 |
INTRODUCTION |
The insulin-secreting
-cell of the endocrine pancreas has a
central role in regulating glucose homeostasis (1, 2). It is now
recognized that
-cell failure is a major contributing factor to type
2 diabetes mellitus, thus emphasizing the importance of elucidating the
normal mechanisms of insulin secretion (3, 4). Glucose oxidation by the
-cell is essential for insulin secretion. In particular,
glucokinase, the first step in glycolysis, has been convincingly shown
to be the
-cell glucose sensor (5).
-Cell metabolism of glucose
results in an increase in the ATP/ADP ratio leading to closure of the
KATP channel, depolarization of the
-cell, and influx of
extracellular Ca2+ through voltage-dependent
Ca2+ channels. The subsequent increase in intracellular
Ca2+ then activates insulin exocytosis. The possibility of
other signaling pathways involved in glucose-induced insulin secretion
has also been suggested (6-11).
Since the discovery of the insulin receptor in insulin-secreting
-cells by Rothenberg and colleagues (12, 13), a rapidly growing body
of evidence indicates that the insulin receptor signaling pathway is
active in pancreatic
-cells (14) and plays an important role in
-cell regulation (4, 12-17). Activation of the
-cell insulin
receptor (IR)1 results in
rapid tyrosine phosphorylation of the IR
-subunit and the IR
substrate proteins (12). Deletion of IR results in neonatal death in
mice (18, 19) and leprechaunism in humans (20). Mice with heterozygous
null alleles of IR and insulin receptor substrate 1 (IRS-1)
(IR/IRS-1+/
) exhibit hyperinsulinemia and
-cell hyperplasia and develop overt diabetes (21). Knockouts of the
IRS-1 and IRS-2 produce different effects. Inactivation of IRS-1
(IRS-1
/
) leads to mild insulin resistance,
hyperinsulinemia, and
-cell hyperplasia with no overt diabetes
syndrome (4, 17, 22). In contrast, inactivation of IRS-2
(IRS-2
/
) results in
-cell failure and
causes type 2 diabetes (17). The differential effects of IRS-1 and
IRS-2 knockout indicate that the two major IR substrates mediate
differential signals in
-cells, but the mechanisms accounting for
such differential regulation and for IRS-1 function are still unknown.
Cellular Ca2+ is a critical element in
-cell regulation.
Rising intracellular Ca2+ ([Ca2+]i)
is an obligated step for glucose induced insulin secretion (1, 23).
Abnormal [Ca2+]i is a common defect in both
insulin-dependent type 1 diabetes and insulin-independent
type 2 diabetes (24). Altered Ca2+ metabolism has also been
reported to affect
-cell function including insulin biosynthesis
(25, 26). The endoplasmic reticulum (ER) plays an important role in the
regulation of intracellular Ca2+ concentrations (27-29).
Endoplasmic reticulum Ca2+-ATPase (SERCA) is the major
calcium pump that sequestrates cytosol Ca2+ into ER lumen
(28, 30). Thapsigargin, a nonphorboid tumor promoter, specifically
inhibits ER Ca2+-ATPase activity (31). Addition of
thapsigargin to pancreatic
-cells leads to elevated cytosol
Ca2+ concentration and enhanced short term
glucose-stimulated insulin secretion (32). Recent data showed that IRSs
may directly interact with ER Ca2+-ATPase (SERCA1 and
SERCA2) in a tyrosine phosphorylation-dependent manner in
muscle and heart (33). This finding suggests that insulin may via
insulin receptor signaling pathway regulate ER Ca2+-ATPase
activity, therefore influencing cellular Ca2+ homeostasis.
It is currently unknown whether insulin exerts any regulatory role in
-cell Ca2+ homeostasis.
To dissect the role of IRS-1 in
-cell function, we have
overexpressed IRS-1 in an insulin-secreting
-cell line. We show that
IRS-1 regulates
-cell Ca2+ homeostasis, insulin
biosynthesis, and
-cell proliferation and that elevated expression
of IRS-1 induces abnormal Ca2+ homeostasis and
-cell dysfunction.
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MATERIALS AND METHODS |
Cell Lines and Culture Media--
The clonal mouse
-cell line
TC6-F7 and culture conditions were previously described (15, 34). In
brief, cells were maintained in high glucose Dulbecco's modified
Eagle's medium (25 mM glucose; Life Technologies, Inc.)
supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 units/ml penicillin, 50 µg/ml streptomycin and incubated at 37 °C
in a 10% CO2/90% air humidified incubator. The Chinese
hamster ovary-T cell line was a kind gift from Dr. R. Roth (Stanford
University School of Medicine, Stanford, CA) and was maintained in F-12
medium with 5% fetal bovine serum, 100 units/ml penicillin, 50 µg/ml
streptomycin at 37 °C in a 5% CO2/95% air atmosphere.
Construction of the IRS-1 Expression Plasmid--
The IRS-1
expression vector was constructed as follows. A 3.9-kilobase DNA
fragment containing the mouse IRS-1 cDNA and the c-Myc epitope tag
was excised from pQE31-mIRS1 (kindly provided by Dr. Ronald Kahn,
Joslin Diabetes Center, Boston, MA) with EcoICRI and
HindIII. The fragment was blunt-ended with Klenow fragment (New England Biolabs, Beverly, MA) and ligated into a SmaI
digested vector pCI-Neo (Promega, Madson, WI). The resulting plasmid
pCMV-IRS1 was purified with QIAGEN Maxi-purification Kit (Qiagen,
Chatsworth, CA). Enzymes for recombinant DNA procedures were from
Promega or New England Biolabs.
