Section 5: ␤-Cell Stimulus-Secretion Coupling:
Hormonal and Pharmacological Modulators
Insulin Receptor Signaling and Sarco/Endoplasmic
Reticulum Calcium ATPase in ␤-Cells
Prabakhar D. Borge, Jacob Moibi, Scott R. Greene, Matteo Trucco, Robert A. Young, Zhiyong Gao,
and Bryan A. Wolf
Glucose is the main physiological secretagogue for insulin secretion by pancreatic ␤-cells, and the major
biochemical mechanisms involved have been elucidated.
In particular, an increase in intracellular calcium is
important for insulin exocytosis. More recently, it has
become apparent that the ␤-cell also has many of the
elements of the insulin receptor signal transduction
pathway, including the insulin receptor and insulin
receptor substrate (IRS) proteins 1 and 2. Studies with
transgenic models have shown that the ␤-cell–selective
insulin receptor knockout and the IRS-1 knockout lead
to reduced glucose-induced insulin secretion. Overexpression of the insulin receptor and IRS-1 in ␤-cells
results in increased insulin secretion and increased
cytosolic Ca2ⴙ. We have thus postulated the existence
of a novel autocrine-positive feedback loop of insulin on
its own secretion involving interaction with the insulin
receptor signal transduction pathway and regulation of
intracellular calcium homeostasis. Our current working
hypothesis is that this glucose-dependent interaction
occurs at the level of IRS-1 and the sarco(endo)plasmic
reticulum calcium ATPase, the calcium pump of the
endoplasmic reticulum. Diabetes 51 (Suppl. 3):
S427–S433, 2002
Glucose-induced insulin secretion by the ␤-cell. The
insulin-secreting ␤-cell of the endocrine pancreas has a
central role in regulating glucose homeostasis (1). 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
(2). ␤-Cell metabolism of glucose results in an increase in
From the Department of Pathology and Laboratory Medicine, the Children’s
Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.
Address correspondence and reprint requests to Dr. Bryan A. Wolf, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, 5135 Main, 34th St. and Civic Center Boulevard, Philadelphia, PA
19104-4399. E-mail: [email protected].
Received for publication 18 March 2002 and accepted in revised form 15
April 2002.
BiP, immunoglobulin binding protein; [Ca2⫹]i, intracellular Ca2⫹ concentration; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; ER,
endoplasmic reticulum; GRP78, glucose-regulated protein 78 kDa; IRS, insulin
receptor substrate; KRB, Krebs-Ringer bicarbonate; SERCA, sarco(endo)plasmic reticulum Ca2⫹-ATPase; Syt3, synaptotagmin III.
The symposium and the publication of this article have been made possible
by an unrestricted educational grant from Servier, Paris.
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
the ATP-to-ADP ratio, leading to closure of the ATPsensitive K⫹ channel, depolarization of the ␤-cell, and
influx of extracellular Ca2⫹ through voltage-dependent
Ca2⫹ channels (3). The subsequent increase in intracellular Ca2⫹ then activates insulin exocytosis (Fig. 1). Most
likely, a number of protein kinases, including the calcium
and calmodulin-dependent protein kinase as well as protein kinase C, are involved in these distal steps (4).
The importance of the ␤-cell in the pathogenesis of type
2 diabetes is now clearly recognized (5). Type 2 diabetes is
characterized by 1) insulin resistance in target tissues of
insulin, such as skeletal muscle and adipocytes for glucose
uptake and liver for glucose production, and 2) impaired
insulin secretion (6). Peripheral tissue and hepatocyte
insulin resistance lead to reduced glucose uptake and
decreased inhibition of hepatic glucose production. To
compensate for insulin resistance and to maintain euglycemia, pancreatic ␤-cell insulin secretion increases significantly, resulting in hyperinsulinemia. Eventually, the
␤-cell fails, and overt disease appears. Type 2 diabetes is
believed to be a polygenic disease that is also affected by
various environmental factors such diet, physical activity,
and age.
Insulin receptor signal transduction pathway in the
␤-cell. Insulin has a central role in regulating cell metabolism, gene expression, growth, and differentiation (7).
