Insulin Secretory Deficiency and Glucose Intolerance in
Rab3A Null Mice*
Kazuro
Yaekura
,
Richard
Julyan
,
Barton L.
Wicksteed
,
Lori B.
Hays
,
Cristina
Alarcon
,
Scott
Sommers
,
Vincent
Poitout
§,
Denis G.
Baskin§,
Yong
Wang¶,
Louis H.
Philipson¶, and
Christopher J.
Rhodes
**
From the
Pacific Northwest Research Institute and
Departments of
Pharmacology and § Medicine,
University of Washington, Seattle, Washington 98122 and the
¶ Department of Medicine, University of Chicago,
Chicago, Illinois 60637
Received for publication, November 6, 2002, and in revised form, December 20, 2002
 |
ABSTRACT |
Insulin secretory dysfunction of the pancreatic
-cell in type-2 diabetes is thought to be due to defective nutrient
sensing and/or deficiencies in the mechanism of insulin exocytosis.
Previous studies have indicated that the GTP-binding protein, Rab3A,
plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A
/
mice develop fasting hyperglycemia and
upon a glucose challenge show significant glucose intolerance coupled
to ablated first-phase insulin release and consequential insufficient
insulin secretion in vivo, without insulin resistance. The
in vivo insulin secretory response to arginine was similar
in Rab3A
/
mice as Rab3A+/+ control animals,
indicating a phenotype reminiscent of insulin secretory dysfunction
found in type-2 diabetes. However, when a second arginine dose was
given 10 min after, there was a negligible insulin secretory response
in Rab3A
/
mice, compared with that in
Rab3A+/+ animals, that was markedly increased above that to
the first arginine stimulus. There was no difference in
-cell mass
or insulin production between Rab3A
/
and
Rab3A+/+ mice. However, in isolated islets,
secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or
fatty acid) was ~60-70% lower in Rab3A
/
islets
compared with Rab3A+/+ controls. Nonetheless, there was a
similar rate of glucose oxidation and glucose-induced rise in cytosolic
[Ca2+]i flux between Rab3A
/
and
Rab3A+/+ islet
-cells, indicating the mechanistic role
of Rab3A lies downstream of generating secondary signals that trigger
insulin release, at the level of secretory granule transport and/or
exocytosis. Thus, Rab3A plays an important in vivo role
facilitating the efficiency of insulin exocytosis, most likely at the
level of replenishing the ready releasable pool of
-granules. Also,
this study indicates, for the first time, that the in vivo
insulin secretory dysfunction found in type-2 diabetes can lie solely
at the level of defective insulin exocytosis.
 |
INTRODUCTION |
Insulin is the major anabolic hormone controlling metabolic
homeostasis, and without an effective supply of insulin diabetes mellitus ensues. Type-1 diabetes occurs as a result of autoimmune destruction of pancreatic
-cells that produce insulin, and type-2 diabetes develops as a result of insulin secretory dysfunction, as well
as insufficient
-cell mass, that no longer compensates for
peripheral insulin resistance (1, 2). The insulin secretory dysfunction
in type-2 diabetes is derived from
-cell secretory abnormalities,
proposed to be either at the level irregular glucose metabolism
required for generating secondary signals necessary to trigger insulin
exocytosis and/or deficiencies in the exocytotic mechanism itself (1,
3). Insulin secretion from the
-cell is highly regulated and only
occurs in response to certain nutrients, hormones, neurotransmitters,
and pharmacological reagents (4). Of these, glucose is the most
physiologically relevant. The secondary signals that emanate from
increased glucose metabolism to stimulate insulin release in
-cells
have been relatively well defined, and of these a rise in cytosolic
[Ca2+]i is a prerequisite (4, 5). Indeed,
increased [Ca2+]i is the necessary signal to
trigger regulated exocytosis in most neuroendocrine cells (6). In
comparison, the mechanism of insulin exocytosis is less well defined.
Several proteins required for insulin exocytosis in
-cells have been
indicated, including SNARE (soluble
N-ethylmaleimide-sensitive factor attachment protein receptors) proteins, analogous to the mechanism of regulated exocytosis in neurons (5). However, how such "exocytotic proteins" interact in
a regulated manner to control insulin exocytosis is currently unclear
(5).