Transfection of
-Cells--
Cells were transfected with
cationic liposome reagent DMRIE-C (Life Technologies, Inc.) as
described before (15). The transfected cells were selected with 500 µg/ml neomycin (Geneticin, Life Technologies, Inc.) for 4 weeks, and
the surviving colonies (defined as passage 4) were individually picked
and transferred to 24-well plates. Then the IRS-1 and c-Myc protein
levels were determined with anti-IRS1 polyclonal antibody (catalog no.
06-248, Upstate Biotechnology, Lake Placid, NY) and anti-c-Myc
monoclonal antibody 9E10 (catalog no. sc-40, Santa Cruz Biotechnology,
Santa Cruz, CA), respectively.
Immunoprecipitation and Western Analysis--
Preparation of
cell lysates, immunoprecipitation, and Western blotting were performed
essentially as described previously (12, 35). Tyrosine-phosphorylated
proteins were detected with rabbit polyclonal anti-phosphotyrosine
antibody K-18 (kindly provided by Dr. P. Rothenberg, University of
Pennsylvania). A secondary antibody, rabbit anti mouse IgG (Sigma,
catalog no. M7023), was used for c-Myc antibody immunoprecipitation and immunoblotting.
Insulin Assay and
-Cell Metabolism (MTT) Assay--
Insulin
content and secretion assays were performed essentially as described
before (12, 35). The metabolic rate of the
-cells was indirectly
measured by the production of formazan, which is produced from 3-(4,
5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) (Sigma)
as the cells metabolize glucose. The MTT assay was performed
essentially as described (36, 37). In brief, the cells were
preincubated in Krebs-Ringer buffer (KRB) (115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 25 mM glucose, and 25 mM Hepes, pH 7.4) for 30 min. Then, MTT was added to a final concentration of 0.5 mg/ml.
Incubation was continued for another 60 min at 37 °C (5%
CO2/95% air). The supernatant was removed, and 500 µl of
2-propanol were added to dissolve the formazan. The optical density of
the solution was measured at 562 nm in a Microkinetics Plate Reader (Fisher).
Measurement of Cellular Nucleotides--
Cellular contents of
nucleotides including ATP were measured by high performance liquid
chromatography (HPLC) as described before (38). In brief, cells were
washed with ice-cold phosphate-buffered saline and extracted with 1 ml
of 5% trichloracetic acid. Cells were scraped, transferred to 1.5-ml
Eppendorf tubes, incubated 5 min at +4 °C, and centrifuged at
14,000 × g (10 min at +4 °C). The supernatant was
collected into borosilicate 12 × 75-mm tubes, extracted three
times with 1 ml of diethyl ether, and centrifuged, and the ether phase
was discarded. Samples (300 µl) and known amounts of standard NAD,
ADP, GDP, ATP, GTP, UTP, CTP, and TTP were analyzed on a Varian HPLC
apparatus (Varian, Sugarland, Texas), using a 250 × 4.6 mm
Partisil SAX (10 µm) column equipped with a guard column. The
gradient of solvent A (1.25 MNaH2PO4, pH 3.8) over 80 min was
as follows: 0 min, 100% water; 1-15 min, 1% A; 25 min, 28% A; 45 min, 32% A; 70 min, 50% A; 70-80 min, 100% A; 80-95 min, 100%
water. The flow rate was 1.5 ml/min. Nucleotides were detected by UV
spectrophotometry (340 nm) and quantitated by comparison to the
corresponding standard curve run in parallel. The typical retention
times were as follows : 8 min for NAD, 10 min for AMP, 35 min for ADP,
27 min for GDP, 32 min for UTP, 34.5 min for CTP, 34.8 min for TTP, 40 min for ATP, and 46 min for GTP.
Cytosolic free Ca2+ Measurement--
For cytosolic
free Ca2+ measurement, the cells were seeded on microscope
glass coverslips (Fisher) coated with poly-D-lysine (Sigma), and grown for 2 days. Cells were then loaded with the calcium
indicator fura-2 for 30 min at 37 °C in 2 ml of KRB plus 0.1% BSA
and supplemented with 2.0 µM fura-2 acetoxymethylester (Molecular Probes, Eugene, OR) and 0.2 mg/ml pluronic F-127 (Molecular Probes). The coverslip with the loaded cells was then mounted in a
perifusion chamber placed on the homeothermic platform of an inverted
Zeiss microscope. The cells were superfused with Krebs-Ringer buffer
(0.1% BSA) at 37 °C at a flow rate of 1.5 ml/min. For experiments using KRB without Ca2+, CaCl2 was omitted and 1 mM EGTA (Sigma) was added to chelate all Ca2+.