Insulin binds the cell surface insulin receptor, which
consists of two extracellular 135-kDa ␣-subunits that bind
insulin and two intracellular 95-kDa ␤-subunits that have
intrinsic tyrosine kinase activity. After the insulin binds to
its receptor, there is autophosphorylation of tyrosyl residues on the ␤-subunits and phosphorylation of cellular
proteins, including insulin receptor substrate (IRS)-1 and
-2 (7). The subsequent cascade of signaling events results
in translocation of the insulin-responsive glucose transporter GLUT4 in muscle and adipocytes.
In 1995, we first reported the presence of the insulin
receptor in insulin-secreting ␤-cells and showed that glucose-induced insulin secretion activates the ␤-cell surface
insulin receptor tyrosine kinase and its intracellular signaling transduction pathway (8,9). In the last few years,
there has been a rapidly growing body of evidence confirming our original findings that the insulin receptor
signaling pathway is active in pancreatic ␤-cells and plays
an important role in ␤-cell regulation (5,8 –15) (Table 1).
S427
INSULIN SIGNALING AND SERCA IN ␤-CELLS
FIG. 1. Model of glucose-induced insulin secretion from ␤-cells. See text for details.
KATP, ATP-sensitive Kⴙ channel; SUR, sulfonylurea receptor.
Activation of the ␤-cell insulin receptor results in rapid
tyrosine phosphorylation of the insulin receptor ␤-subunit
and the IRS proteins (8). Deletion of insulin receptor
results in neonatal death in mice (16) and leprechaunism
in humans (17). Mice with heterozygous null alleles of
insulin receptor and IRS-1 (IR/IRS-1⫹/⫺) exhibit hyperinsulinemia and ␤-cell hyperplasia, and they develop overt
diabetes (18). Knockouts of IRS-1 and -2 produce different
effects. Inactivation of IRS-1 (IRS-1⫺/⫺) leads to mild
insulin resistance, hyperinsulinemia, and ␤-cell hyperplasia with no overt diabetes syndrome (5,13). In contrast,
inactivation of IRS-2 (IRS-2⫺/⫺) results in ␤-cell failure
and causes type 2 diabetes (13,14). The differential effects
of IRS-1 and -2 knockout indicate the two major IRSs
mediate differential signals in ␤-cells. However, the mechanisms accounting for such differential regulation and for
IRS-1 function in the ␤-cell are still unknown.
Our prior studies (Table 2) have shown that in ␤-cells
stably overexpressing twofold IRS-1, Ca2⫹ homeostasis
was perturbed, with an increase in cytosolic Ca2⫹ and
increased insulin secretion (19). Further investigation revealed that the increase in cytosolic Ca2⫹ was due to
inhibition of sarco(endo)plasmic reticulum Ca2⫹-ATPase
(SERCA), a protein responsible for Ca2⫹ uptake into the
endoplasmic reticulum (ER) lumen. When the control
␤-cell line was treated with thapsigargin, a SERCA inhibitor, the cytosolic Ca2⫹ levels increased to the same level
as the ␤-IRS1 cell line. Furthermore, ER calcium uptake in
a digitonin-permeabilized cell system was reduced in the
␤-IRS1 cells compared with controls. This increase in
cytosolic Ca2⫹ was also seen in a cell line overexpressing
the insulin receptor, but not in a cell line overexpressing a
kinase-deficient mutant of the insulin receptor (11). We
thus postulated the existence of a novel autocrine loop of
insulin on insulin secretion from the ␤-cell, mediated by
activation of the insulin receptor signaling pathway and
IRS-1. In isolated mouse islets, insulin treatment of islets
caused an increase in intracellular Ca2⫹ within minutes,
with concomitant insulin exocytosis (20 –22). In addition,
studies with a ␤-cell–specific insulin receptor knockout
mouse and IRS-1– deficient ␤-cell lines show that glucoseinduced insulin secretion is lost under those conditions
(15,23). Thus, there is a convergence of data obtained in
animal and cellular models that demonstrate the existence
TABLE 1