One protein implicated to play a role in control of insulin exocytosis
is the small GTP-binding protein, Rab3A (7). In
-cells, Rab3A is
located on the cytosolic face of
-granules where it is probably
involved in control of
-granule transport and/or exocytosis (8, 9).
Members of the Rab protein family are key to directing vesicular
transport in eukaryotic cells (10). In Rab3A
/
mice,
there is a defect in recruiting synaptic vesicles for exocytosis in
hippocampal neurons (11), and it has been indicated that Rab3A plays a
role in the later stages of synaptic vesicle exocytosis controlling the
efficiency of neurotransmitter release (12). However, although there
are similarities, the mechanism of synaptic vesicle exocytosis is
distinct from that for large dense-core granules (13). Nonetheless,
given the proposed key role that Rab3A plays in controlling insulin
exocytosis, we examined whether there was a deficient insulin secretory
phenotype of Rab3A
/
mice that would not only better
characterize the mechanism of exocytosis in
-cells, but also reveal
novel insight into insulin secretory dysfunction found in type-2 diabetes.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The EasyTagTM
Expre35S35S protein labeling mix from
PerkinElmer Life Sciences, containing 73% of
L-[35S]methionine, was used for islet protein
synthesis radiolabeling. Uridine
5'-[
-32P]trisphosphate (3000 Ci/mmol) was purchased
from Amersham Biosciences. D-[U-14C]glucose (250-360 mCi/mmol) was
purchased from PerkinElmer Life Sciences. GLP-17-36
was purchased from Bachem Inc. (King of Prussia, PA). Rab3A polyclonal
antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and
VAMP-2 antibody was from Calbiochem. The anti-rabbit IgG-horseradish
peroxidase conjugate was from Jackson ImmunoResearch (West Grove, PA).
All other reagents were of analytical grade and obtained from either
Sigma or Fisher.
Animals--
The Rab3A+/+ on a B6 background
(B6129SF2/J) and Rab3A
/
mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). Mice were housed on a 12-h
light/dark cycle and were allowed free access to standard mouse food
and water. Mice were used at 12-20 weeks of age.
Glucose, Arginine, and Insulin Tolerance Tests--
Glucose (1 mg/g), arginine (1 mg/g), and insulin (0.75 milliunits/g) tolerance
tests were performed on 15-17-week-old Rab3A
/
and
Rab3A+/+ mice after an overnight fast by intraperitoneal
injection dose relative to body weight as described (14). Blood samples
were obtained from the tail vein at the times indicated after the
glucose injection. Blood glucose concentrations were measured with a
HemoCue blood glucose analyzer (HemoCue AB, Ängelholm, Sweden),
and plasma insulin levels measured by enzyme-linked immunosorbent
assay (Crystal Chem, Chicago, IL).
Islet Isolation and in Vitro Insulin Secretion
Analysis--
Pancreatic mouse islets were isolated by collagenase
digestion, and insulin secretory activity examined in static or
perifusion incubation studies of isolated islets as described
previously (15), in response to various concentrations glucose, 1 nM GLP-1, 5 µM glyburide, or 125 µM oleate complexed to 1% (w/v)
BSA.1
Histological Analyses--
Pancreata from 16-week
Rab3A
/
and Rab3A+/+ mice were removed and
fixed in 4% paraformaldehyde in 0.1 M phosphate buffer,
rinsed in ethanol, and embedded in paraffin. Serial sections (10 µm) were stained with hematoxyline and eosin or immunostained with either
anti-insulin or a combination of anti-glucagon/anti-somatostatin antibodies for visualization of islet
-cells and
-/
-cells
using a Leica confocal microscope as described previously (16). For each section the number and cross-sectional islet area, and the number
and size of
-cells and
-/
-cells per islet were assessed (2).
Measurement of Glucose Oxidation--
Glucose oxidation assays
were performed as described previously (17). Briefly, groups of 40-50
islets were suspended in 200 µl of Krebs-Ringer bicarbonate buffer,
16 mM HEPES, and 0.1% (w/v) BSA containing 2.8 mM glucose or 16.7 mM glucose in rounded bottom
cups sealed with a rubber-sleeved stopper. Then, ~2 × 105 cpm of [U-14C]glucose in samples
containing glucose as a substrate was added to the islet suspensions.