Ca2+ measurement is as follows and was also described in
detail elsewhere (37, 39). The microscope was used with a × 40 oil objective. Fura-2 was successively excited at 334 and 380 nm by
means of two narrow band-pass filters. The emitted fluorescence was
filtered through a 520 nm filter, captured with an Attofluor CCD video camera at a resolution of 512 × 480 pixels, digitized into 256 gray levels and analyzed with version 6.00 of the Attofluor RatioVision software (Atto Instruments, Rockville, MD). The concentration of
Ca2+ at each pixel was calculated by comparing the ratio of
fluorescence to an in vitro two-point calibration curve. The
Ca2+ concentration is presented by averaging the values of
all pixels of a cell body. Data were collected from at least 10 individual cells in each measurement at an interval of 4.5 s.
The endoplasmic reticulum Ca2+-ATPase inhibitor
thapsigargin was purchased from Biomol (Plymouth Meeting, PA) and
dissolved in dimethyl sulfoxide.
Measurement of Endoplasmic Reticulum Ca2+
Uptake--
Endoplasmic reticulum Ca2+ uptake was measured
as described (40, 41). Briefly, the cells were trypsinized and washed
twice with KRB without Ca2+ or glucose (0.1% BSA). Then,
they were permeabilized with 20 µg/ml digitonin (Sigma) in KRB
without Ca2+ or glucose (0.1% BSA) at 37 °C for 10 min.
The cells were then incubated for 30 min in TES buffer (100 mM TES, pH 7.2, 100 mM KCl, 2.5 mM
MgCl2, 0.2 mM EGTA, 5 µCi/ml of
45Ca (ICN, Lisle, IL), 5 µCi/ml of
3H2O (ICN) for diffusible space correction, 5 µg/ml of ruthenium red, 0.1% BSA) at the indicated Ca2+
concentration. The incubation medium was removed by aspiration, and the
cells were solubilized with 100 µl of 1 M NaOH and
neutralized with 100 µl of 1 M HCl. Radioactivity content
of the endoplasmic reticulum was measured by liquid scintillation
spectrometry. Calcium concentrations were titrated with a calcium
electrode (Orion, Beverly, MA). Calculation of endoplasmic reticulum
calcium uptake was as described before (41).
Pulse-Chase Labeling of
-Cells and Determination of Insulin
Biosynthesis--
The rate of insulin biosynthesis was determined with
[3H]leucine pulse-chase labeling assay. The cells were
seeded in 6-well culture dishes at 3 × 105 cells/well
and grown for 2 days in high glucose Dulbecco's modified Eagle's
medium (Life Technologies, Inc.). The cells were then washed twice with
KRB buffer and labeled with 40 µCi/ml of
L-[3,4,5-3H(N)]leucine (6660 GBq/mmol, NEN Life Science Products) in KRB buffer supplemented with
0.25% BSA and 0 or 16 mM glucose. After being labeled for
30 min, the cells were chased with 1 mM cold leucine for 30 min at the same glucose concentration as used in the labeling process.
The cells were then extracted with 1 M acetic acid and
sonicated. One hundred µl of the clarified acetic acid extract were
used for trichloroacetic acid precipitation as described (15) to
determine the total cellular content of 3H-labeled
proteins. The 3H-labeled insulin immunoreactive material
was immunoprecipitated with 3 µl of undiluted guinea pig anti-bovine
insulin serum (ICN) and protein A-Sepharose CL-4B beads (Amersham
Pharmacia Biotech). The radioactivity was determined by liquid
scintillation counting. Normal guinea pig serum (Linco, St. Charles,
MO) was used as background control. Recovery of insulin immunoreactive
material was more than 90% under the assay condition as determined
with 125I-labeled insulin.
RNA Protection Assay--
RNA protection assays were performed
according to Ambion's protocol (Ambion, Austin, TX). Briefly, the
cells were grown in culture medium for 2 days and then trypsinized and
washed twice with Dulbecco's phosphate-buffered saline. To 4 × 106 cells, 0.4 ml of Ambion's lysis/denaturation solution
(direct protect kit, Ambion) were added to lyse the cells. An insulin probe was generated by in vitro transcription using
pGEM-rPPI (gift of Dr. Christopher Rhodes, Joslin Diabetes Center,
Boston, MA) as a template. For internal control, a probe for 18 S RNA was obtained by in vitro transcription using pT7RNA18S
(Ambion) as a template. Labeling of the 18 S RNA probe was done with
[32P]UTP in the presence of 500 µM cold UTP
to lower the specific activity. The labeled transcripts were gel
purified as described in Ambion's manual. The insulin probe was 430 bases in length and completely complementary to the insulin mRNA.
The 18 S rRNA probe was 116 nucleotides in length, 80 of which are
complementary to 18 S rRNA (Ambion). Hybridization, RNase and protease
digestion, and separation detection were carried out as per Ambion's
instructions. The results were quantified with a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
Data Analysis--
Data were analyzed by paired t
test or one-way or two-way ANOVA; p
0.05 was
considered significant. Nonlinear regression and curve fitting were
performed with PRISM 2.01 (GraphPad Software, San Diego, CA).
 |
RESULTS |
Overexpression of IRS-1 in
-Cells--
We overexpressed IRS-1
in a clonal
-cell line
TC6-F7. The exogenous IRS-1 had a c-Myc
tag at its C terminus and migrated slightly slower than the endogenous
IRS-1 in SDS-gels (Fig. 1A, lanes
1, 3, and 4).
