From the
Mastoparan, a tetradecapeptide found in wasp venom that
stimulates G-proteins, increases insulin secretion from
Insulin secretion from
In addition to small
GTP-binding proteins, heterotrimeric GTP-binding proteins have also
been implicated in regulating insulin secretion by pancreatic
In addition to
epinephrine, a variety of other compounds are thought to modulate
insulin secretion by acting through heterotrimeric G-proteins (23). One
class of such compounds consists of amphiphilic peptides. A well known
member of this class is mastoparan, a tetradecapeptide found in wasp
venom. Mastoparan stimulates insulin secretion from
To better understand if mastoparan stimulates
insulin secretion from the
Inserts were transferred to LR White, 100% ethanol
(1:1) for 2 h and through three changes of 100% LR White (Electron
Microscopy Sciences, Fort Washington, PA) for 1 h, overnight, and for 1
h the next morning. The membrane on which the cells were attached was
removed from the plastic insert with a scalpel. The membrane was gently
rolled, with cells on the inside, into a roll approximately 2 mm in
diameter. The membrane was secured in two places by tying a short
length of hair around the roll. The roll was cut between the two ties
and excess membrane on the outer side of the ties was removed and
discarded. Membranes were placed in gelatin capsules partially filled
with LR White resin with the cut end oriented toward the bottom of the
capsule. Capsules were filled with resin, capped, and cured for 18 h at
60 °C. Thin sections (600-700 Å thick) were cut
perpendicular to the plane of the membrane and mounted on nickel grids.
In this study, we have presented data that strongly suggest
that mastoparan stimulates insulin secretion from permeabilized
In
addition to its specific effect on G-proteins at relatively low
concentrations, mastoparan has also been shown to increase secretion at
much higher concentrations due to a toxic effect ascribed to membrane
lysis
(52, 53) . At very high concentrations, mastoparan
has been claimed to be toxic to
Although heterotrimeric
G-proteins are believed to have a role in modulating insulin secretion
from pancreatic
Recently,
Olszewski et al.(22) have shown that the small
monomeric GTP-binding protein Rab3A induces insulin exocytosis
independent of calcium by interacting with specific cytosolic proteins
and that this interaction may be part of a preexocytotic protein
complex
(22) . Rab3A binds to these inhibitory proteins, called
REEP-1 and REEP-2, causing them to dissociate from the complex and
allowing exocytosis to proceed
(22) . In our study, the effects
of mastoparan to stimulate exocytosis are independent of cytosolic
proteins, since digitonin-permeabilization of
The true effectors
coupled to G
We thank Donna Berner and Frances Chilsholm for help
in performing the insulin RIA (Diabetes Endocrine Research Center). We
also thank Chris Major and Misha Amagasu for excellent technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-cells. In
this study, we have examined the role of heterotrimeric G-proteins in
mastoparan-induced insulin secretion from the insulin-secreting
-cell line
-TC3. Mastoparan stimulated insulin secretion in a
dose-dependent manner from digitonin-permeabilized
-TC3 cells.
Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan
X also stimulated secretion. Mastoparan 17, an inactive
analogue of mastoparan, did not increase insulin secretion from
permeabilized
-TC3 cells. Mastoparan-induced insulin secretion
from permeabilized
-TC3 cells was inhibited by pretreatment of the
cells with pertussis toxin, suggesting that mastoparan-induced insulin
secretion is mediated through a pertussis toxin-sensitive G-protein
present distally in exocytosis. Enriched insulin secretory granules
(ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western
immunoblotting performed on
-TC3 homogenate and ISG demonstrated
that G
was dramatically enriched in ISG. Levels of
G
and G
were comparable in homogenate
and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis
toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated
GTPase activity in the ISG, while the inactive analogue mastoparan 17
had no effect. Selective localization of G
to ISG was
confirmed with electron microscopic immunocytochemistry in
-TC3
cells and
-cells from rat pancreas. In contrast to G
and G
, G
was clearly localized
to the ISG. Together, these data suggest that mastoparan may act
through the heterotrimeric G-protein G
located in the
ISG of
-cells to stimulate insulin secretion.
-cells can be stimulated by
different types of secretagogues
(1) . D-Glucose, a fuel
secretagogue, is the major physiological
stimulus
(2, 3) . The mechanism by which glucose-induced
insulin release occurs is not completely elucidated, although glucose
oxidation is essential
(3, 4, 5, 6) .
Glucokinase is believed to act as a glucose sensor, with
phosphorylation of glucose to glucose 6-phosphate serving as the
rate-limiting step in glucose oxidation
(7) . While inhibition of
glucose oxidation inhibits insulin release, the details of the
mechanism coupling glucose oxidation to insulin secretion are less
clear. It is currently believed that oxidation of fuel secretagogues
increases intracellular levels of ATP (8), although this view has been
challenged by some groups
(9) . An increased ATP/ADP ratio is
believed to close K
channels at the
plasma membrane, resulting in decreased K
efflux and
subsequent depolarization of the
-cell
(10, 11, 12) . Depolarization then
activates voltage-dependent Ca
channels, causing an
influx of extracellular Ca
into the
-cell and an
increase in intracellular Ca
levels
(6, 13) . In addition, it has recently been
shown that nutrient secretagogues increase
-cell malonyl-CoA
levels, which leads to increased cytosolic long-chain acyl-CoA esters
that positively modulate insulin secretion (14-16). While
increased intracellular Ca
activates protein kinases
such as the Ca
- and calmodulin-dependent protein
kinase
(17, 18, 19, 20, 21) , the
mechanism by which insulin exocytosis occurs is not completely
elucidated. Over the past few years, factors other than calcium have
been proposed to be involved, including GTP and small monomeric
GTP-binding proteins such as Rab3A
(22) .