The cups were then sealed within a 20-ml borosilicate glass
scintillation vial using a rubber-sleeved stopper and incubated for
2 h at 37 °C. Then islet-cell oxidation was halted by the
addition of 20 µl of 100 mM sodium phosphate buffer (pH
6.0) containing 100 µM rotenone to the islets in the cups via the sleeved stoppers. Afterward, 100 µl of HClO4 was
added to the islets in the cup, and 300 µl of 1 M
benzethonium was added to the bottom of the scintillation vials via the
appropriate sleeved stoppers. The samples then were incubated for an
additional 2 h at 37 °C in a shaking water bath. Then the seals
were removed, the cups were discarded, and 10 ml of scintillation
mixture was added to the vials. The vials then were kept at 25 °C
overnight before scintillation counting.
Proinsulin Biosynthesis and Preproinsulin mRNA
Analysis--
Freshly isolated islets from 16-week
Rab3A
/
and Rab3A+/+ mice were incubated for
1 h at 37 °C in 200 µl of Krebs-Ringer buffer (pH 7.4)
containing a basal 2.8 mM or stimulatory 16.7 mM glucose and 0.1% (w/v) BSA. Messenger RNA levels were
analyzed by the RNase protection assay, as described previously (18).
Immunoprecipitaton analysis of proinsulin biosynthesis in isolated
islets pulse-radiolabeled with [35S]methionine was as
described previously (18).
Standard Wide-field Epifluorescence Imaging--
Dual-wavelength
excitation microspectrophotometry was used to measure
[Ca2+]i as described previously (19). Isolated
islets from 18-week Rab3A
/
and Rab3A+/+
mice were loaded with Fura-2 by a 25-min incubation at 37 °C in
Krebs-Ringer buffer containing basal 2.8 glucose and 5 µM Fura-2/AM (Molecular Probes Inc., Eugene, OR)
and then placed into a temperature-controlled perfusion chamber
(Medical Systems Inc.) mounted on an inverted epifluorescence
microscope (Diaphot, Nikon, Inc.) and perifused by a continuous flow
(rate: 2.5 ml/min) of 5% CO2-bubbled Krebs-Ringer buffer
at 37 °C. Groups of islets are visualized with a ×20 quartz objective. Fura-2 dual wavelength excitation at 340 and 380 nm, and
detection of single wavelength emission at 510 nm was accomplished using the Metafluor/Metamorph system (Universal Imaging Corp.); images
were collected with an intensified CCD camera.
Statistical Analysis--
Where appropriate, results are
expressed as a mean ± S.E. Statistical analysis was performed by
unpaired Student's t test or repeated measure analysis of
variance, where p < 0.05 was considered significant.
 |
RESULTS |
Pancreatic
-cells are located in the islets of Langerhans of
the endocrine pancreas. By immunoblot analysis it was first confirmed
that Rab3A protein was indeed absent in isolated Rab3A
/
mice versus Rab3A+/+ control mice compared with
other
-granule proteins involved in insulin exocytosis, such as
VAMP-2 (Fig. 1). At >3 months old, a
glucose-intolerant phenotype emerged in Rab3A
/
mice.
Male Rab3A
/
mice at 4 months old exhibited significant
(p
0.05) fasting hyperglycemia (127.4 ± 3.0 mg/dl; n = 25) compared with male Rab3A+/+
animals (102.2 ± 4.1 mg/dl; n = 15). Fasting
blood glucose levels did not significantly differ in female
Rab3A
/
mice compared with Rab3A+/+ controls
(108.0 ± 4.2 mg/dl; n = 22 versus
102.9 ± 3.2 mg/dl; n = 11, respectively). It is
not uncommon for a diabetic/glucose-intolerant phenotype to be much
more severe in male versus female transgenic mouse models of
this age (20). Intraperitoneal glucose tolerance tests (IPGTT)
indicated that male Rab3A
/
mice were significantly
glucose-intolerant showing an increased excursion in blood glucose in
response to a glucose load compared with control Rab3A+/+
animals (Fig. 2A). Fasting
insulin levels in male Rab3A
/
mice were lower (349 ± 36 pg/ml; n = 16) but not statistically significantly different from male Rab3A+/+ mice (428 ± 56 pg/ml; n = 12). However, in response to a glucose challenge of an IPGTT, Rab3A
/
mice showed a severe
decrease in first phase plasma insulin secretory response accompanied
by a marked ~75% decrease in plasma insulin levels compared with
Rab3A+/+ mice (Fig. 2B). An intraperitoneal
insulin tolerance tests (IPITT) indicated that insulin sensitivity was
unaltered in either male Rab3A
/
mice (Fig.