-IRS1-A, one of the 11 stable
transfectants tested, expressed IRS-1 protein two times (199 ± 36%) more than the controls (p = 0.02) (Fig.
1B). Fifty percent of the increase was contributed by the
exogenous IRS1-Myc; the other 50% was from the endogenous IRS-1.
Addition of 100 nM insulin led to rapid tyrosine
phosphorylation of proteins in the 160-180 kDa range that co-migrated
with the IRS-1 protein (Fig. 1C, upper gel, lanes 2, 4, and
6) (also see Ref. 12). A 120-kDa protein (p120) was also
heavily tyrosine-phosphorylated in the
-cells. The abundance of p120
and its extent of tyrosine phosphorylation are relatively stable in the
three
-cell lines (Fig. 1C and data not shown), and it
was therefore used to normalize the level of IRS-1 phosphorylation. Normalized level of IRS-1 tyrosine phosphorylation in the
IRS1-A cells was 2-fold higher than that in the control cells (Fig. 1, C, upper gel, lane 4, and D). This is
proportional to the elevated IRS-1 protein level in the
IRS1-A
cells. These data demonstrated that the excess IRS-1 was
tyrosine-phosphorylated to the same extent as the endogenous substrate
upon insulin stimulation.

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Fig. 1.
Western analysis of the IRS-1 protein and
tyrosine phosphorylation of IRS-1. A, Western analysis.
Cells were lysed as described under "Materials and Methods." For
c-Myc epitope detection (lanes 1 and 2), 250 µg
of total protein were immunoprecipitated and blotted with c-Myc
antibody 9E10. For IRS-1 detection (lanes 3-6), 16 µg of
total protein were loaded in duplicate and blotted with IRS-1 antibody.
Positions of IRS1-Myc and endogenous IRS-1 are indicated to the right.
Eleven clones were analyzed. Data shown are from a representative clone
IRS1-A. B, quantitation of IRS-1 protein. IRS-1 protein
levels by immunoblots were quantitated with a PhosphorImager and
expressed as a percentage of the IRS-1 level in parental TC6-F7
cells. Data are shown as mean ± S.E., n = 6, from
three independent experiments. *, p = 0.02. C, tyrosine phosphorylation. Cells were treated with or
without insulin (15). Twenty µg of total protein were loaded onto SDS
gels for phosphotyrosine and IRS-1 Western analysis.
Anti-phosphotyrosine ( -PY) detection is shown in the
upper panel. The IRS-1 protein was detected in duplicate
blots with -IRS1 antibody and is shown in the lower
panel. Chinese hamster ovary-T is a cell line overexpressing the
insulin receptor and is used as positive control (lanes 7 and 8). D, quantitation of IRS-1 tyrosine
phosphorylation. Radioactivity of the protein bands was quantitated
with a PhosphorImager. The IRS-1 signal was normalized to the p120 band
(IRS1/p120) to correct for small sample loading variation. Data were
collected from two experiments with n = 6 and are shown
as mean ± S.E. **, p = 0.001. Cell lines were as
follows: IRS1-A, stable transfectant expressing the tagged IRS1-Myc
protein; TC6-F7, parental cell; NEO, TC6-F7 cells transfected
with vector only.
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Down-regulation of Insulin Content and Secretion by Overexpression
of IRS-1--
Nine of the transfected clones that showed detectable
c-Myc signal and increased IRS-1 levels, including
IRS1-A, were
tested for their insulin content. They all exhibited lowered insulin content. Data from the
IRS1-A clone are shown in Fig.
2. The
IRS1-A cells had significantly
lowered insulin content: 29.0 ± 2.9 ng/105 cells
versus 114 ± 2.2 ng/105 cells (Neo
control), p = 0.0001 (Fig. 2A). Net insulin
secretion was also reduced 61% at 0 mM glucose
(G0) and 58% at 15 mM glucose (G15)
(Fig. 2B). Insulin secretion of both
IRS1-A and control cell lines was glucose- and extracellular
calcium-dependent. Removal of extracellular
Ca2+ and addition of 1 mM EGTA completely
abolished glucose-stimulated insulin secretion. Addition of either the
phosphatidylinositol 3-kinase inhibitor wortmannin (100 nM)
or the p70 ribosomal S6 protein kinase inhibitor rapamycin (100 nM) did not change insulin content and secretion of
IRS1-A cells (data not shown).

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Fig. 2.
Insulin contents and glucose-stimulated
insulin secretion. Insulin contents and secretion were assayed as
described under "Materials and Methods." A, insulin
content; B, glucose-stimulated insulin secretion. Open
bars, 0 mM glucose; filled bars, 15 mM glucose. All data represent means ± S.E. from at
least two independent experiments each performed in triplicate. Cell
number was determined by cell counting and measurement of cellular DNA
content. **, p = 0.0001. C, fractional
insulin secretion. Secreted insulin at each glucose concentration was
normalized with total cellular insulin content (secreted insulin/total
insulin) and expressed as a percentage. The difference between the two
fractional secretion curves is significant as analyzed with the ANOVA
test (p < 0.05).
