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Effects of cytochalasins B and D on alloxan inhibition of insulin release
BIOCHEMICAL
Vol. 66, No. 4, 1975
EFFECTS
OF CYTOCMLASINS
Michael
McDaniel,
B AND
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
D ON ALLOXAN
Catherine
Joan
Roth,
Department Washington School St. Louis,
Received
August
INHIBITION
Fink,
Gwen
OF Fyfe
INSULIN and
RELEASE
Paul
Lacy
of
Pathology University of Medicine Missouri 63110
14, 1975
SUMMARY: In isolated rat islets, cytochalasin-B, which potentiates glucoseinduced insulin release and inhibits hexose transport, provided immediate protection from the inhibitory effect of alloxan on glucose-induced insulin release. Cytochalasin-D, which also potentiates glucose-induced insulin release, but exerts no detectable effect on hexose transport, provided no protection from the action of alloxan. These results indicate 1) the protective action of cytochalasin-B against the effect of alloxan is mediated on the beta-cell membrane in proximity to the hexose transport site, and 2) the ability of cytochalasin-B and D to potentiate glucose-induced insulin release appears unrelated to an interaction with the hexose transport site. The
--in
minutes
vitro
abolishes
concomitant of
exposure subsequent
exposure
either
of
D-glucose,
protection
of
from
the
inhibitory
3-O-methyl-D-glucose,
carrier
for
facilitated
ose ccl
exert
a direct
transport
(3),
1 types
microfilamentous In
(7-9)
-
cell
lines,
similar The sin-D compare
on
into
effect
on to
system
and
it
of
shown
effect
share
been
glucose-induced does
not
disruption
of
concentrations almost
a common
by hexose
D-
hexosehas
been
of
hex-
inhibition transport
shown
to
in
disrupt in
transport
in
microfilamentous
other
the
release
hexose
the
complete
hexoses,
insulin
inhibit
five
However,
Cytochalasin-B
on
has
(1).
high
membrane
inhibitory
potentiate
produces
to
for
protective
(2).
beta-cell
alloxan
provides
The
cytochalasin-B
islets certain
system
(10). the
present
glucose-induced
cytochalasin-B
D-mannose
beta-cell
the
its
with
alloxan. been
the
cytochalasin-D
cytochalasin-6
both
of
to release
alloxan or
entry
addition,
contrast,
purpose
to
have
In
although to
islets
effect
similar
(4-6).
islets insulin
3-O-methyl-D-glucose
and
to
pancreatic
glucose-induced isolated
glucose
shown
isolated
study insulin
and
0 as
Copyright o 1975 by Academic Press, Inc. All rights of reproductiorz irz arzy fbrm reserved.
was
to
release
protective
determine and
agents
1089
the
effect
hexose
transport,
against
alloxan
of
cytochalaand
inhibition
to of
Vol. 66, No. 4, 1975
glucose-induced
BIOCHEMICAL
insulin
release
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
isolated
MATERIAL
islets.
AND
METHODS
Perifusion of isolated Islets: Islets were isolated from the pancreas of male Wistar rats (2OO-3OO gm) using the collagenase technique described previously (II). The perifusion system utilized-was identical to that described in studies on alloxan inhibition of insulin release, b vitro (1). lnsul in was determined by the radioimmunoassay of Wright et al. musing crystalline pork insulin as the standard and []25] I labeled pork insulin (Cambridge Nuclear Pharmaceutical Co.) as the tracer. Exposure to A 1 loxan : was added to previously to the perifusion of
MO.) prior
Alioxan monohydrate warmed and gassed the islets to yield
(Sigma perifusion a final
Chemical Co., St. Louis, medium immediately concentration of 20 mg/lOOml.