2C), indicative of minimal insulin resistance in these animals.

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Fig. 1.
Confirmation of Rab3A gene knockout in
Rab3A /
mouse islets and brain. Equivalent total protein (25 µg)
containing lysates of isolated islets and brain from
Rab3A / and Rab3A+/+ mice were analyzed for
Rab3A, and VAMP-2 as a control, protein expression by immunoblot
analysis as described previously (15).
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Fig. 2.
Glucose intolerance and insulin secretory
insufficiency, but normal insulin sensitivity in
Rab3A /
mice. A, intraperitoneal glucose (1 mg/g) tolerance
tests (IPGTT) were performed on 16-h fasted Rab3A / and
Rab3A+/+ mice and glucose levels measured as described
under "Experimental Procedures." A mean ± S.E. of blood
glucose levels are shown, where represents Rab3A /
mice (n = 24), and represents Rab3A+/+
mice (n = 18). B, plasma insulin levels were
also measured during an IPGTT. A mean ± S.E. of plasma insulin
levels are shown, where represents Rab3A / mice
(n = 18), and represents Rab3A+/+ mice
(n = 14). C, IPITT were performed on 16-h
fasted Rab3A / and Rab3A+/+ mice as
described under "Experimental Procedures." A mean ± S.E. of
glucose levels during the IPITT are shown, where represents
Rab3A / mice (n = 15), and represents Rab3A+/+ mice (n = 12).
|
|
In contrast to the IPGTT, an intraperitoneal arginine stimulus (1 mg/g)
showed no apparent deficiency in the in vivo insulin secretory response in Rab3A
/
versus
Rab3A+/+ control mice (Fig.
3A). However, when a second
intraperitoneal dose (1 mg/g) was given 10 min after the first, the
subsequent insulin secretory response to arginine was negated
(p
0.05) in Rab3A
/
mice
compared with an enhanced response in Rab3A+/+
control animals (Fig. 3B). Blood glucose levels in
Rab3A
/
or Rab3A+/+ mice did not appreciably
alter during the in vivo arginine stimulus (Fig.
3C).

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Fig. 3.
Normal in vivo insulin
secretory response to a single arginine stimulus, but ablation of such
a response to a second consecutive arginine stimulus in
Rab3A /
mice. A, a single intraperitoneal arginine (1 mg/g)
stimulation test was performed on 16-h fasted Rab3A /
and Rab3A+/+ mice and plasma insulin levels measured as
described under "Experimental Procedures." A mean ± S.E. of
plasma insulin levels are shown, where represents
Rab3A / mice (n = 16), and represents Rab3A+/+ mice (n = 14).
B, a double intraperitoneal arginine stimulation test was
performed on 16-h fasted Rab3A / and
Rab3A+/+ mice 10 min apart, each a 1 mg/g dose of arginine
as indicated by the arrows, and plasma insulin levels measured. A
mean ± S.E. of plasma insulin levels are shown, where represents Rab3A / mice (n = 16), and
represents Rab3A+/+ mice (n = 14).
C, blood glucose levels were also measured during the double
intraperitoneal arginine stimulation test. A mean ± S.E. of blood
glucose levels are shown, where represents Rab3A /
mice (n = 8), and represents Rab3A+/+
mice (n = 7).
|
|
The insulin secretory-deficient phenotype of the Rab3A
/
mice did not appear to be based at the level of defective
(pro)insulin production. Isolated pancreatic islets from
Rab3A
/
mice had similar insulin content stores to that
of Rab3A+/+ mouse islets (Fig.
4A). Likewise, preproinsulin
mRNA levels were equivalent in islets from Rab3A
/
versus Rab3A+/+ mice (Fig. 4B), and
translational control of glucose-induced proinsulin biosynthesis was
unaffected in isolated Rab3A
/
mouse islets (Fig.
