPK activation had no effect on glucose-induced INS-1
cell proliferation. Thus, whereas the PI 3'-kinase branch of mitogenic
signaling pathways may be required for glucose-induced INS-1 cell
replication, that via MAPK may not, despite glucose-induced activation
of MAPK in
-cells (7, 8) (Fig. 7). However, this did not appear to be the case for IGF-I where inhibition of MEK/MAPK significantly inhibited glucose-dependent IGF-I-induced INS cell
mitogenesis. It follows that these inhibitor studies revealed that
there are differences between glucose and IGF-I signaling to induce
-cell mitogenesis. Nonetheless, a degree of caution should be taken in interpreting such inhibitor studies, since these pharmacological reagents often act via secondary mechanisms and as such can be misleading. Thus, such inhibitor experiments are better supported with
alternative biochemical evidence, and hence in this study IRS/Shc-mediated signal transduction pathways were directly examined in
INS-1 cells.
Increasing the glucose concentration from a basal 3 mM to a
stimulatory 15 mM in the absence of IGF-I resulted in
activation of IRS-mediated signaling pathways independent of a growth
factor stimulus. Glucose induced an increased association of PI
3'-kinase with IRS-2 in INS-1 cells, but that with IRS-1 could not be
detected. This was supportive of recent observations of the key role
for IRS-2 in
-cell mitogenesis, in that there is a specific increase in IRS-2 expression but not that of IRS-1 in pancreatic
-cell lines
(40), and that
-cell mass in vivo is markedly reduced in
IRS-2 knockout mice yet increased in IRS-1-deficient mice (41). Glucose-stimulated IRS-2/PI 3'-kinase association in INS-1 cells correlated with an increase in tyrosine phosphorylation (19, 20), as
indicated by glucose-induced increase of PI 3'-kinase in
anti-phosphotyrosine immunoprecipitates (Fig. 5F). However, it is unlikely that glucose-induced PI 3'-kinase activation in INS-1
cells is exclusively mediated via IRS-2. Glucose also increased an
association of PI 3'-kinase with a 60-kDa tyrosine-phosphorylated protein (pp60; Fig. 5C). The identity of pp60 in INS-1 cells
has yet to uncovered, however, it is quite possible that this may represent the truncated member of the IRS family, IRS-3, that associates with PI 3'-kinase in a tyrosine phosphorylation-dependent manner resulting in PI 3'-kinase activation (42, 43). Notwithstanding, it was found that glucose could induce an activation of
p70S6K which lies downstream of PI 3'-kinase activation
(19-21). The p70S6K activation correlated with the glucose
concentration dependence for stimulation of [3H]thymidine
incorporation in INS-1 cells (Fig. 1 verses Fig. 8). Thus,
these data are consistent with the notion that glucose can activate the
PI 3'-kinase/p70S6K branch of IRS-mediated signaling
pathways in pancreatic
-cells, which is required for glucose
increased
-cell proliferation.
Glucose induced recruitment of mSOS to p85 PI 3'-kinase
immunoprecipitates (Fig. 5B). This was not necessarily due
to a direct association of p85 PI 3'-kinase and mSOS, but rather via
Grb2 interaction with tyrosine-phosphorylated IRS which
co-immunoprecipitated with p85 PI 3'-kinase as components of an
activated IRS signaling complex (19, 20). MAPK is activated downstream
of Grb2/mSOS association with IRS, so this complemented the observation
of glucose-induced activation of MAPK (7, 8) (Fig. 7). Notwithstanding, MAPK can also be activated via an IRS-independent pathway involving tyrosine phosphorylation of the adaptor molecule Shc (17, 19, 20).
Glucose independently induced an increase in Grb2/mSOS association with
Shc (Fig. 6), that would also contribute to downstream activation of
MAPK (7, 8) (Fig. 7). However, it should be noted that the
concentration dependence for glucose-stimulated activation of MAPK
(Fig. 7) did not correlate with glucose stimulation of
[3H]thymidine incorporation in INS-1 cells (Fig. 1). This
suggested that MAPK activation was not necessarily required for
glucose-induced
-cell proliferation, in agreement with the finding
that MEK inhibition did not affect glucose-induced
[3H]thymidine incorporation into INS-1 cells.