-cells
(23, 24, 25) . Epinephrine is an
adrenergic agonist that inhibits insulin secretion by acting through a
heterotrimeric G-protein
(23, 26, 27) . Although
epinephrine acts through a G-protein-coupled adrenergic receptor
located on the plasma membrane of the
-cell to decrease cAMP
accumulation, this is not thought to account entirely for its
inhibition of insulin secretion
(26) . The ability of epinephrine
to inhibit insulin secretion is believed to be due to its action on the
distal portion of the insulin exocytotic pathway, since epinephrine is
capable of inhibiting calcium-induced insulin secretion from
permeabilized
-cells
(27) . Epinephrine is thought to
inhibit insulin release by acting through a pertussis toxin-sensitive
G-protein. Epinephrine inhibition of insulin secretion is itself
inhibited by pretreatment of cells with pertussis toxin, which ADP
ribosylates pertussis toxin-sensitive G-protein
subunits at a
cysteine residue located four amino acids from the carboxyl
terminus
(26) . This ADP ribosylation renders the G-protein
subunit incapable of interacting with its receptor.
-cells (28,
29) and also stimulates secretion from many other cell types such as
platelets
(30, 31, 32) , neutrophils
(33) ,
and pneumocytes
(34) . Mastoparan has been shown to increase the
GTPase activity of many G-proteins including G
and
G
(35, 36) . Mastoparan is thought to
stimulate these G-proteins by inserting itself into the membrane
adjacent to G-proteins and mimicking the normal interaction that occurs
between G-protein-coupled receptors and their respective
G-proteins
(37, 38) . Mastoparan is believed to bind to
the carboxyl terminus of the G-protein
subunit, resulting in
and GDP dissociation from the
subunit, causing the
subunit to assume the active
configuration
(39, 40) . Mastoparan, however, unlike
epinephrine, stimulates insulin secretion from
-cells, suggesting
that G-proteins, in addition to having a role in negatively modulating
insulin secretion, may also positively modulate insulin
secretion
(28, 29) . The identity of the G-protein
through which mastoparan stimulates insulin secretion from the
-cell is unknown.
-cell by acting distally in exocytosis,
we have examined the effects of mastoparan and its analogues on insulin
secretion from digitonin-permeabilized
-cells. We have also
examined the effects of various mastoparan analogues on GTPase activity
of insulin secretory granules (ISG)
(
)
prepared
from
-cells and have shown that the ability of mastoparan
analogues to stimulate ISG GTPase activity correlates with their
ability to stimulate insulin secretion. Finally, we have used G-protein
antisera for Western blotting and electron microscopic
immunocytochemistry in order to determine which G-protein is localized
to the ISG of
-cells and is a target for mastoparan.
Materials
Tissue culture medium (CMRL-1066) and 1 M HEPES were
from Life Technologies, Inc. Newborn bovine serum was from Hazleton
Biologics (Lenexa, KS). -TC3 cells (passage 34) were obtained
through the University of Pennsylvania Diabetes Center from Dr. D.
Hanahan (University of California, San Francisco). The following
compounds were purchased from Sigma: D-glucose, carbachol,
Hanks' balanced salt solution, penicillin, streptomycin,
glutamine, Ficoll, bovine serum albumin, ovalbumin, ATP, GTP, NAD,
thymidine, and creatine phosphate. Pertussis toxin was purchased from
List Biological Laboratories (Campbell, CA). Mastoparan; its active
analogues mastoparan 7, mastoparan 8, and mastoparan X; and
its inactive analogue mastoparan 17 were purchased from Peninsula
Laboratories (Belmont, CA). Creatine kinase and adenylyl
imidodiphosphate were purchased from Boehringer Mannheim. Digitonin was
obtained from Waco Pure Chemical Industries.
[
P]GTP (10 Ci/mmol) and
[
P]NAD (1000 Ci/mmol) were purchased from
Amersham Corp.
I-Protein A was purchased from ICN
Radiochemicals (Irvine, CA). Antisera to selected G-proteins were
raised by injecting New Zealand White rabbits with synthetic
decapeptides corresponding to the carboxyl-terminal amino acid
sequences of the respective G-proteins as described previously
(41-43). In this study, the following antisera were used for
Western blotting and electron microscopic immunocytochemical analysis
of rat pancreas: anti-G
8730 (immunogen (KLH)KNNLKDCGLF),
anti-G
9072 (immunogen (KLH)ANNLRGCGLY), and anti-G
946 (immunogen (KLH)QLNLKEYNLV). These same antisera, along with
anti-G
/G
5296 (immunogen bovine brain
G
) were used for electron microscopic immunocytochemical
analysis of
-TC3 cells.
Methods
-TC3 Cell Line Culture
-TC3 cells were
cultured in 6-well plates or 15-cm dishes in the presence of RPMI 1640
(11 mM glucose) supplemented with 10% fetal bovine serum,
penicillin (75 µg/ml), streptomycin (50 µg/ml), and 2
mML-glutamine. Cells were trypsinized and subcloned
weekly. Medium was changed twice weekly and the day prior to an
experiment. Insulin secretory capacity of the
-TC3 cells in
response to glucose and carbachol was monitored regularly. Cells were
used between passages 40 and 55.