4C). Immunohisotchemical examination of pancreata from
Rab3A
/
and Rab3A+/+ mice indicated no
discernable difference in islet architecture, with insulin-expressing
-cells in the central core of an islet and glucagon expressing
-cells and somatostatin expressing
-cells around the periphery
(Fig. 4). Further analysis of pancreatic serial sections showed no
obvious change between Rab3A
/
and Rab3A+/+
mice in islet number per pancreas, islet size, number of
-cells, and
non-
-cells per islet or size of
-cells and non-
-cells per islet (data not shown). The weight and size of pancreata from Rab3A
/
and Rab3A+/+ mice were equivalent,
and as such it is reasonable to deduce that there was no change in
pancreatic
-cell mass between Rab3A
/
and
Rab3A+/+ mice. It follows that the insulin
secretory-deficient phenotype of Rab3A
/
mice does not
reside at the level of insufficient insulin production.

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Fig. 4.
Insulin production is unaffected in
Rab3A /
mice. A, total insulin content was measured in
isolated Rab3A / (open bar) and
Rab3A+/+ (closed bar) mouse islets by enzyme-linked
immunosorbent assay (n 18). B,
preproinsulin mRNA and, as a control, glyceraldehyde phosphate
dehydrogenase (GAPDH) mRNA levels were measured by
nuclease protection assay in isolated islets from
Rab3A / and Rab3A+/+ mouse islets incubated
for 1 h at basal 2.8 mM or stimulatory 16.7 mM glucose as described under "Experimental
Procedures." A representative autoradiograph analysis is shown.
C, proinsulin biosynthesis was analyzed by
immunoprecipitation of [35S]proinsulin from
lysates of isolated islets from Rab3A / and
Rab3A+/+ mouse islets incubated for 1 h at basal 2.8 mM or stimulatory 16.7 mM glucose, the last 20 min of which was in the presence of [35S]methionine as
described under "Experimental Procedures." A representative
fluorograph analysis is shown. D, analysis of 10-µm serial
sections of pancreata from Rab3A / and
Rab3A+/+ mice, stained with hematoxyline and eosin
(H & E; left panels) or immunofluoresence for
glucagons/somatostatin (center panels) and insulin
(right panels). A representative image of an islet in
pancreatic serial sections is shown.
|
|
An increase in
-cell glucose metabolism is required to generate
secondary signals for glucose-induced insulin secretion (4, 21) and
proinsulin biosynthesis (17, 21). We compared the rate of glucose
oxidation in isolated islets from Rab3A
/
and
Rab3A+/+ mice and found that there was no significant
difference in glucose oxidation at either a basal 2.8 mM or
stimulatory 16.7 mM glucose concentration (Fig.
5). As such, the insulin secretory
deficiency in Rab3A
/
mouse
-cells does not lie at
the level of defective glucose metabolism.

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Fig. 5.
Glucose oxidation is unaffected in
Rab3A /
islet -cells. Isolated islets from
Rab3A / and Rab3A+/+ mice were analyzed for
glucose oxidation rates at a basal 2.8 mM (open
bars) or stimulatory 16.7 mM glucose (hatched
bars) induced as described under "Experimental Procedures."
Results are shown as a mean ± S.E. of three experiments performed
in duplicate.
|
|
Downstream of increased glucose metabolism, a rise in cytosolic
[Ca2+]i in pancreatic
-cells is essential to
trigger glucose-induced insulin secretion (4, 5, 7). We examined
whether the insulin secretory-deficient phenotype of
Rab3A
/
mice was due to a defect in generating secondary
signals required to trigger insulin exocytosis by measuring
glucose-induced increases in
-cell [Ca2+]i.
Changes in cytosolic [Ca2+]i was monitored by
Fura-2 fluorescence imaging as described previously (19). Increasing
the glucose concentration from a basal 2 mM glucose to a
stimulatory 14 mM glucose induced a significant rise in
cytosolic [Ca2+]i, after a short lag period, that
was equivalent in isolated islet
-cells from Rab3A
/
and Rab3A+/+ mice (Fig. 6).