Effect of manipulation of the insulin receptor signaling pathway on insulin secretion
Gene alteration
IRS 1 knockout (IRS-1⫺/⫺)
Heterozygous IR and IRS-1
knockout (IR⫹/⫺/IRS-1⫹/⫺)
IRS-1– and ␤-cell specific
glucokinase knockout
(IRS-1⫺/⫺/GK⫹/⫺)
IRS-2 knockout (IRS-2⫺/⫺)
Pancreatic-specific IR
knockout (IR⫺/⫺)
Heterozygous IR, IRS-1, and
IRS-2 knockout
(IR/IRS-1/IRS-2⫹/⫺)
Effect on insulin secretion
Reduced glucose-stimulated
insulin secretion
Effect on ␤-cell
mass
␤-Cell hyperplasia
␤-Cell hyperplasia
Reduced glucose-stimulated
insulin secretion
␤-Cell hyperplasia
No secretory defects
Reduced ␤-cell
mass, impaired
survival at
differentiation
Decrease in islet
size
Loss of acute first-phase
glucose-stimulated insulin
secretion response
␤-Cell hyperplasia
Diabetic phenotype
References
Insulin resistance:
hyperinsulinemia, no
overt diabetes
Insulin resistance:
overt type 2 diabetes
Insulin resistance:
hyperinsulinemia,
overt type 2 diabetes
Insulin resistance:
␤-cell failure, overt
type 2 diabetes
43,44
Hyperinsulinemia,
impaired glucose
tolerance
Insulin resistance:
overt type 2 diabetes
15,23
18
45
13
46
IR, insulin receptor.
S428
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
P.D. BORGE AND ASSOCIATES
TABLE 2
Effect of overexpression of the insulin receptor and IRS-1 on
␤-cell function
Glucose metabolism
Insulin secretion
Insulin content
Insulin gene expression
Insulin biosynthesis
␤-Cell growth
Cytosolic Ca2⫹
Insulin receptor
overexpression
(reference 11)
IRS-1
overexpression
(reference 19)
No change
Increased
Increased
Increased
No change
No change
Increased
No change
Increased
Decreased
No change
Decreased
Decreased
Increased
of a positive feedback loop of insulin on glucose-induced
insulin secretion that is mediated by an interaction of the
insulin signal transduction pathway with the Ca2⫹-homeostatic mechanisms of the ␤-cell.
Role of SERCA. Cellular Ca2⫹ is a critical element in
␤-cell function (1,24,25). Abnormal intracellular concentration of Ca2⫹ ([Ca2⫹]i) is a common defect in both type
1 and type 2 diabetes (26). Altered Ca2⫹ metabolism has
also been reported to affect ␤-cell function, including
insulin biosynthesis (27). The ER plays an important role
in the regulation of [Ca2⫹]i (24). Typically, basal Ca2⫹
concentrations are in the 80 –100 nmol/l range, and after
glucose stimulation they increase three- to fivefold. Once
the stimulation is removed, the ER sequesters excess
cytosolic Ca2⫹, and the Ca2⫹ levels return to baseline.
SERCA is the major calcium pump that sequestrates
cytosolic Ca2⫹ into ER lumen. Its biochemical characteristics have been extensively described, and its implication
in the regulation of intracellular Ca2⫹ homeostasis is
recognized. Thapsigargin, a nonphorboid tumor promoter,
specifically inhibits ER Ca2⫹-ATPase activity and leads to
elevated cytosolic Ca2⫹ in ␤-cells and enhanced short-term
glucose-stimulated insulin secretion (28).
SERCA2 and -3 have been found in ␤-cells (28 –31). Both
SERCA2 and -3 gene transcripts have splicing variants
resulting in distinct protein isoforms. SERCA2a is the
muscle isoform and is expressed in slow-twitch skeletal
muscle. SERCA2b differs from SERCA2a only in the
COOH-terminal part and is widely expressed in nonmuscle
tissues (32). SERCA3 is coexpressed with SERCA2b in
nonmuscle tissues and has three splicing isoforms:
SERCA3a, -b, and -c (33). SERCA3 has a lower apparent
affinity for Ca2⫹ than other members of the SERCA family
(34). SERCA3b and -c have even lower apparent affinities
for Ca2⫹ than SERCA3a (33). Recent studies show that
loss of SERCA activities and reduction of SERCA3 gene
expression in ␤-cells are associated with diabetes in db/db
mice (30) and GK rats (35). Finally, Varadi et al. (36) have
identified four rare missense mutations of the SERCA3
gene in type 2 diabetic patients recruited for the U.K.