Pretreatment of
Approximately 24 h prior to the experiment, -TC3 cells with Pertussis
Toxin
-TC3
cells in either 6-well plates or 15-cm dishes were cultured in RPMI
1640 supplemented with 50 ng/ml pertussis toxin.
Permeabilization of
-TC3 cells
-TC3
cells in 6-well plates were washed 3 times with 2 ml of Krebs-HEPES
buffer (25 mM HEPES, pH 7.40, 115 mM NaCl, 24
mM NaHCO
, 5 mM KCl, 2.5 mM
CaCl
, 1 mM MgCl
, and 0.1% bovine serum
albumin). Cells in monolayers were permeabilized in the same media
supplemented with 20 µg/ml digitonin for 10 min at 37 °C under
an atmosphere of 95% air, 5% CO
(44) . Using this
method, greater than 99% permeabilization of
-TC3 cells was
achieved as assessed by trypan blue exclusion. Lactate dehydrogenase
release into the permeabilization medium was found to become maximal at
a digitonin concentration of 20 µg/ml.
Incubation of Digitonin-permeabilized
Permeabilized cells were washed 3 times with 2
ml of TES buffer (50 mM TES, pH 7.40, 100 mM KCl, 1
mM EGTA, 2 mM MgCl-TC3 for
Insulin Secretion
, and 0.1% bovine serum
albumin). Cells were incubated for 60 min at 37 °C under an
atmosphere of 95% air, 5% CO
with fresh buffer supplemented
with 1 mM ATP and the appropriate secretagogue. In selected
experiments, an ATP regenerating system consisting of 5 mM
creatine phosphate and 0.2 mg/ml creatine phosphokinase was included.
At the end of the incubation period, a sample of the supernatant was
removed for insulin measurement by radioimmunoassay
(45) .
Subcellular Fractionation of
-TC3
Cells
-TC3 cells were fractionated by sequential
centrifugation using a sucrose/nycodenz gradient as described
previously
(46) . 10-20 confluent 15-cm dishes of
-TC3
cells were washed 3 times with ice-cold 50 mM MES, pH 7.20,
containing 0.25 M sucrose and 1 mM EGTA. Cells were
scraped and resuspended in 1 ml of the same buffer. Cells were then
mechanically homogenized with 10 strokes of a motor-driven Teflon
pestle in a Potter-Elvehjem homogenizing tube. The homogenate was
centrifuged (600
g, 5 min) to remove intact cells and
nuclear debris. The supernatant was saved, and the pellet was
homogenized twice more as above. Supernatants were combined and
transferred to a 13-ml Beckman ultracentrifuge tube already underlaid
with stepwise gradients (4 ml) of nycodenz stock/homogenization buffer
in the following ratios: 16:84, 32:68, 64:36. Tubes were centrifuged at
100,000
g (SW-40 rotor, Beckman TL-100 centrifuge) for
60 min to yield a band at the lower interface corresponding to ISG.
Granules were harvested, resuspended in a 15-ml Corex tube in 14 ml of
10 mM MES, pH 6.50 containing 0.25 M sucrose, and
centrifuged at 27,000
g (Beckman J2-21
centrifuge, JA-20 rotor) for 20 min. ISG were resuspended in
100-200 µl of 10 mM MES, pH 7.40, supplemented with
1 mM EDTA. Insulin enrichment of the ISG relative to the
respective homogenate was systematically checked. Enzyme marker studies
were performed as described previously
(47) .
Western Blotting of
For these experiments, 2-25 µg of
protein in 20 µl of loading buffer (70 mM Tris, pH 6.7, 16
M urea, 6.0% SDS, 100 mM dithiothreitol, 0.005%
bromphenol blue) were loaded onto 10% SDS-polyacrylamide gel
electrophoresis gels. Colored rainbow molecular weight markers
(Amersham Corp.) were also run on each gel. Proteins were separated for
1 h at 175 V at room temperature using a Bio-Rad Mini-PROTEAN II dual
slab cell. Proteins were transferred to nitrocellulose paper (Hybond C,
Amersham Corp.) for 1.5 h at 100 V at 4 °C using a Bio-Rad mini
Trans-Blot electrophoretic transfer cell. Nitrocellulose blots were
blocked overnight in blocking buffer (1% ovalbumin, 3% bovine serum
albumin, 10 mM Tris, pH 7.40, 150 mM NaCl, and 0.1%
sodium azide) at 4 °C. The next day, blots were probed with
appropriate antisera at a dilution of 1:100 in blocking buffer for 3 h
at room temperature. Blots were washed twice with 10 mM Tris,
pH 7.40, 150 mM NaCl, and 0.1% sodium azide (TNA) for 10 min,
once with TNA supplemented with 0.05% Nonidet P-40 for 5 min, and twice
more with TNA for 10 min. Blots were incubated with 5 µCi of
-TC3 Fractions with Antibodies
to G-Proteins
I-protein A in 10 ml of blocking buffer for 1 h at room
temperature. Blots were washed again as above, air dried, and exposed
on a PhosphorImager cassette (Molecular Dynamics) overnight at room
temperature. The following day, radioactivity bound to the blot was
quantitated using a Molecular Dynamics PhosphorImager and ImageQuant
software.