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Although the net amount of insulin secreted by the IRS-1-overproducing
cells was reduced as mentioned above, glucose-stimulated fractional
insulin secretion (the ratio of secreted insulin/total insulin content,
expressed as a percentage) was significantly increased in
IRS1-A
cells (Fig. 2C). This increased fractional insulin secretion
was glucose-dependent. At 0 mM glucose,
fractional insulin secretion of the
IRS1-A cells (4.3 ± 0.9%)
was not significantly different from that of the Neo control (3.0 ± 0.6%) (p > 0.5). At glucose concentrations above 1 mM (stimulatory glucose concentrations for this
-cell
line), fractional insulin secretions were increased more than 2-fold
compared with the Neo control (Fig. 2C) (p < 0.04).
These data indicate that overexpression of IRS-1 may enhance the
capacity of the
-cell to secrete insulin under stimulatory glucose
concentrations. Neither wortmannin (100 nM) nor rapamycin (100 nM) affected glucose-stimulated fractional insulin
secretion (data not shown).
Glucose Responsiveness of the
-Cells--
To determine whether
the reduced insulin content and secretion were due to reduced glucose
sensitivity, we examined glucose responsiveness of the
IRS-1-overproducing
-cells. Glucose strongly stimulates
-cell
metabolism, and its effect is reflected by the MTT assay (36, 37). As
shown in Fig. 3A, addition of
15 mM glucose doubled MTT values in both the
IRS1-A and
the Neo control cells. No significant difference was detected between
the two cell lines.

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Fig. 3.
Glucose responsiveness of the
-cells. A, MTT assay; B,
cellular ATP content. Cellular ATP content was measured by HPLC as
described under "Materials and Methods" and elsewhere (38).
Open bars, 0 mM glucose; filled bars,
15 mM glucose. C, glucose-dependent
insulin secretion. Insulin secretion at different glucose
concentrations were normalized with the maximal insulin secretion at 30 mM glucose and expressed as a percentage of the maximum.
Glucose response curves from the two cell lines were not significantly
different (one-way ANOVA test, p > 0.28). All data
represent means ± S.E. from at least two independent experiments,
each performed in triplicate.
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Glucose metabolism also results in the production of ATP. An increased
cellular ATP/ADP ratio is a critical event triggering glucose-stimulated insulin secretion. To determine whether IRS-1 overexpression affects the ability of the
-cell to produce ATP upon
glucose stimulation, we measured cellular nucleotide contents using
HPLC. As indicated in Fig. 3B, glucose increased cellular ATP content in both Neo control and
IRS1-A cells: 1.7 ± 0.3- and 2.0 ± 0.4-fold increase respectively. No statistical
difference was detected (p > 0.05).
Glucose-stimulated insulin secretion was also measured at different
glucose concentrations ranging from 0 to 30 mM. Insulin secretion at 30 mM glucose was set as the maximum. Insulin
secretion at other glucose concentrations was expressed as a percentage of the maximum. The data were fitted to generate sigmoidal
glucose-response curves for the
IRS1-A and Neo control cells (Fig.
3C). No significant difference was detected between the two
cell lines (two-way ANOVA test, p > 0.28). These data
indicate that overexpression of IRS-1 did not change
-cell glucose
sensitivity or its glucose metabolism in general.
Down-regulation of Insulin Biosynthesis at Translational
Level--
IRS-1-overproducing cells exhibited a reduced rate of
insulin biosynthesis both in the absence or the presence of 16 mM glucose. Addition of 16 mM glucose increased
(pro)insulin as well as the total protein biosynthesis in
-cells
(Fig. 4A).
[3H]Leucine incorporation into total protein at 0 mM glucose (G0) was 3.4 ± 0.4 × 106 dpm/mg of protein (
IRS1-A) and 3.0 ± 0.3 × 106 dpm/mg of protein (control), and at 16 mM glucose (G16), it was 5.7 ± 0.5 × 106 dpm/mg protein (
IRS1-A) and 5.6 ± 0.5 × 106 dpm/mg of protein (control). However, insulin
biosynthesis was significantly reduced as judged by insulin-specific
fractional biosynthesis (insulin/total trichloroacetic
acid-precipitable proteins) (Fig. 4B) at both basal and
glucose stimulated states. Insulin biosynthesis was reduced 66% in the
IRS1-A cells compared with the control. This down-regulation is
specific to the translational regulation. Insulin mRNA levels in
IRS1-A and control cells were similar as measured with the RNA
protection assay (42, 43): 100 ± 19% (control) versus
132 ± 60% (
IRS1-A) (n = 10, p > 0.1). These data demonstrated that overexpression of IRS-1 in
-cells decreased the rate of insulin biosynthesis at the
translational level but did not change the steady-state insulin
mRNA level.

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Fig. 4.
Insulin biosynthesis. A,
glucose-stimulated insulin biosynthesis. Insulin biosynthesis was
measured with [3H]leucine pulse-chase assay.
Insulin-specific radioactivity was immunoprecipitated with anti-insulin
antibody (15). For each cell line, the rate of insulin-specific
[3H]leucine incorporation at 0 mM glucose was
set as basal level. With 16 mM glucose stimulation, the
rate of insulin biosynthesis increased 2-fold in both cell lines.