Prospective Diabetes Study: Gln1083 His, Val6483 Met,
Arg6743 Cys, and Ile7533 Leu, suggesting that the
SERCA3 gene locus contributes to genetic susceptibility to
type 2 diabetes.
Interaction of IRS-1 and SERCA. IRSs may directly
interact with ER Ca2⫹-ATPase (SERCA1 and -2) in a
tyrosine phosphorylation– dependent manner in muscle
and heart (37). This finding suggests that insulin may
regulate ER Ca2⫹-ATPase activity, therefore influencing
cellular Ca2⫹ homeostasis. To dissect the role of IRS-1 in
␤-cell function, we have overexpressed IRS-1 in an insulinsecreting ␤-cell line (Table 2). We showed 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 (19). We suggested a novel functional link
between the IRS-1 signaling pathway and the stimulussecretion pathway in ␤-cells, which we believe to be
physiologically significant. Under basal conditions in the
␤-cell, this pathway is not activated. However, once glucose or other secretagogues stimulate insulin secretion,
the released insulin will feedback to the ␤-cell insulin
receptor and activate the associated signal transduction
pathway. Increased signaling results in IRS-1 tyrosine
FIG. 2. Insulin receptor signaling in the
␤-cell. In this scheme, insulin has a positive
autocrine feedback loop on its own secretion
in the presence of glucose. Secreted insulin
binds to the insulin receptor present on the
plasma membrane of the ␤-cells, which initiates a signal transduction cascade mediated
by the IRS proteins. Tyrosine phosphorylation of IRS-1 results in inhibition of the
activity of SERCA located in the ER, which in
turn causes an increase in cytosolic calcium
and insulin secretion. Grb2, growth factor
receptor– bound protein 2; KATP, ATP-sensitive Kⴙ channel; PI3K, phophatidylinositol
3-kinase; SUR, sulfonylurea receptor.
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
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INSULIN SIGNALING AND SERCA IN ␤-CELLS
phosphorylation and subsequent inhibition of ER Ca2⫹
uptake. Decreased Ca2⫹ fluxes into the ER can then
increase cytosolic Ca2⫹ and further facilitate the maintenance of increased Ca2⫹ levels due to secretagogueinduced Ca2⫹ influx from the extracellular space (Fig. 2).
More recently, we have used three complementary
techniques to show that IRS-1 colocalizes to intracellular
membranes in pancreatic ␤-cell lines (P.D.B., B.A.W.,
unpublished observations). Subcellular fractionation of
the ␤TC6-F7 and the ␤-IRS1 cell lines showed that IRS
proteins localized to the intracellular membranes in the
high-speed pellet. Furthermore, overexpressed IRS-1 in
the ␤-IRS1 cell line was primarily in the intracellular
membrane fraction. Colocalization of IRS-1 with the ER
marker protein immunoglobulin binding protein (BiP)/
glucose-regulated protein 78 kDa (GRP78) was demonstrated by confocal microscopy. Although the signals for
the IRS proteins and ER marker clearly overlapped, there
was some additional immunofluorescence staining in
other portions of the cell. Finally, immunoelectron microscopy experiments showed that IRS-1 is present in ERderived microsomal vesicles from ␤-cell lines, as
confirmed by its coimmunostaining with BiP/GRP78 and
SERCA3b. Taken together, these data present a strong
case for IRS protein localization to intracellular membranes, in particular the ER, in pancreatic ␤-cell lines. The
lack of complete localization of IRS-1 and -2 to the ER by
confocal microscopy indicates that IRS proteins may
distribute to several pools within the cell. Considering the
dynamic nature of insulin receptor signaling, it is possible
that IRS proteins translocate to several locations in the
cell in response to upstream signals. However, the majority of both phosphorylated and nonphosphorylated IRS
proteins are maintained in intracellular membranes. IRS
proteins in this pool can interact with other proteins in the
same location and potentially form large protein complexes that are insoluble in detergents. An example of this
comes from one study reporting that IRS-1 can bind to the
␴3A subunit of the AP-3 adaptor protein complex, resulting in intracellular membrane localization (38). The localization of IRS proteins to the ER increases the likelihood
of a direct interaction with SERCA isoforms in ␤-cells.