Measurement of GTPase Activity
The reaction was
initiated by the addition of 5 µg of sample protein to 100 µl
of 25 mM HEPES, pH 7.20, supplemented with 1 mM
dithiothreitol, 1 mM EGTA, 20 µM
MgCl, 1 mM ATP, 1 mM adenylyl
imidodiphosphate, 5 mM creatine phosphate, 0.2 mg/ml creatine
phosphokinase, 0.2% bovine serum albumin, 100 nM
[
-
P]GTP, and varying concentrations of
mastoparan or mastoparan analogues in a 1.5-ml snap-top Eppendorf tube.
Tubes were incubated at 30 °C in a shaking water bath for 5 min.
The reaction was stopped by the addition of 900 µl of ice-cold
trichloroacetic acid, pH 2.0, supplemented with 5% charcoal by weight,
which was kept continuously stirring on ice. Tubes were centrifuged at
10,000
g for 30 min at 4 °C, and
P
in the supernatant was quantitated by liquid
scintillation spectrophotometry. Blanks determined in the presence of
50 µM GTP were routinely subtracted, and GTPase activity
was calculated as pmol of GTP hydrolyzed per mg of protein/min. Under
these conditions, GTPase activity was linear at 30 min.
Pertussis Toxin-catalyzed ADP-ribosylation
Assay
The reaction was initiated by the addition of 5 µg of
protein to 100 µl of 25 mM HEPES, pH 7.20, supplemented
with 1 mM EDTA, 1 mM ATP, 0.1 mM GTP, 0.1%
Lubrol, 0.02% bovine serum albumin, 10 mM thymidine, 10
µM [P]NAD, and 5 µg/ml
activated pertussis toxin. Tubes were incubated at 30 °C in a
shaking water bath for 30 min. The reaction was stopped by the addition
of 900 µl of ice-cold 10% trichloroacetic acid, pH 2.0. Tubes were
centrifuged at 10,000
g for 20 min at 4 °C, and
membrane pellets were washed once with 1 ml of 10% trichloroacetic
acid, pH 2.0. Membrane pellets were then suspended in 1 ml of ice-cold
diethyl ether. Ether was evaporated in a Savant concentrator connected
to a -90 °C cold trap, and membrane pellets were resuspended
in 30 µl of loading buffer supplemented with 15 mg/ml
dithiothreitol. Samples were then run on 10% SDS-polyacrylamide gels as
described above. Gels were dried and exposed on a PhosphorImager
cassette overnight. The following day, radioactivity bound to the blot
was quantitated using a Molecular Dynamics PhosphorImager and
ImageQuant software.
-TC3 Cell Processing for Immunoelectron
Microscopy
-TC3 cells were plated on Costar Transwell cell
culture inserts 5 days before use and cultured as described above.
Medium was changed the day prior to fixation. On the day of fixation,
the medium was aspirated, and 2 ml of ice-cold phosphate-buffered
saline (PBS), pH 7.40, was added to wash the cells. Inserts were
removed and transferred to a culture plate containing PBS for 1-2
min. PBS was aspirated, and 2 ml of GPA fixative containing 1%
glutaraldehyde and 0.2% picric acid in pH 7.40 PBS at 4 °C was
added. Inserts were transferred to culture plates containing fixative.
The cells were allowed to fix for 2-18 h at 4 °C. Cells were
dehydrated with 70, 80, and 90% ethanol at 4 °C for 30 min. Cells
were stained with 1% eosin B (to permit visualization of the cells) in
100% ethanol for 8 min at 24 °C and washed in 100% ethanol
3-4 times.
Tissue Processing for Immunoelectron
Microscopy
Pancreata were excised from male Sprague-Dawley rats
and minced in the GPA fixative. Tissue was fixed 2-18 h,
dehydrated, infiltrated, and embedded in LR White resin as described
above. Thick sections were cut from randomly selected tissue blocks and
stained with 1% toluidine blue in 1% sodium borate to select blocks
with islets. Thin sections (600-700 Å thick) were cut from
the selected blocks and mounted on nickel grids.
Immunostaining and Electron Microscopy
Thin
sections of -TC3 cells and rat pancreas were floated section-side
down on small drops of 1% ovalbumin, 0.2% cold water fish skin gelatin
in PBS for 1 h at 24 °C to reduce nonspecific immunoglobulin
absorption. Sections of
-TC3 cells were incubated with G-protein
antisera diluted one-tenth to one-thousandth with PBS overnight at 4
°C in a humidified chamber. Sections were washed by transferring
the grids through small drops of 20 mM Tris-HCl-buffered
saline with 0.02% Tween-20, pH 7.40, (TBST) four times for 5 min, each
at 24 °C.
-TC3 cells were incubated with 15-nm diameter
gold-labeled protein A prepared as described
(48, 49, 50) for 1 h at 24 °C to detect bound immunoglobulin. Two
staining procedures were used to identify
-cells and
-cells
and, by deduction,
-cells, in islets. In the first, serial
sections were cut, and different sections were stained with
anti-insulin, anti-glucagon, or anti-G-protein antisera, and the
antibodies were detected with gold-labeled protein A as described
above. Alternatively, sections were co-incubated overnight at 4 °C
with anti-G-protein Ig complexed to 18-nm diameter colloidal gold and
anti-insulin or anti-glucagon Ig complexed to 8-nm diameter colloidal
gold. All gold-labeled immunoglobulins were prepared as described
previously
(49, 50) . All sections were washed twice with
TBST, twice with deionized water, and then stained with 2% aqueous
uranyl acetate for 3 min.
Other Methods
Insulin radioimmunoassay was
performed by the University of Pennsylvania Diabetes Endocrine Research
Center. Protein concentrations of -TC3 cell granule fractions and
homogenates were calculated using a bicinchoninic acid microassay with
bovine serum albumin as the standard
(51) .