B, fractional insulin biosynthesis (% of trichloroacetic
acid). Insulin-specific 3H-counts were divided by total
trichloroacetic acid-precipitable 3H-counts under each
condition. Open bars 0 mM glucose; solid
bars, 16 mM glucose. Data are presented as mean ± S.E., n = 7. *, p < 0.001.
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Increased
-Cell Cytosol Ca2+ Level--
An increase
in intracellular Ca2+ is recognized as a key obligatory
step in insulin secretion and has been implicated mechanistically in
insulin release (1, 23). Calcium content in ER has been shown to affect
insulin biosynthesis. Reduced ER Ca2+ content results in
decreased rate of translation initiation (25). It is currently unknown
whether insulin receptor signaling affects
-cell Ca2+
homeostasis. To investigate that, we measured cytosolic
Ca2+ levels in
IRS1-A and control cells using fura-2
Ca2+ indicator as described under "Materials and
Methods." As shown in Fig.
5A, cytosolic free
[Ca2+] in the
IRS1-A cells was increased more than
3-fold both in basal (G0) and 15 mM glucose-stimulated
(G15) conditions: 278 ± 39 nM (
IRS1-A)
versus 81 ± 18 nM (control) at G0
(p < 0.001) and 739 ± 121 nM(
IRS1-A) versus 209 ± 42 nM (control) at G15 (p < 0.001). The peak
value upon glucose stimulation, however, was not different between the
two cell lines: 1886 ± 614 nM (
IRS1-A) versus 1634 ± 512 nM (control).

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Fig. 5.
Cytosol Ca2+ measurements.
Cytosol [Ca2+] was measured as described under
"Materials and Methods" and by Gao et al. (37) under the
conditions indicated by the perifusion procedure bar shown
above the curves. A, effect of IRS-1 on cytosol
free [Ca2+]. Cytosol [Ca2+] in IRS1-A
cells and control cells was measured at 0 mM glucose
(G0) for 3 min and then shifted to 15 mM glucose
plus 1 mM carbachol (G15+CCH). Data are
expressed as mean ± S.E., n = 30. B,
effect of the insulin receptor on cytosol free [Ca2+].
The IR-overexpressing cell lines were established as described (15).
TC6-F7 and NEO are parental and vector control cells, respectively.
AK-S2, transfected TC6-F7 cells overexpressing the kinase-inactive
insulin receptor AK1018; IR-S2, transfectants overexpressing the wild
type insulin receptor. For each cell line, at least 30 individual cells
were measured in three different experiments. Differences among the
cell lines were analyzed by one-way ANOVA. **, p < 0.01. C, the effect of thapsigargin on free cytosol
[Ca2+]. The cells were perifused with KRB without glucose
(G0) for 1 min, shifted to KRB plus 200 nM
thapsigargin (G0+THP) for 5 min, and then incubated with 15 mM glucose, 1 mM carbachol and 200 nM thapsigargin (G15+CCH+THP). Data were
obtained from two experiments with at least 20 individual cells
measured. D, effect of thapsigargin. Cytosol
[Ca2+] for the first 60 s (at the G0
condition in B) and from 240 to 360 s
(G0+THP) were averaged and are presented as mean ± S.E.: Cytosol [Ca2+] was significantly different (**,
p < 0.01) between two cell lines at 0 mM
but not at 200 nM thapsigargin.
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To determine whether this increased cytosolic [Ca2+] is
specific to insulin receptor signaling, we examined cytosolic
Ca2+ levels in the insulin receptor-overproducing cells
(Fig. 5B). We have previously established
-cell lines
overproducing the wild type IR (the IR-S2 cells) or the
kinase-deficient IR (the AK-S2 cells) (15). Compared with control
cells, expression level of IR was 4-fold and 2-fold higher in the IR-S2
and AK-S2 cells, respectively (15). Cytosolic [Ca2+] in
control cell lines
TC6-F7 and NEO was at 90 ± 9 and 88 ± 8 nM, respectively. The cells overexpressing
the kinase-deficient insulin receptor (AK-S2) had a
[Ca2+] of 92 ± 11 nM similar to the
controls. The cells overproducing the wild type IR (IR-S2) had a
significantly elevated Ca2+ level: 135 ± 16 nM (p < 0.01 compared with controls, a
50% increase). These data demonstrated that elevated cytosol
[Ca2+] is insulin receptor kinase-dependent.
Overexpression of IR and IRS-1 both increased cytosol
[Ca2+] in
-cells.
ER Ca2+-ATPase plays an important role in regulating
cytosol [Ca2+]. It sequestrates cytosolic
Ca2+ into endoplasmic reticulum in an
ATP-dependent manner (44-46) therefore lowering
[Ca2+]i. Thapsigargin is a specific inhibitor of
the ER Ca2+-ATPase (31, 47). Addition of thapsigargin to
the
-cells prevented ER Ca2+ uptake, therefore leading
to elevated cytosol free [Ca2+] (32). As shown in Fig.
4C, addition of 200 nM thapsigargin to the
control
-cell
TC6-F7 led to an increase in
[Ca2+]i: from the basal level of 107 ± 7 nM to 184 ± 11 nM (p < 0.05) (Fig. 5, C and D). In the
IRS1-A cells,
however, thapsigargin had no effect on cytosol free
[Ca2+]: 223 ± 35 nM (basal)
versus 194 ± 36 nM (200 nM
thapsigargin). Because addition of thapsigargin raised
[Ca2+]i to the same level as that caused by
overexpression of IRS-1, these data indicated that altered ER
Ca2+ uptake may be the major cause of elevated
[Ca2+]i in IRS-1-overproducing cells.