Transport Studies : Hexose transport in isolated islets was determined with a double-labeled isotope procedure described previously (3). This non-wash procedure is a modification of the original technique described by Hellman et al. (13). [3H]Sucrose, which does not penetr te the beta-ccl 1 membrane, w s used as an extracellylar marker in determining &] O-glu[ 18 CID-glucose uptake (14). cose-U and [ HIsucrose were supplied by New England Nuclear. insulin Static Incubation of Islets: isolated islets was determined in the following transfered into incubation vials (11 mm IO x Krebs-bicarbonate media. The vials were then which were stoppered, and maintained at 37°C and at pH 7.4 by continuous gassing with 95% media changes were made using a constriction scope.
release following static incubation Twenty-five islets were manner. 20 mm height) containing 200 ul of transfered to scintillation vials in a Oubnoff shaking water bath 02 and 5% CO2. All subsequent pipette and a dissecting micro-
Cytochalasin B and 0: Cytochalasin B and 0 were obtained from Aldrich Chemical Co., Mi Iwaukee, Wise. The cytochalasins were dissolved in dimethylsulfoxide (Fisher Scientific Co.) to provide stock solutions which when added to the perifusion media at 0.4% dimethylsulfoxide provided final cytochalasin conOimethylsulfoxide at 0.4% had no effect on either centrations of l-30 ug/ml. hexose transport, glucose-induced insulin release or protection against alloxan. Cytochalasin-B and 0 did not alter the rate of decomposition of alloxan as determined by the procedure of Patterson et al. (15). RESULTS Glucose-Induced on
the
pattern
of
isolated
Insulin of
islets
and
experimental
ml)
for
to
glucose
Release:
glucose-induced is
At
50 minutes. (3.0
imental
chamber
exposed
to
glucose
insulin
shown
chambers
mg/ml) resulted (3.0
in were
the and
The
end
Figure
1.
perifused of
this
mg/ml)
an
cytochalasin-0
as
In
study,
this media
pretreatment
enhancement alone
of
release
with
cytochalasin-0 in
effect
(15
ug/ml)
of
insulin
(control
1090
chambers
(15
determined
by
the
perifusion
in
the
control
islets
containing period, for
glucose
(0.3
exposure
60
release
18.7
of
minutes
in
compared + 2.9
ug/ml)
mg/
islets the to
mU insulin/
experislets
of
Vol.
66, No.
BIOCHEMICAL
4, 1975
AND
BIOPHYSICAL
GLUCOSE 03
1
0
IO
30
40
50 60 MINUTES
70
of
Cytochalasin-D
on
90
100
110
with glucose (0.3 mg/ml) for 50 minfor 60 minutes. The experimental 0.3 mq/ml) for 50 minutes. then lasin-I? (1s ug/mi) for 60 minutes.
I
(
I
Table Effect
80
1
(-) were perfused to qlucose (3.0 mq/ml) perfused with glucose (3.0 mg/ml) and cytocha
mm
1
C”L
Figure The control chambers exposed utes, then ) were chambers ( exposed to glucose N=4.
COMMUNICATIONS
mp/ml
I
‘20
RESEARCH
Glucose
(3
Cytochalasin-D uq/ml
0 mg/ml)
Induced
% increase insulin
in
Release
of control release
10
+ 114 (8) + 90 (8)
:z
+ +
5
lnsul
89 go
(lo) (IO)
Islets were both preincubated 30 minutes with glucose (0.3 mg/ml) and incubated 30 minutes with glucose (3.0 mg/ml) in the presence and absence of cytochalasin-D The number of individual observations are in parentheses and the S.E.M. in all cases was less than 10% of the mean.
100
islets
as
experimental of
1 and
compared
29.1
chambers). 3 ug/ml
in
glucose-induced of
concentrations
the
5-30
Transport: on
D-glucose
3.5
mU
of
perifusion
insulin/l00
islets
release.
With
(Table The
transport
islets,
static
no
was
.005, at
of observed
in
the
concentrations
potentiating
incubation
release
p<
cytochalasin-D
exerted
insulin
ug/ml
to
system
glucose-induced of
D-Glucose
f
Exposure
insulin
potentiation
determined
to
effect isolated at
on islets,
cytochalasin-D
1).
effect in
of isolated
1091
cytochalasin-D islets
at (Table
3 and 2).
15 ug/ml In
this
was study,
Vol.