Data Analysis
Results are expressed as the mean
± S.E. Statistical analysis was performed using version 5.0 of
SPSS for Windows. Data were analyzed by one-way analysis of variance or
analysis of covariance as appropriate followed by multiple comparisons
between means using the least significant difference test. A
probability of p < 0.05 was considered statistically
significant.
Choice of
An insulinoma cell
line was used to study mastoparan-induced insulin secretion and the
effects of mastoparan on secretory granule GTPase activity because of
the large amount of cells necessary to perform these experiments.
Between 30 and 60 rats would need to be sacrificed to obtain the same
number of cells provided by 20 15-cm dishes of -TC3 Cells
-TC3 cells. In
addition,
-TC3 cells provide a pure population of
-cells
(which respond to glucose and carbachol with robust insulin
secretion)
(25) , whereas islets obtained from rats constitute a
mixed population of endocrine and other cell types. Finally,
-TC3
cells grown in monolayers can be more uniformly permeabilized than
islets, which consist of aggregates of thousands of cells.
Effect of Mastoparan and Mastoparan Analogues on Insulin
Secretion from Digitonin-permeabilized
In this study,
digitonin-permeabilized -TC3 Cells and on
-TC3
Cell Insulin Secretory Granule GTPase Activity
-TC3 cells were used as a model system to
study insulin exocytosis. Mastoparan is a peptide that mimics an
activated receptor
(35, 36, 37, 38) by
causing exchange of GDP for GTP on the G-protein
subunit
concomitant with dissociation of
from
(35, 39, 40).
The
subunit, having assumed an active configuration, can interact
with the appropriate effector, as can
in certain instances.
Mastoparan stimulated insulin secretion from digitonin-permeabilized
-TC3 cells in a dose-dependent manner (Fig. 1A).
Mastoparan-induced insulin secretion was largely dependent on ATP being
present. Removal of 1 mM ATP from the incubation buffer
decreased mastoparan-induced insulin secretion from 493.10 ±
32.65% of control to 209.16 ± 8.50% of control (n = 6). Inclusion of an ATP regenerating system
(18) in addition to the presence of 1 mM ATP did not
significantly affect mastoparan-induced secretion (n =
6). Mastoparan-induced insulin secretion was not significantly
increased or decreased by the addition of 2.5 mM CaCl
to the incubation buffer (n = 6). The active
mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X stimulated insulin secretion to 517 ± 29, 501 ± 55,
and 412 ± 57% of control, respectively (p < 0.05
versus control), while mastoparan 17, an inactive analogue of
mastoparan, had no significant effect (Fig. 1B). The
inclusion of 1 mM GTP or 10 µM GTP
S in the
incubation buffer did not significantly augment mastoparan-induced
insulin secretion (n = 6). This could be due to the
possibility that permeabilization of the cells does not completely
remove GTP from cellular membranes. Alternatively, the GTP hydrolyzed
by the G-protein(s) stimulated by mastoparan could reside inside an
intracellular structure, such as the insulin secretory granule, which
is not permeabilized by digitonin
(18) .
Figure 1:
Effect of mastoparan and its analogues
on insulin secretion from digitonin-permeabilized -TC3 cells and
GTPase activity in
-TC3 insulin secretory granules. Panel
A, dose curve of mastoparan on insulin secretion from
digitonin-permeabilized
-TC3 cells.
-TC3 cells were grown in
6-well plates in RPMI, and the medium was changed the day prior to the
experiment. On the day of the experiment, cells were washed 3 times
with Krebs-HEPES buffer and permeabilized for 10 min with 20 µg/ml
digitonin in the same buffer. Cells were washed twice in 50 mM
TES buffer, pH 7.40, supplemented with 100 mM KCl, 2
mM MgCl
, 1 mM EGTA, and 0.1% bovine serum
albumin. Cells were incubated for 60 min in the same buffer further
supplemented with 1 mM ATP and 0-50 µM
mastoparan. At the end of the incubation, a sample of the supernatant
was removed for insulin assay. Results are shown as the mean ±
S.E. of insulin secretion expressed as a percent of control from eight
observations. Basal insulin secretion was 60.8 ± 3.7 microunits
insulin/well/min. Panel B, effect of mastoparan analogues on
insulin secretion from digitonin-permeabilized
-TC3 cells.
-TC3 cells grown in 6-well plates were permeabilized as in
A. Cells were washed twice in 50 mM TES, pH 7.40,
supplemented with 100 mM KCl, 2 mM MgCl
,
1 mM EGTA, and 0.1% bovine serum albumin. Cells were incubated
for 60 min in the same buffer further supplemented with 1 mM
ATP and 10 µM mastoparan or mastoparan analogues. At the
end of the incubation, a sample of the supernatant was removed for
insulin assay. Results are shown as the mean ± S.E. of insulin
secretion expressed as a percent of control from 8-20
observations. Panel C, stimulation of GTPase activity in
-TC3 cell-enriched insulin secretory granule fraction with
mastoparan and mastoparan analogues. Enriched ISG fractions were
prepared from
-TC3 cells grown in 15-cm dishes. GTPase activity in
ISG prepared from
-TC3 cells was measured by incubating 5 µg
of granule protein with 100 nM [
P]GTP
in the presence or absence of 50 µM mastoparan or various
mastoparan analogues for 5 min as described under ``Experimental
Procedures.'' Blanks determined in the presence of 50
µM GTP were routinely subtracted. Basal ISG GTPase
activity was 1.66 ± 0.45 pmol/mg of protein/min. Results are
shown as the mean ± S.E. of GTPase activity expressed as a
percent of control from six observations.