Inhibition of Endoplasmic Reticulum Ca2+
Uptake--
To determine whether endoplasmic reticulum
Ca2+ uptake is affected by IRS-1, we directly measured ER
Ca2+ uptake with permeabilized
-cells. Calcium uptake by
endoplasmic reticulum can be directly measured with radioactive
45Ca2+. We assayed ER Ca2+ uptake
at two Ca2+ concentrations: 100 nM (basal
condition) and 500 nM (equivalent to glucose-stimulated
-cells). As shown in Fig. 6,
Ca2+ uptake in
IRS1-A was significantly reduced at both
Ca2+ concentrations compared with control. At 100 nM [Ca2+], ER Ca2+ uptake for the
control and
IRS1-A cell was 4.28 ± 0.89 and 2.77 ± 0.22 nmol/mg of protein, respectively (a reduction of 35%)
(p < 0.05). At 500 nM
[Ca2+], the uptake was 11.42 ± 1.65 (control) and
7.15 ± 0.90 (
IRS1-A) nmol/mg of protein (p = 0.01), a reduction of 37%. Similar ER Ca2+ uptake results
were also obtained from two additional IRS-1-overproducing clones (data
not shown). These data clearly demonstrated that overexpression of
IRS-1 reduced endoplasmic reticulum Ca2+ uptake and
therefore lowered ER Ca2+ content.

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Fig. 6.
Endoplasmic reticulum Ca2+
uptake. ATP-dependent endoplasmic reticulum
Ca2+ uptake was measured with permeabilized -cells as
described under "Materials and Methods." Open bars, the
NEO control cells; solid bars, IRS1-A cells. Data shown
are mean ± S.E. from two experiments each in triplicate. *,
p = 0.04; **, p = 0.01.
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Inhibition of
-Cell Proliferation--
The insulin receptor
signaling pathway is also implicated in mitogenic regulation (49, 50).
To determine how overexpression of IRS-1 affects
-cells growth, we
used the [3H]thymidine incorporation assay (51) to assess
-cell proliferation (Fig. 7).
IRS1-A cells exhibited a 32 ± 4% (n = 6)
decrease in the rate of cell proliferation as measured by
[3H]thymidine incorporation (6,842 ± 513 dpm/µg
DNA) compared with the passage-matched
TC6-F7 control cells
(10,114 ± 890 dpm/µg DNA) (p = 0.02) in the
presence of 10% serum. In the absence of serum, the growth rate of
IRS1-A was reduced 40% compared with the control (1,895 ± 167 dpm/µg DNA,
IRS1-A versus 3,183 ± 274 dpm/µg
DNA, control; n = 6) (p = 0.007). No
increase in
-cell apoptosis was observed when measured with terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (52)
(data not shown). These data demonstrated that IRS-1 negatively
regulates
-cell proliferation.

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Fig. 7.
-Cell proliferation assay.
-Cell proliferation was determined by incorporation of
[3H]thymidine in the presence (solid bars) or
the absence (open bars) of 10% fetal bovine serum
(FBS). Data are shown as mean ± S.E.,
n = 12. *, p = 0.02; **,
p = 0.007.
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DISCUSSION |
Earlier studies had suggested that the insulin receptor may be
present in
-cells and that it could function to regulate insulin secretion. For many years, however, this concept was viewed as controversial in the absence of definitive evidence identifying the
insulin receptor in the
-cells. Our group has recently demonstrated that the various components of the insulin receptor signaling pathway
are indeed present in
-cells, including the insulin receptor and
IRS-1 (12, 13). Furthermore, we had shown that glucose-induced insulin
secretion activates the
-cell surface insulin receptor tyrosine
kinase and its intracellular signal transduction pathway and had
proposed that this represented a novel autocrine mechanism for the
regulation of
-cell function (12). However, the physiological role
of this pathway in the
-cell has been difficult to elucidate because
the
-cell is not a classical insulin target tissue, and there is
scant evidence that insulin regulates its own secretion. Very recently,
it has been shown that one role of the insulin receptor signaling
pathway in
-cells is regulation of
-cell growth because
disruption of IRS-2 leads to
-cell deficiency at birth and diabetes,
and it has been proposed that IRS-2-dependent signaling
pathways are involved in
-cell neogenesis, proliferation, and
survival (17). In contrast, mice heterozygous for null alleles of the
insulin receptor and IRS-1 become diabetic and develop
-cell
hyperplasia (21). Other studies have also shown that insulin receptor
signaling in the
-cell can regulate insulin gene transcription, as
well as autoregulation of protein synthesis via PHAS-1 phosphorylation
(14, 16). Thus, the insulin receptor signaling pathway of the
-cell
appears to have multiple physiological effects.