66, No. 4,1975
cytochalasin-D
was
as
during
the
at
either
3 or
to
had
mg/ml)
for
minutes
while
still
were
perfused
in
a similar
mu
28.1
+ 2.3
insulin/100
tions sure exposure substantial
islets of
to
the
for
the
alloxan
protection
Effect
against
of
and the
Cytochalasin
Expe ri men ta 1 Condition
in
the
these
effect
Table
2
D and
B on
pmoles
of
D-glucose
I-
(control
22.6
2
1.7
condipre-expo-
simultaneous
minutes alloxan
also (control
provided chamber
Transport
D-glucose/islet/min.
control
23.6
+
1.1
(12)
cy to-D 3 ug/ml
24.3
2
1.2
(12)
control
20.1
+ 2.7
(16)
cyto-D 15 ug/ml
20.6
+
(16)
islets were both preincubated and incubated for one minutes The final of cytocha las i n-D. [lk,llabeled D-glucose (sp. 2.0 mCi/mm). Mean sp. act. in parentheses.
alloxan
minute the
five
chambers Cytocha
experiments,
forty-five
for
inhibitory
of
for
control
of
conditions,
cytochalasin-B
alloxan
chamber
series
and
alloxan.
experimental
of
experimental
to
effect
of
subsequent
ug/ml)
The
the
a second
the
exposed
of
ability
on
(15
omission
against
Under
well
cytochalasin-D
alloxan
Islets
then
the
omission
as
The
cytochalasin-8.
for
In
of
2.
islets/hour;
cytochalasin-8. to
Fig.
and
protection
p > .2).
to
cytochalasin-B
of
minutes
Alloxan:
effect
containing
20
islets
D Against
in
except
complete
except
islets
shown
COMMUNICATIONS
transport.
inhibitory
presence
manner
of of
D-glucose
minutes,
mU insulin/100
identical
period
B and
forty-five
islets/hour,
were of
media
in
almost
is
RESEARCH
Exposure
on
the
release
five
provided
effect
against
with
BIOPHYSICAL
period.
Cytochalasin
protect
perifused
(1.0
no
of
insulin were
chamber
a preincubation
incubation
Actions
glucose-fnduced
asin-
during
present
15 ug/ml
cytochalasin-B
D-glucose
AND
one-minute
Protective
chambers
BIOCHEMICAL
1.1
for 20 minutes at 20°C in a glucose-free medium with glucose (3 mg/ml) in the presence and absence incubation medium contained 3 mg/ml uniformly act. 0.6 mCi/mm) and 1.8 mg/ml (6,6’ - [3HI-sucrose, The number of individual observations are f S.E.M.
1092
BIOCHEMICAL
Vol. 66, No. 4, 1975
GLUCOSE mg/ml 401 C& C+-B
I C”tO-B
1
AND BIOPHYSICAL RESEARCH COMMUNICATIONS *
1Or
Figure
5
I
2
The control chambers (-) were perfused with glucose (1 mg/ml) and cytochalasin(15 ug/ml) for 45 minutes, followed by cytochalasin-B alone for 5 minutes, and then exposed to glucose (5 mg/ml ) for 60 minutes. The experimental chambers (---) were perfused with glucose (1 mg/ml) and cytochalasin-B (15 ug/ml) for 45 minutes, followed by cytochalasin-B and alloxan (20 mg/lOO for 5 minutes, and then exposed to glucose (5 mg/ml) for 60 minutes. N=4.
21.9
+ 2.0
100
islets/hour, The
mU insulin/100
the I5
under
of
from
the
Omitting
cytochalasin-D
and
to
those
against
and
to
alloxan.
of
alloxan
as
19.9
3.5 mU insulin/
+
with
cytochalasin-D
in
3
exposing not
was
not
in
ug/ml)
present
for
during
protect
(control 2.2
and did
(I5
did
chambers
also
alloxan Islets
was
Figure
period
of
cytochalasin-B.
cytochalasin-D
shown
5 minutes
effect
for
Cytochalasin-D
pretreatment for
the
used
experimental
the
alloxan
not
protect
exposure,
islets/hour;
islets/hour).
to
preincubated
period
action
mU insulin/l00
alloxan(results
were
alloxan
exposure
islets
similar
chambers preceding
five-minute
4.3
cytochalasin-D
conditions
experimental minutes
chamber
p >.5).
ability
evaluated
experimental
islets/hour;
ml)
the
the
chamber,
27.9
+ mU insulin/100 the
provide
islets
to
protection
against
shown). DISCUSSION
The
present
enhancement
of
effective asin-B
studies glucose-induced
concentration potentiates
demonstrated
that
insulin for
glucose-induced
cytochalasin-D
release
cytochalasin-D insulin
1093
produces
from
isolated
ranged
from
release
at
5-30
rat ug/ml.