Indeed, IRS proteins have previously been shown to bind
to several SERCA isoforms, including the ␤-cell isoform
SERCA2b (37), and the pancreatic ␤-cell SERCA isoform
SERCA3b coimmunoprecipitates with IRS-1 when expressed in a CHO T-cell line (P.D.B. and B.A.W., unpublished observations). Furthermore, this interaction is
enhanced when IRS-1 is phosphorylated by insulin stimulation. Overexpression of IRS-1 in this system results in
further enhancement of IRS-1 binding to SERCA3b, which
could explain why IRS-1 overexpression in ␤-IRS1 cells
inhibits SERCA function (19).
In contrast to our findings, a recent study demonstrates
that insulin can cause ␤-cell hyperpolarization and diminish cytosolic calcium oscillations in mouse islets incubated in 10 mmol/l glucose via an increase in whole-cell K⫹
conductance (39). This effect could be mediated through a
phosphatidylinositol 3-kinase– dependent pathway. However, in another study, mouse islets incubated with the
insulin-mimetic compound L-783,281 had increased insulin
release at 11 mmol/l glucose (40). There was no change in
S430
the pulsatile release of insulin under these conditions.
Treatment with the insulin-mimetic at 3 mmol/l glucose
decreased the pulse frequency of insulin release, but not
the amount. Although Aspinwall et al. (21) showed that
insulin-induced insulin exocytosis could occur at 3 mmol/l
glucose, their experiments measured exocytosis events
and not actual insulin release, which may have been
minimal at that glucose concentration. Thus, although
insulin can effect the electrophysiology of the ␤-cell in a
negative fashion, it can also increase insulin release. Most
likely, the effects of insulin depend on the extracellular
glucose concentration.
Overexpression of SERCA isoforms in ␤-cells. Because of the importance of SERCA as a downstream
effector of IRS-1, we have studied the effect of overexpressing SERCA isoforms in ␤-cells (J.M., Z.G., and B.A.W.,
unpublished observations). cDNAs encoding rat SERCA2b
and mouse SERCA3b were cloned by PCR into a Histagged destination vector using Gateway cloning technology (Life Technologies). The Gateway cloning system uses
phage ␭– based site-specific recombination instead of restriction endonucleases and ligases, and it was completed
in a two-step reaction, with the initial step generating an
entry clone. The template plasmids were obtained from
Dr. J. Lytton (pMT2mSERCA3b; University of Calgary,
Alberta, Canada) and F. Wuytack (pMT2rSERCA2b; Katholieke Universiteit Leuven, Leuven, Belgium). Amplification
of the cDNAs was performed in a two-step PCR. PCR
fragments were gel-purified and subcloned into a Gateway
entry vector (pDONR201; Life Technologies) using BP
Clonase enzyme mix and proteinase K solution. After
transformation into library efficiency DH5␣ and grown on
kanamycin plates, positive clones were sequenced. Plasmid DNAs from clones with correct nucleotide sequence
were purified by Qiagen column chromatography. The
final step in the cloning process was the creation of
expression clones by combining the entry clones
(pDONR.serca2b/pDONR.serca3b) with the destination
vector pDEST26 containing an NH2-terminal histidine fusion protein (Life Technologies). Correct clones were
identified by sequencing and then amplified. The expression constructs were driven by a cytomegalovirus (CMV)
promoter. ␤-TC6 insulinoma cells were transfected with
plasmid DNAs using either FUGENE-6 transfection reagent (Roche Diagnostics, Indianapolis, IN) or by electroporation technique (Gene Pulser II RF Module; BioRad)
and were selected for growth in the presence of 300 ␮g/ml
Geneticin (Life Technologies, Grand Island, NY). Single
colonies of the Geneticin-resistant cells were expanded
and were tested for overexpression of SERCA2b and -3b
by Western blotting, using anti-His and anti-SERCA2b and
-3b peptide antibodies.