To identify the role of IRS-1 in insulin secreting
-cells, we
overexpressed IRS-1 in a clonal
-cell line
TC6-F7. Our data demonstrates that IRS-1 is involved in regulating Ca2+
homeostasis, insulin secretion, insulin biosynthesis, and
-cell proliferation and that elevated expression of IRS-1 induces
-cell failure. This is the first study to demonstrate that a 2-fold overexpression of IRS-1 in
-cells increases
-cell cytosol
Ca2+ levels and reduces ER Ca2+ content. These
findings are significant because the ER is one of the major
compartments for intracellular Ca2+ storage. Elegant
earlier experiments have shown that the ER is actively involved in
regulating intracellular Ca2+ in the nanomolar range (27,
28). In
-cells, it is widely established that glucose stimulation
results in an increase in intracellular Ca2+, a required
step for insulin secretion. Typically, basal Ca2+
concentrations are in the 80-100 nM range, and following
glucose stimulation, they increase 3-5-fold. Once the stimulation is
removed, the ER sequesters excess cytosolic Ca2+, and the
Ca2+ levels return to baseline. The ER
Ca2+-ATPase is the main pump responsible for
Ca2+ uptake into the ER. Its biochemical characteristics
have been extensively described, and it is implicated in the regulation of intracellular Ca2+ homeostasis. Recently, two isoforms
of the Ca2+-ATPase, SERCA2 and SERCA3, have been localized
to the islet. Furthermore, SERCA3 expression is reduced in the GK rat,
a nonobese model of type 2 diabetes (44-46).
Our observations that thapsigargin, an ER
Ca2+-ATPase-specific inhibitor, increased cytosolic
Ca2+ levels in the control cells, but not in the
IRS-1-overproducing cells (Fig. 5, C and D)
suggest that ER Ca2+-ATPase in the
IRS1-A cells could
have been suppressed by IRS-1 overproduction. This is also strongly
supported by the observed decrease in
IRS1-A ER Ca2+
uptake (an indirect measurement of Ca2+-ATPase) and the
fact that Ca2+ release from the ER (induced by A23187, data
not shown) is not changed in
IRS1-A cells. A possible explanation
for IRS-1-induced inhibition of ER Ca2+-ATPase is based on
an elegant study by Kahn and co-workers (33), who have identified
SERCA1 in skeletal muscle and SERCA2 in cardiac muscle as novel IRS-1-
and IRS-2-binding proteins. Importantly, this interaction is
insulin-dependent (maximal at 100 nM insulin and at 5 min of stimulation), and requires tyrosine phosphorylation of
IRS-1. Whether there is a similar interaction between IRS-1 and the
-cell ER Ca2+-ATPase still remains to be determined,
although our data suggest that such an interaction could result in
decreased ER Ca2+-ATPase activity, leading to reduced ER
Ca2+ uptake in
-cells and increased cytosolic
Ca2+ concentrations.
Our study provides a novel functional link between the IRS-1 signaling
pathway and the stimulus-secretion pathway in
-cells, which we
believe to be physiologically significant. We postulate that under
basal conditions in the
-cell this pathway is not activated.
However, once glucose or other secretagogues stimulate insulin
secretion, the released insulin will feed back to the
-cell insulin
receptor and activates the associated signal transduction pathway.
Increased signaling results in IRS-1 tyrosine phosphorylation (12) and
subsequent inhibition of ER Ca2+ uptake as shown by this
study. Decreased Ca2+ fluxes into the ER can then increase
cytosolic Ca2+ and further facilitate the maintenance of
increased Ca2+ levels due to secretagogue-induced
Ca2+ influx from the extracellular space. We believe that
our studies have identified a novel pathway of autocrine regulation of
intracellular Ca2+ homeostasis and insulin secretion in the
-cell of the endocrine pancreas.
Insulin secretion and cellular insulin content were significantly
reduced in the cells overproducing IRS-1. This is most likely the
consequence of reduced insulin biosynthesis. Elevated expression of
IRS-1 inhibits insulin biosynthesis at the translational level as
measured by the [3H]leucine labeling assay. This
inhibition is likely due to the reduced ER [Ca2+] found
in the IRS-1-overproducing cells. A similar finding that ER
Ca2+ affects insulin biosynthesis has been reported before
(25, 26). The insulin mRNA level in the IRS-1-overexpressing cells was similar to the controls as measured with RNA protection assay. It
suggests that IRS-1 may not be involved in mediating activation of
insulin gene transcription in
IRS1-A cells. A recent report suggests
that IRS-2 may mediate signals for insulin gene transcription (16).
Augmented expression of IRS-1 also leads to inhibition of
-cell
growth.
IRS1-A cells exhibited a 32 ± 4% decrease in the rate
of cell proliferation as measured by [3H]thymidine
incorporation compared with the control. These observations are
supported by the finding that loss of IRS-1 function leads to
-cell
hyperplasia and hyperinsulinemia in the IRS-1 knockout animals (21,
48).
-Cell function is also regulated by the insulin receptor
substrate IRS-2. Loss of IRS-2 function results in
-cell failure,
including reduced
-cell growth and decreased insulin secretion.
Deletion of IRS-2 leads to development of diabetes in transgenic mice
(4, 17). It appears that insulin receptor signaling differentially
regulates
-cell function via different substrates. The net output of
insulin receptor signaling in
-cells may therefore depend on the
relative strength of different substrate signals. Abnormalities in the
insulin receptor substrates, e.g. altered IRSs expression
levels, may directly contribute to
-cell failure in diabetes.