concentrations
significant islets,
The
Cytochalof
5-15
+
Vol. 66, No. 4, 1975
BIOCHEMICAL
0
20
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,” .. ..----. 60 MINUTES
40
Figure
__..- r--L- -__... 80 100
3
were perfused with glucose (1 mg/ml) for 30 minutes, The control chambers (-) followed by cytochalasin-D (IS ug/ml) and glucose (1 mg/ml) for 1.5 minutes, cytochalasin-D alone for 5 minutes, and then exposed to glucose (5 mg/ml) for The experimental chambers (---) 60 minutes. were perfused with glucose (I mg/ ml) for 30 minutes, followed by cytochalasin-D (15 ug/ml) and glucose (I mg/ ml) for 15 minutes, then cytochalasin-D and alloxan (20 mg/lOO ml) for 5 minutes, and then exposed to glucose (5 mg/ml) for 60 minutes. N=4.
w/ml
(9).
Cytochalasin-8
located
beneath
similar
morphological
is
reported In
more
plasma
effective of
in
capacity
to
cytochalasin-D
at
3 and
respective and
Cytochalasin-B
has
including cytochalasin-B
and
cytochalasin-B
inhibits
hexose
shown
of
hexose
glucose-induced by
insulin cytochalasin
to
lines,
with
hexose
failed
to of
for
The D may
in
1094
to
present D-glucose both
release, that
to
ability
its
release. cell
lines, Since
(3).
appear
of
on
many
islets
unrelated
by
the
insulin
transport
would
(10).
represent
insulin
related
effect
glucose-induced
enhancement be
In
any
basis
characterized
transport.
pancreatic
it is
been
exert
a molar
cytochalasin-B
cytochalasin-D
glucose-induced transport,
on
than has
hexose in
system produces
and
changes
interfere
cytochalasin-B
B and
cell
cytochalasin-D
inhibit
microfilamentous
Cytochalasin-D
(IO),
ug/ml
the
cells.
visible
transport
hexose
release.
disrupt
of
levels
D potentiate
by
beta
concentrations
been
transport
to
a variety
I5
potentiating
inhibition
of
producing
limited
non-potentiating
release
in
lines
These
shown
membrane
cell
transport.
of
been
changes
a variety
relatively study,
the
has
and
only
the
inhibition to
glucose-induced
a primary
effect
both
enhance insulin
on
the
micro-
a
BIOCHEMICAL
Vol. 66, No. 4, 1975
filamentous
system
of
Cytochalasin-6 conditions the
of
action
protective with
site
action the
hexose
Previous does
flux tions to
tritiated
alloxan.
alloxan
not
Thus, insulin
site
on
into
the
the ccl
subsequently
hexose of
in
islets
rat
I via
of the
exerts
involved
in
Supported
an
in
a direct
beta
interaction
concept
is
of
alteration
Zawalich,
et
the
per
alloxan the in
primary
inhibiting
I membrane,
transport
system
alloxan In
anaerobic
addition,
glycolytic
observed in
no
islets of
alteraexposed
action
factors: with
and/or
b)
without
on
to
of
glucose-induced
following proximity
ccl
interact
islets
site
the
se. in
involve
effect
also
(18)
al.
a
by
(16).
in
that
is
rat
that with
supported
which
indicate
which
inhibitory
insulin
to
[3H]5-D-glucose
may
hexose
appear
would
transport no
with
it
from
of
employed
transport,
derived
action
the
those
protection
transport,
exposure
system,
a gluco-receptor surface
to
provide
all
D-glucose
due
vitro
and
carrier
mechanism
release with
alloxan,
formation
to
under
inhibit
This
observed
observations
the
the
interacts
site
HZ0 These
is
to
unable
3-O-methyl-D-glucose
(17)
exposed
--in
(7,8).
al.
alloxan
3-O-methyl-D-glucose,
that or
not
et
of
similar
membrane. and
indicate
was
is
cell
Hellerstrzm
islets in
the
D-glucose
and
in
on
Orci
effect
D-glucose
cytochalasin-B
site.
toxic
does
of
carrier
by
conditions
inhibit
D-glucose
inhibit
Gunnarsson
with
of
studies
not
the
cytochalasin-D
does
transport
suggested
cytochalasin-D
cytochalasin-B
hexose
as
against
alloxan
Since
protective
cell
However,
effect
cytochalasin-B. whereas
beta
protected
employed.
against
the
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a)
a hexose alloxan
damaging
insulin
release
transport is
it, via
alloxan
transported and
an
then
intra-cellula
release.
part
by
NIH
grants
AM
01226, AM
06181
and
AM 03373
REFERENCES 1.