In order to determine
if mastoparan stimulated a G-protein present in ISG, highly enriched
ISG were prepared by sucrose/nycodenz ultracentrifugation. ISG
fractions were significantly enriched 6.0 ± 0.96-fold in insulin
content relative to homogenate (p < 0.05, n = 5) and had no significant contamination with mitochondria
or endoplasmic reticulum as assessed by enzyme markers. ISG fractions,
however, were also enriched with the plasma membrane 5` nucleotidase.
Electron microscopy of the prepared ISG fraction (not shown)
demonstrated that it consisted mostly of insulin secretory granules
with few scattered organelles present. GTPase activity in ISG was
measured in the presence or absence of mastoparan and mastoparan
analogues. ISG were highly enriched in GTPase activity compared with
the homogenate. GTPase activity in the homogenate was 0.12 pmol/mg/min
compared with 1.66 pmol/mg/min in the ISG. Mastoparan increased
homogenate GTPase activity to 0.49 pmol/mg/min and increased ISG GTPase
activity to 10.09 pmol/mg/min (n = 6). Mastoparan
increased GTPase activity of ISG in a dose-dependent manner at
concentrations of 0-50 µM, with maximal stimulation
at 50 µM (n = 6). Similar to their effects
on secretion, mastoparan, mastoparan 7, and mastoparan 8 stimulated ISG
GTPase activity 497 ± 56, 770 ± 40, and 908 ± 48%,
respectively (p < 0.05 versus control). The
inactive mastoparan analogue mastoparan 17 had no significant effect on
ISG GTPase activity (Fig. 1C). Importantly, in control
experiments, mastoparan did not promote lysis of the isolated insulin
secretory granules as assessed by measuring insulin release directly
from the granules (n = 3) as well as by electron
microscopic analysis of the granules (n = 3).
Effect of Pertussis Toxin Pretreatment on
Mastoparan-induced Insulin Secretion from Digitonin-permeabilized
The above data
strongly suggested that the effect of mastoparan to increase insulin
secretion from permeabilized -TC3 Cells and on Mastoparan-induced Increases in
-TC3 Cell
Insulin Secretory Granule GTPase Activity
-cells was specific and not
attributable to lytic effects of the peptide that have been reported at
higher concentrations on intact
cells
(52, 53, 54) . Furthermore, pertussis toxin
pretreatment decreased mastoparan-induced insulin secretion by
permeabilized
-TC3 cells from 347 ± 17% of control (p < 0.05 versus control) to 219 ± 9% (p < 0.05 versus control, p < 0.05 versus mastoparan-stimulated insulin secretion) (Fig. 2A).
Pretreatment of
-TC3 cells with pertussis toxin (24 h) reduced
mastoparan-induced ISG GTPase activity from 479 ± 141% of
control (p < 0.05 versus control) to 129 ±
33% (Fig. 2A). ISG from untreated
-TC3 cells
contained a 40-41-kDa substrate that was clearly ADP ribosylated
in vitro by pertussis toxin. In contrast, when
-TC3 cells
had been incubated 24 h with pertussis toxin, ADP-ribosylation of this
substrate was markedly diminished (Fig. 2B), verifying
that pertussis toxin pretreatment of
-TC3 cells resulted in ADP
ribosylation of a pertussis toxin-sensitive ISG G-protein.
Figure 2:
Effect of pertussis toxin on
mastoparan-induced insulin secretion from digitonin-permeabilized
-TC3 cells and on mastoparan-induced increases in ISG GTPase
activity. Panel A, effect of pertussis toxin pretreatment on
mastoparan-induced insulin secretion from digitonin-permeabilized
-TC3 cells and on mastoparan-induced insulin secretory granule
GTPase activity.
-TC3 cells in 6-well plates were incubated 24 h
± 50 ng/ml pertussis toxin. Cells were permeabilized as in Fig.
1 and washed twice in 50 mM TES buffer, pH 7.40, supplemented
with 100 mM KCl, 2 mM MgCl
, 1 mM
EGTA, and 0.1% bovine serum albumin. Cells were incubated for 60 min in
the same buffer further supplemented with 1 mM ATP and 10
µM mastoparan. At the end of the incubation, a sample of
the supernatant was removed for insulin assay. Results are shown as the
mean ± S.E. from 15 observations. For GTPase measurements,
enriched ISG fractions were prepared from
-TC3 cells in 15-cm
dishes incubated 24 h with or without 50 ng/ml pertussis toxin. GTPase
activity in ISG prepared from
-TC3 cells was measured as in Fig.
1. Data represent the mean ± S.E. from six observations.
Panel B, pertussis toxin-catalyzed ADP-ribosylation of a
G-protein present in
-TC3 cell granule fraction. Pertussis
toxin-catalyzed ADP ribosylation of G-proteins was measured in
-TC3 cell ISG as described above. The reaction was initiated by
the addition of 5 µg of granule protein to 100 µl of 25
mM HEPES, pH 7.40, supplemented with 1 mM EDTA, 1
mM ATP, 0.1 mM GTP, 0.1% Lubrol, 0.02% bovine serum
albumin, 10 mM thymidine, 10 µM
[
P]NAD, and 5 µg/ml activated pertussis
toxin. Following a 30-min incubation at 30 °C, membranes were
analyzed by SDS-polyacrylamide gel electrophoresis. PTX,
pertussis toxin pretreatment.