IGF-I-induced INS-1 cell mitogenesis was glucose-dependent. For the
moment, it is uncertain what the critical signaling factor(s) downstream of glucose metabolism might be that provides a platform for
IGF-I to provoke a mitogenic response in
-cells. However, the
addition of IGF-I tended to potentiate glucose-induced activation of
IRS/Shc signal transduction pathways in INS-1 cells. Phosphotyrosine immunoblot analysis of PI 3'-kinase immunoprecipitates revealed that
IGF-I induced tyrosine phosphorylation of a 95-kDa protein (Fig.
5C). This phosphotyrosine protein was not detectable in the
absence IGF-I, and as such, it was likely the 95-kDa
-subunit of the
IGF-I receptor (17). At a stimulatory 15 mM glucose, an
increase in tyrosine phosphorylation of the IGF-I receptor
-subunit
was detected in PI 3'-kinase immunoprecipitates, compared with that at
a basal 3 mM glucose. This may be due to either increased association of the IGF-I receptor with a PI 3'-kinase
immunoprecipitated signaling complex or increased tyrosine
phosphorylation of the IGF-I receptor at a stimulatory glucose
concentration. However, increases in extracellular glucose
concentration are unlikely to affect binding of IGF-I to its receptor.
Thus, in considering that glucose is required for IGF-I-induced INS-1
cell proliferation, it is more likely that glucose facilitates
recruitment of IRS/PI 3'-kinase to an activated tyrosine-phosphorylated
IGF-I receptor during formation of an activated IRS signaling complex
(17, 19, 20). This notion was supported in that, IGF-I further increased the association of PI 3'-kinase with IRS-2, mSOS (presumably via Grb2 (17, 19, 20)), and pp60 (Fig. 5, A-C), as well as
Shc with Grb2/mSOS (Fig. 6), especially at a basal 3 mM
glucose. Furthermore, downstream activation of p70S6K was
enhanced by IGF-I at physiological glucose concentrations between 6 and
18 mM (Fig. 8), which correlated with the extent of
IGF-I-induced INS cell proliferation rate (Fig. 1). However, unlike
p70S6K activation, IGF-I-induced MAPK activation did not
correlate with IGF-I-stimulated INS-1 cell proliferation (Fig. 7
verses Fig. 1). Nonetheless, inhibition of MAPK activation
led to a marked inhibition of IGF-I-induced INS-1 cell proliferation in
contrast to that by glucose alone (Table I). Thus, some MAPK activity was likely required in order to facilitate IGF-I increased
-cell proliferation, but activation of MAPK alone was not sufficient to
provoke a mitogenic response.
In summary, this study establishes that certain elements of the
mitogenic signal transduction pathway are present in pancreatic
-cells, and can be stimulated by glucose ± IGF-I leading to
downstream activation of MAPK and p70S6K. Although, MAPK
activity was likely required, activation of PI 3'-kinase was an
essential element for glucose/IGF-I-induced
-cell proliferation. It
will be important in future studies to identify the appropriate
transcription factors relevant to
-cell mitogenesis that are
activated downstream of MAPK and p70S6K, and the
"signaling factor" which renders IGF-I signaling in
-cells
dependent on glucose metabolism. Notwithstanding, only a limited number
of mitogenic signal transduction elements have been examined in this
study, and other factors should not be ruled out (36, 44, 45). This is
an important consideration, especially as there is likely interaction
between certain mitogenic signal transduction pathways in
-cells as
in other mammalian cells (46). In the light of the apparent tight
regulation of adult
-cell mitogenesis (2-4, 6), it is probably
synergy between different signaling pathways that irreversibly commits
the
-cell into a growth phase of the cell cycle.
We thank Dr. M. Cobb for the
anti-erk1/erk2-antibody.