However, there was a subtle difference in subsequent glucose-induced
oscillations in [Ca2+]i. Although the amplitude
of the [Ca2+]i oscillations did not appreciably
change the frequency of [Ca2+]i, oscillations
were slower in Rab3A
/
compared with
Rab3A+/+ islet
-cells (Fig. 6, A versus
B). Nonetheless, despite this disparity in glucose-induced
[Ca2+]i oscillations the total increase in
[Ca2+]i was only 4% lower in
Rab3A
/
islet
-cells. This was reaffirmed in that
depolarization of islet
-cells with 30 mM KCl at 2 mM glucose gave an identical rapid increase in cytosolic
[Ca2+]i in both in Rab3A
/
and
Rab3A+/+ islet
-cells, which promptly returned to basal
cytosolic [Ca2+]i levels on removal of the
stimulus (Fig. 6). As such, there was not a noticeable defect in
inducing a rise in cytosolic [Ca2+]i in
Rab3A
/
mouse islet
-cells that was sufficient to
explain the insulin secretory deficiency in vivo.

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Fig. 6.
Glucose- and depolarization-induced rise in
total [Ca2+]i is unaffected in
Rab3A /
islet -cells. Isolated islets from
Rab3A / and Rab3A+/+ mice were analyzed for
a 14 mM glucose-induced and 30 mM KCl-induced
(to cause -cell depolarization) increase in cytosolic
[Ca2+]i as analyzed by the Fura-2 indicator as
described under "Experimental Procedures." A represenative trace
for changes in Fura-2 indicated [Ca2+]i is shown
of an islet -cell from >40 -cells analyzed from at least 20 islets, in two individual experiments.
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|
Characterization of regulated insulin secretion from isolated islets
indicated a defect downstream of generating secondary signals in
Rab3A
/
islet
-cells, at the level of insulin
exocytosis. In static in vitro incubation experiments, basal
insulin secretion from isolated Rab3A
/
mouse islets was
equivalent to control Rab3A+/+ mouse islets (Fig.
7, A and B).
However, secretagogue-stimulated insulin release was compromised in
Rab3A
/
islets. Qualitatively, the glucose dose-response
pattern for stimulated insulin secretion from Rab3A
/
and Rab3A+/+ islets was similar, with a typical threshold
glucose concentration between 5 and 6 mM glucose reaching a
maximum insulin secretory rate at >15 mM glucose (Fig.
7A). However, Rab3A
/
islets secreted only
30-40% of the amount of insulin secreted by control
Rab3A+/+ islets (Fig. 7A). Likewise, the
potentiation of glucose-induced insulin secretion by GLP-1, the
sulfonylurea, glyburide, or the fatty acid, oleate, was decreased by
~60% in Rab3A
/
islets compared with control
Rab3A+/+ islets (Fig. 7B). Examination of the
biphasic pattern of insulin secretion in perifused islets from
Rab3A
/
mice indicated the kinetics for
glucose-stimulated insulin secretion to be comparable with
Rab3A+/+ islets; however, a major abnormality was found in
a blunted first phase of insulin release (between 3 and 13 min) that
consequentially diminished the second phase in Rab3A
/
islets (Fig. 7C). As a result, the accumulated
glucose-induced insulin secretion over 40 min in perifused
Rab3A
/
islets was significantly reduced by ~70%
compared with control Rab3A+/+ islets (Fig.
7D).

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Fig. 7.
Secetagogue-induced insulin secretion is
impaired from isolated islets of
Rab3A /
versus Rab3A+/+ control
islets. A, insulin secretion from isolated islets
incubated for 1 h at 37 °C was assessed over a range of glucose
concentrations. Results are shown as a mean ± S.E., where represents Rab3A / mice (n = 8), and represents Rab3A+/+ mice (n = 8).
B, insulin secretion from isolated islets incubated for
1 h at 37 °C was assessed at either a basal 2.8 mM
glucose or stimulatory 16.7 mM glucose in the additional
presence of 1 nM GLP-1, 5 µM glyburide, or
125 µM oleate complexed to 1% (w/v) BSA as indicated.
Results are shown as a mean ± S.E., where open
bars represents Rab3A / mice
(n = 6), and closed bars represents
Rab3A+/+ mice (n = 6). C,
insulin secretion from isolated islets was assessed in perifusion
incubation experiments at either a basal 2.8 mM glucose or
stimulatory 16.7 mM glucose as indicated. Results are shown
as a mean ± S.E., where represents Rab3A /
mice (n = 10), and represents Rab3A+/+
mice (n = 10). D, total insulin secretion
from 16.7 mM glucose-induced insulin secretion from
perifused isolated Rab3A / (open bar)
versus Rab3A+/+ (closed bar)
mouse islets as indicated under the curve (AUC) derived from
C. Results are shown as a mean ± S.E., where represents Rab3A / mice (n = 10),
and represents Rab3A+/+ mice (n = 10).