G-protein Localization in Insulin Secretory Granules of
The data shown in Fig. 1and
Fig. 2
suggested that mastoparan-induced insulin secretion from
permeabilized -TC3 Cells
-TC3 cells was mediated through a pertussis
toxin-sensitive heterotrimeric G-protein located in the ISG. In order
to determine if the pertussis toxin-sensitive G-proteins G
or G
were enriched in the ISG, Western blotting was
performed on homogenate and ISG with antibodies directed against
G
and G
, as well as
G
. Western blotting with quantitative PhosphorImager
analysis demonstrated highly significant enrichment of G
in ISG (6.5 ± 1.6-fold enrichment, p < 0.05
versus homogenate), but not of G
(2.7
± 0.5-fold enrichment, nonsignificant compared with homogenate)
and G
(1.9 ± 0.3-fold enrichment,
nonsignificant compared with homogenate) (Fig. 3).
Figure 3:
Western blotting showing the relative
enrichment of G in the enriched insulin secretory
granule fractions of
-TC3 cells. To determine the cellular
localization of G
, G
, and G
,
subcellular fractions were prepared from
-TC3 cells using a
sucrose/nycodenz-based sequential centrifugation procedure. Secretory
granule fractions were prepared that were routinely enriched 6.0
± 0.96-fold in insulin content relative to the homogenate.
G-proteins were detected by Western blotting using antisera directed
against their respective
subunits. Equivalent amounts
(2.5-25 µg) of granule and homogenate protein were separated,
transferred to nitrocellulose, and probed with the respective
antibodies. Blots were washed and incubated with
I-protein A. Radioactivity bound to the blot was
quantitated with PhosphorImager analysis. The enrichment of various
G-proteins in secretory granules was calculated relative to the
homogenate. A representative analysis from five independent experiments
is shown in panelA (G, granule fraction;
H, homogenate). PanelB shows the -fold
enrichment in insulin and G-proteins in ISG compared with homogenate,
expressed as mean ± S.E. from five independent experiments. ISG
were consistently significantly enriched in insulin content and
G
, but not in G
and
G
.
Immunoelectron Microscopy of
Although the
ISG fractions were consistently highly enriched in insulin,
biochemically we could not exclude some degree of contamination with
plasma membrane. In order to address this issue, electron microscopic
immunocytochemistry of -TC3 Cells and
-Cells from Rat Islets for Selected G-proteins
-TC3 cells and of rat endocrine pancreas
was performed. Sections of rat pancreas or
-TC3 cells grown on
filters were fixed, and electron microscopic immunocytochemistry was
performed using rabbit polyclonal antisera directed against the
subunits of the respective G-proteins. In selected instances, electron
microscopic immunocytochemical analysis included identification of
-cells and
-cells with anti-glucagon or anti-insulin
antibodies, respectively, as described under ``Methods.'' In
rat pancreatic islets,
-cell secretory granules that stained
positively for insulin also stained positively for G
(antisera 8730) as shown in Fig. 4A. The majority
of gold particles complexed to the anti-G
Ig appeared
to be at the periphery of the ISG. In contrast, neither
anti-G
or anti-G
antisera (9072 and
946 respectively) were detected in the
-cell secretory granules
(not shown). No G
was detected in the granules of
cells presumed to be
-cells based on the lack of insulin or
glucagon immunostaining (Fig. 4B).
Figure 4:
Electron microscopic immunocytochemistry
of -cells from rat pancreas and
-TC3 cells for
G
and insulin. Minced rat pancreatic tissue was fixed,
dehydrated, infiltrated, and embedded. Thick sections were cut and
stained with 1% toluidine blue in 1% sodium borate to select blocks
with islets. A and B, rat pancreas was co-incubated
with anti-G
(8730) Ig complexed to 18-nm diameter
colloidal gold and anti-insulin Ig complexed to 8-nm colloidal gold as
described under ``Methods.'' The majority of the large gold
particles (G
) colocalized to the ISG, which were also
stained with colloidal gold-labeled anti-insulin Ig (small particles).
B, in the same section as A, immunostaining of the
-cells was insignificant. Sections of
-TC3 cells were
incubated with anti-G
/G
(5296)
(C), anti-G
(9072) (D), or anti
G
(946) (E) and detected with 15-nm diameter
colloidal gold-labeled protein A as described under
``Methods.'' G
and G
(D and E, respectively) were localized to the plasma
membranes and microvilli and dispersed throughout the
-TC3 cells.
The anti-G
/G
antibody labeled the ISG
of the
-TC3 cells. Because anti-G
failed to stain
the granules, this suggests that the
anti-G
/G
antibody recognized
G
in the ISG.
In -TC3
cells, immunolabeling with anti-insulin and anti-glucagon antibodies
demonstrated only insulin-containing granules in all cells examined
(not shown), therefore, dual labeling, as shown in
Fig. 4A, was not necessary. Antiserum 5296, which
recognizes both G
and G
equally,
localized to the ISG of the
-TC3 cells (Fig. 4C).
We believe this demonstrates primarily G
in the ISG
because antibodies to G
only(9072) did not associate
with the granules as shown in Fig. 4D. Both G
and G
(Fig. 4, D and E,
respectively) were found on the membranes and dispersed throughout the
-TC3 cells.