Sequencing information showed that we successfully
generated the expression clones of pDT-SERCA2b and
pDT-SERCA3b transgenes under the control of a CMV
promoter. We then used the constructs to create stable
expression in ␤-TC6 insulin-secreting cells and quantify
the endogenous as well as the His-tagged SERCA protein
levels. Analysis of SERCA2b cell lysates showed that the
protein is expressed in the nontransfected wild type, in
cells transfected with the empty vector (control), as well
as in cells with the pDT-SERCA2b construct (Fig. 3A).
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
P.D. BORGE AND ASSOCIATES
FIG. 3. SERCA overexpression in ␤-cells and CHO cells. A: SERCA2b
levels measured by immunoblotting in ␤TC6 insulin-secreting cells
transfected with SERCA2b. pDT, empty vector; pS2b, clones stably
transfected with SERCA2b; WT, wild type. B: SERCA3b levels measured by immunoblotting in ␤TC6 and CHO cells transfected with
SERCA3b. Cho3b, CHO cells transfected with SERCA3b; ChoT, parent
CHO T-cell line; pDT, empty vector; pDT-S3bb, ␤-cell clones stably
transfected with SERCA3b. C: Anti-His immunoprecipitation and
SERCA3b immunoblotting of ␤-TC6 and CHO cells transfected with
His-tagged SERCA3b.
However, further analysis showed that the level of expression was not significantly different in control cells and the
cells stably transfected with pDT-SERCA2b. Furthermore,
immunoprecipitation of the His-tagged SERCA2b protein
using anti-His antibody and immunoblotting with either
anti-His or anti-SERCA2b revealed lack of expression of
the pDT-SERCA2b construct to any sufficient amount that
can be detected using the protocol used (data not shown).
Western blot analysis of analysis of SERCA3b also did not
show any significant difference in expression levels between the neo controls and the stable cell lines (Fig. 3B,
lanes 3–7). Furthermore, immunoprecipitation complex
data showed that the SERCA3b protein was also not
expressed to any significant extent in the stably transfected ␤-cells compared with the neo controls (Fig. 3C).
To demonstrate that the constructs can be expressed at
all, we transiently or stably transfected CHO cells (which
do not express SERCA3b) with the pDT-SERCA3b plasmid. As shown in Fig. 3B (lanes 1 and 2) and Fig. 3C (lanes
1, 2, 5, and 6), SERCA3b was robustly overexpressed in
CHO cells after transfection (Cho3b), whereas the protein
was not detected in the control nontransfected cells. The
fact that ␤-TC6 cells transfected with SERCA3b survived
antibiotic selection (compared with nontransfected ␤-TC6
cells that died under the same conditions) suggests that
these cells did in fact incorporate the SERCA DNA into
their genome. However, the lack expression of the protein
in these cells (as compared with CHO) may suggest a
teleological tight regulatory control necessary for the
maintenance of calcium homeostasis in the insulin-secreting cells.
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
To determine the effect of overexpression of the SERCA
proteins on cytosolic Ca2⫹, CHO cells stably transfected
with pDT-SERCA3b were plated on 25-mm glass coverslips. After 48 h, the cells were loaded with Fura 2-AM
(acetoxymethylester) in Ca2⫹-free Krebs-Ringer bicarbonate (KRB) (no EGTA) in the presence or absence of 100
nmol/l insulin. After a 40-min incubation, cells were
washed with prewarmed KRB and perifused on top for 10
min with KRB (G15) with or without insulin. We observed
no difference between the SERCA3b-expressing cells and
the vector controls. Thus, it appears that the contribution
of SERCA3b to total calcium regulation in this system is
minimal.