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|
 |
DISCUSSION |
Rab3A
/
mice are glucose-intolerant and exhibit
loss of first-phase insulin secretory response to glucose, but the
insulin secretory response to a single dose of arginine was similar in
Rab3A
/
versus Rab3A+/+ control
mice, which are typical characteristics of insulin secretory dysfunction in type-2 diabetes (1, 3). This insulin secretory dysfunction in the Rab3A
/
mice did not appear to be
driven by peripheral insulin resistance, but rather by a primary defect
in the pancreatic
-cell. This was reaffirmed in that stimulated
insulin secretion from Rab3A
/
mouse isolated islets
in vitro was markedly decreased compared with control
Rab3A+/+ islets. However, since Rab3A
/
mice
may have impaired secretory function in other neuroendocrine cells
(11), it is possible that the
-cell secretory defect could be
secondary, particularly when considering hypothalamic or adrenergic
cells. However, when isolated islets from Rab3A
/
mice
are cultured for 24 or 48 h, that would give time to recover from
any in vivo influence of circulating factors, the insulin secretory response to glucose ± glyburide, GLP-1, or oleate
remained 60-70% inhibited compared with similarly cultured
Rab3A+/+ control islets (data not shown), as found in
freshly isolated islets (Fig. 7). Moreover, if the blunted insulin
secretory response in
-cells were secondary to increased
-adrenergic activity, one would also expect to see a blunted first
phase insulin secretory response to arginine in vivo (22,
23), a decrease in glucose- and K+-induced rise in
cytosolic [Ca2+]i (24, 25), as well as
glucose-induced proinsulin biosynthesis (26). However, there were no
differences found in these parameters between Rab3A
/
and Rab3A+/+ control mouse islets (Figs. 3A, 4,
and 6). Thus, the most likely scenario is that the insulin secretory
dysfunction in the Rab3A
/
mice is due to a primary
defect in the
-cell itself.
There was no difference in pancreatic
-cell mass in between
Rab3A
/
versus Rab3A+/+ mice and
neither was insulin production and intracellular stores of insulin
affected in Rab3A
/
mouse islets. As such, the insulin
secretory deficiency in Rab3A
/
mice was not due to an
insufficient store or supply of insulin. Glucose metabolism is a
prerequisite to generate secondary signals for glucose-induced insulin
secretion and proinsulin biosynthesis, but rates of glucose oxidation
were similar in Rab3A
/
and Rab3A+/+ mouse
islets (Fig. 5). This indicated that insulin secretory dysfunction in
Rab3A
/
mice was not due to defective
-cell glucose
sensing or metabolism. Indeed, the observation that glucose-induced
translational control of proinsulin biosynthesis was unaffected in
Rab3A
/
indicated that stimulus-response coupling
mechanisms were intact in Rab3A
/
-cells. This was
reaffirmed in that glucose- and K+-induced depolarization
of Rab3A
/
islet
-cells caused an increase in
cytosolic [Ca2+]i similar to that in
Rab3A+/+ islet
-cells. As such, the defect in insulin
secretion in Rab3A
/
mouse
-cells most likely lies
downstream of generating secondary signals, at the level of insulin
exocytosis. A defective exocytosis mechanism in Rab3A
/
islet
-cells would also be consistent with the observation that the
insulin secretory response to all secretagogues tested in vitro was reduced by
50% in Rab3A
/
islets,
despite these secretagogues stimulating insulin secretion via distinct
signaling mechanisms (4).