-TC3 cells by activating the heterotrimeric G-protein
G
, which is localized to the ISG. Mastoparans constitute a
family of peptides whose activities are measured in terms of their
ability to accelerate guanine nucleotide exchange; regulatory activity
requires that the peptides be both amphiphilic and
cationic
(55) . Helix-breaking residues or charged residues on
the hydrophobic face of the helix severely diminish activity, as is the
case for mastoparan 17, which has a lysyl residue on the hydrophobic
side of the helix
(55) . Mastoparan has previously been shown to
cause secretion from a variety of cell types (28, 30-34, 56). The
exact mechanism of action of the tetradecapeptide is not elucidated.
Mastoparan is believed to exert its effects by acting through
G-proteins present in membranes
(35, 36) . It is thought
that mastoparan intercalates into the membrane and mimics the
receptor-G-protein interaction
(37, 38) . In this manner,
mastoparan binds to the carboxyl terminus of the G-protein and
activates it by causing dissociation of the
subunits and GDP
from the
subunit
(35, 39, 40) . The
subunit assumes the active configuration and interacts with its
effector. Supportive data for this mechanism of action stem mainly from
experiments demonstrating that mastoparan increases the rate of guanine
nucleotide exchange, while not affecting the intrinsic rate of GTP
hydrolysis by G-protein
subunits
(35, 40) .
-cells due to lysis of the plasma
membrane (54). In our experiments, several observations demonstrated
that mastoparan acts selectively through heterotrimeric G-proteins to
stimulate insulin secretion from
-TC3 cells rather than merely
causing insulin release due to lytic toxicity. First, the effect of
mastoparan was evident at relatively low concentrations of the
tetradecapeptide. Second, the ability of mastoparan to stimulate
insulin secretion from permeabilized
-TC3 cells was attenuated by
pretreatment of the cells with pertussis toxin. Third, no effect on
insulin secretion from permeabilized
-TC3 cells was seen with the
inactive analogue of mastoparan, mastoparan 17, while active analogues
of mastoparan such as mastoparan 7, mastoparan 8, and mastoparan X caused robust insulin secretion. Fourth, only mastoparan analogues
that stimulated GTPase activity in the enriched insulin secretory
granule fraction were able to stimulate insulin secretion from
permeabilized
-TC3 cells. Fifth, the heterotrimeric G-protein
G
, a target of mastoparan, was localized to ISG both by
Western blotting and electron microscopic immunocytochemistry. Sixth,
mastoparan had no direct lytic effect on ISG as assessed by electron
microscopic analysis and insulin release. Taken together, these
observations suggest that nonspecific lytic properties of the
tetradecapeptide are not responsible for its ability to increase
insulin secretion from permeabilized
-TC3 cells. Rather, these
data suggest that the ability of the tetradecapeptide to increase
insulin secretion is due to its stimulation of GTPase activity in ISG.
The above data suggest that mastoparan-induced increases in GTPase
activity and insulin exocytosis are attributable to G
activity. However, other G-proteins associated with ISG, the
plasma membrane, or other intracellular loci could contribute to the
GTPase activity since pertussis toxin pretreatment diminution of
mastoparan-induced insulin secretion was less than its diminution of
mastoparan-induced ISG GTPase activity.
-cells, their exact localization within the
-cell and their mechanism of action has remained
uncertain
(57) . Our observation by immunoelectron microscopy
that G
is selectively localized to the ISG of
-cells represents the first direct detection of G
in the insulin secretory granule. G-proteins have multiple roles
in insulin secretion. In addition to the classical receptor-coupling
roles at the plasma membrane, there is increasing evidence that
heterotrimeric G-proteins are also involved in the distal steps of
insulin exocytosis, both as inhibitors and activators of insulin
secretion. Thus, epinephrine, galanin, and somatostatin inhibit insulin
secretion and activate an unidentified pertussis toxin-sensitive
G-protein present distally to the generation of intracellular
messengers
(26, 27, 58, 59) . In
contrast, guanine nucleotides have been shown to stimulate insulin
secretion from permeabilized
-cells independent of calcium (60).
Furthermore, it was recently suggested that the putative stimulatory
G-protein of exocytosis may be activated by mastoparan (29). Our data,
which localize G
to the ISG and strongly suggest
mastoparan-induced activation of G
, support a role for
G
in insulin exocytosis. These observations are
consistent with the known roles of G
in secretion and
vesicular trafficking
(61, 62, 63) .
-cells results in
the loss of most cytosolic proteins
(18) . It is difficult at the
present time to identify at which level of the cascade of insulin
exocytosis G
functions, however, it is reasonable to
suggest that it may be involved in a very distal step of exocytosis.
Further work will be required to understand what effectors are coupled
to G
in the ISG. One possible effector could be
phospholipase A
. It has been shown that vesicle fusion with
plasma membrane requires phospholipase A
activity and the
presence of unsaturated fatty acids such as arachidonic
acid
(64, 65) . Of interest, arachidonic acid has also
been shown to inhibit GTPase activity in ISG
(66) . Thus, a
possible effector of G
could be a phospholipase
A
isoform localized to the ISG. G-protein activation of
this enzyme would produce arachidonic acid, which could then, by
feedback, inhibit granule GTPase activity.
in ISG remain to be identified. Whatever
they may be, our data suggest that activation of the heterotrimeric
G-protein G
in the ISG is a crucial and distal event of
insulin exocytosis that is important in the fusion of the insulin
secretory granule with the
-cell plasma membrane.
S, guanosine
5`-O-(thiotriphosphate).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.