Real-time monitoring of secretory granule trafficking
in ␤-cells using enhanced green fluorescent protein
(EGFP)-tagged synaptotagmin III. Synaptotagmins are
a family of 11 isoforms of a Ca2⫹-mediated phospholipid
binding protein originally identified in neuron secretory
granules (41). Synaptotagmin III (Syt3) has a single transmembrane domain and two Ca2⫹ regulatory C2 domains
that are thought to regulate membrane fusion and membrane budding reactions involved in exocytosis; Syt3 is
sensitive to submicromolar levels of Ca2⫹. This and other
evidence strongly suggests that Syt3 is one of, if not the,
Ca2⫹ sensor in the exocytosis of insulin secretory granules
(42). To further dissect the role of the insulin receptor
signal transduction pathway in insulin exocytosis, we
wished to develop a cell-biological technique to monitor
movement and exocytosis of insulin secretory granules in
real time, by creating a fusion protein of Syt3 and EGFP
(M.T., Z.G., B.A.W., unpublished observations). The Syt3
cDNA was generously provided by Dr. Thomas Südhof
(University of Texas Southwestern Medical Center). This
DNA sequence was inserted into two plasmids that would
produce fusion proteins with the EGFP tag at either the
COOH terminus or the NH2 terminus of the Syt3 protein.
The vectors in which Syt3 DNA was inserted were
pEGFP-C3 (Clontech, Palo Alto, CA) cut with Xma-1
restriction enzyme, resulting in the fusion protein referred
to as Fsyt3, and pEGFP-N3 (Clontech) cut with EcoR-1
restriction enzyme, resulting in the fusion protein referred
to as Syt3F. An pEGFP-C3 empty vector was used as
control and referred to as pEGFP.
␤-TC6 cells were stably transfected with the constructs
using FuGene. Cells transfected with the Fsyt3 plasmid
were referred to as Fsyt3 cells, those transfected with the
Syt3F plasmid were referred to as Syt3F cells, and those
transfected with empty vector were referred to as EGFP
cells. Western blot analysis of the EGFP and Fsyt3 cell
lines demonstrated overexpression of the constructs. Immunogold electron microscopy of the transfected cells
demonstrated colocalization of Syt2 with insulin secretory
granules. When viewed under a confocal microscope,
there was a clear difference in the distribution of fluorescence in the EGFP cells as opposed to the Fsyt3 and Syt3F
cells. The fluorescence within the EGFP cells is diffuse and
spreads throughout the entire cell. In the cells expressing
the fusion protein, however, the fluorescence is concentrated into dense clusters. Actual vesicular trafficking
appeared to be a rather rare event, however. Movies of
⬃60 cells were constructed during the course of this
study, but only a handful of these movies clearly showed
S431
INSULIN SIGNALING AND SERCA IN ␤-CELLS
what appears to be vesicular trafficking. Much optimization, however, is needed to make this technique a useful
and efficient tool.
CONCLUSIONS
In conclusion, we propose the existence of a novel positive-feedback pathway in which insulin can regulate insulin secretion in pancreatic ␤-cells. A key component of this
pathway is interaction between IRS-1 and SERCA, which
regulates intracellular Ca2⫹. IRS-1 is present in the ER and
can directly bind to the ␤-cell isoforms of SERCA, specifically SERCA3b. Insulin stimulation results in increased
binding of IRS-1 to SERCA3b, which inhibits the Ca2⫹ATPase, increases cytosolic Ca2⫹, and augments fractional
insulin secretion. Importantly, this positive-feedback loop
on insulin secretion is dependent on the presence of
glucose. It is conceivable that glucose or products of
glucose oxidation may regulate the interaction of IRS-1
and SERCA by some means yet to be determined. Although the physiological significance of this positive feedback loop is not completely elucidated, it could serve to
prime islets for optimal insulin release.
ACKNOWLEDGMENTS
This work was supported by grants from the American
Diabetes Association and the National Institutes of Health
(DK49814). The Radioimmunoassay Core and the Biomedical Imaging Core of the Penn Diabetes Center are supported by the National Institutes of Health (DK19525).
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