In pancreatic
-cells, Rab3A is mostly located on the cytosolic face
of
-granule membranes and has been implicated to play a role in
control of insulin exocytosis (8, 9), as it does for
Ca2+-dependent exocytosis in other
neuroendocrine cell types (10). Here, we find that the absence of Rab3A
in
-cells decreases secretagogue-induced insulin secretion in
vivo and in vitro, reaffirming an important role of
Rab3A in control of insulin exocytosis. Rab GTP-binding proteins
specifically direct vesicular transport by an interaction with a
particular "effector protein," characteristic of an individual Rab
protein and neuroendocrine cell type (10). Rab3A has been shown to
interact with several candidate effector proteins (10), but the
Rab3A-calmodulin interaction appears most pertinent in control of
Ca2+-regulated insulin exocytosis (27). Recently, it has
been found that the Rab3A-calmodulin interaction on
-granules
provides a platform for local activation of the
Ca2+/calmodulin-dependent phosphoprotein
phosphatase, calcineurin, in response to increased
[Ca2+]i (15, 28). Activation of calcineurin on
-granules then leads to dephosphorylation activation of the
ATP-dependent motor, kinesin, and subsequent transport of
-granules to a "readily releasable pool" docked at the
-cell
plasma membrane committed to undergo exocytosis (15). Therefore, in the
absence of Rab3A, calmodulin cannot be readily sequestered to the
-granule membrane for local activation of calcineurin and kinesin in
response to elevated [Ca2+]i, so that
-granule
transport becomes much less efficient, the number of
-granules
recruited to a readily releasable pool is reduced, and consequently
insulin exocytosis is impaired. A defect in replenishing the readily
releasable pool of
-granules in
-cells of Rab3A
/
mice would be consistent with the observations of a blunted first-phase glucose-induced insulin release in vitro and in
vivo, as well as a severely diminished insulin secretory response
to a second arginine stimulus where the prior arginine stimulus would
have emptied the readily releasable pool of
-granules. Moreover,
such a mechanism is also consistent with observations of a decay in synaptic transmissions in hippocampal neurons of Rab3A
/
mice in response to multiple repeat Ca2+-induced
stimulations, which are normally sustained as found in Rab3A+/+ mice (11).
Our study of the Rab3A
/
insulin secretory phenotype
indicates that the root of
-cell secretory dysfunction in type-2
diabetes does not necessarily lie at the level of glucose-sensing and
-cell metabolism from which secondary signals emanate
(e.g. a rise in [Ca2+]i), but rather
at the level of a defective insulin exocytosis mechanism. This would be
symptomatic of insulin secretory dysfunction in type-2 diabetes arising
from an "overworked"
-cell trying hard, but not quite
succeeding, to compensate for peripheral insulin resistance (3). In the
face of persistent hyperglycemia in type-2 diabetes, the
-cell is
chronically stimulated to produce and secrete much higher amounts of
insulin than usual, and as such there is a higher rate of
-granule
turnover. This, in turn, compromises
-granule transport that
depletes the ready releasable pool of
-granules, so that insulin
secretory insufficiency develops leading to
-cell secretory
dysfunction (29). Such is the case with Rab3A
/
mice,
best illustrated by a severe reduction in the in vivo
insulin secretory response to a second consecutive arginine stimulus
that is contrastingly augmented in normal Rab3A+/+ animals.
However, there are no reports in the literature of an insulin secretory
response to such a double consecutive arginine stimulus test in human
type-2 diabetics, but if pursued could well reveal a similar insulin
secretory insufficiency found in Rab3A
/
mice that would
be supportive of insulin secretory dysfunction in human type-2 diabetes
arising from deficiencies in the insulin exocytotic mechanism due to
-cell exhaustion (3, 29). In this regard, it should be noted that if
the
-cell is induced to rest its secretory activity in type-2
diabetics, normal insulin secretory function can be recovered (30).
Notwithstanding, the glucose-intolerant/insulin secretory deficient
phenotype of Rab3A
/
mice, in the absence of insulin
resistance or changes in
-cell mass, emphasizes the important
contribution that insulin secretory dysfunction makes in the
pathogenesis of type-2 diabetes and further suggests that protection of
-cell function is a worthy consideration for the treatment of type-2
diabetes (2).
 |
ACKNOWLEDGEMENT |
We thank Cynthia Jacobs for the preparation of
this manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health DK47919 and DK5061.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Pacific Northwest
Research Inst., 720 Broadway, Seattle, WA 98122. Tel.:
206-860-6777; Fax: 206-726-1202; E-mail: cjr@pnri.org.
Published, JBC Papers in Press, January 1, 2003, DOI 10.1074/jbc.M211352200
 |
ABBREVIATIONS |
The abbreviations used are:
BSA, bovine
serum albumin;
IPGTT, intraperitoneal glucose tolerance test(s);
IPITT, intraperitoneal insulin tolerance test(s);
GLP-1, glucagon-like
peptide-1.
 |
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