2. 3.
Tomita,
T.,
Lacy,
P.E.,
Matschinsky,
F.M.
and
McDaniel,
M.L.
(1974)
23, 517-524. He1 lman, McDaniel, Diabetologia
B., Sehlin, H. and T;iljedal, M.L., King, S., Anderson, 10, 303-308.
I. S.,
1095
(1973) Fink,
J.
Pfliigers Arch. and Lacy, P.E.
340, (1974)
Diabetes
51-58.
Vol. 66, No. 4, 1975
4. 5.
Kletzien, 2964-2966. Estensen,
R.F., R.D.
BIOCHEMICAL
Perdue, and
J.F.
Plegemann,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and P.
Springer, (1972)
A. Proc.
(1972) Nat.
J. Acad.
Biol.
Chem.
Sci.
247,
(Wash.)
69,
1430-1434. 6. i: 9. IO. 11.
12.
Mizel, Orci Malaisse, 594-604. Lacy, Miranda, Biol. Lacy, Wright,
S.B. and Gabbay, L., W.J.,
Wilson, K.H. Hager,
L.J. (1972) and Malaisse, D. and Orci,
J.
Biol. Chem. 247, W.J. (1972) Science L. (1972) Diabetes
4102-4105.
175,
1128-1130.
(suppl.
Klein, N.J. and Fink, C.J. (1973) Endocrinology P. E., A., Godman, G., Deitch, A. and Tanenbaum, S. (1974) 61, 481-500. P-E. and Kostianovsky, M. (1967) Diabetes 16, 35-39. and Sussman, K.E. P.H., Makuiu, D.R., Vichick, D.
2) 92, J.
(1971)
21, 1458-1468. Cell
Diabetes
20, 33-45. 13. 14. 15. 16. 17. 18.
Sehlin, S. and T;iljedal, 1.8. (1971) Biochim. Biophys. Acta Hellman, B., 241, 147-154. Bloom, G.D., Hellman, B., Idahl, L., Lernmark, A., Sehlin, J. and Tgljedal, I. (1972) Biochem. J. 129, 241-254. Patterson, J-W., LazarMnl, A. and Levey, S. (1949) J. Bioi. Chem. 177, 187-196. McDaniel, M.L., Anderson, S., Fink, C., Roth, C. and Lacy, P.E. (1975) Endocrinology 97, 68-75. Gunnarsson, R. and HellerstrEm, C. (1973) Horm. Metab. Res. 5, 404-409. Zawalich, W.S., Karl, R.C., Ferrendelli, J.A. and Matschinsky, F.M. (1975) Diabetes 24, (suppl. 2) 405.
1096
Vol. 66, No. 4, 1975
EFFECTS
OF CYTOCMLASINS
Michael
McDaniel,
B AND
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
D ON ALLOXAN
Catherine
Joan
Roth,
Department Washington School St. Louis,
Received
August
INHIBITION
Fink,
Gwen
OF Fyfe
INSULIN and
RELEASE
Paul
Lacy
of
Pathology University of Medicine Missouri 63110
14, 1975
SUMMARY: In isolated rat islets, cytochalasin-B, which potentiates glucoseinduced insulin release and inhibits hexose transport, provided immediate protection from the inhibitory effect of alloxan on glucose-induced insulin release. Cytochalasin-D, which also potentiates glucose-induced insulin release, but exerts no detectable effect on hexose transport, provided no protection from the action of alloxan. These results indicate 1) the protective action of cytochalasin-B against the effect of alloxan is mediated on the beta-cell membrane in proximity to the hexose transport site, and 2) the ability of cytochalasin-B and D to potentiate glucose-induced insulin release appears unrelated to an interaction with the hexose transport site. The
--in
minutes
vitro
abolishes
concomitant of
exposure subsequent
exposure
either
of
D-glucose,
protection
of
from
the
inhibitory
3-O-methyl-D-glucose,
carrier
for
facilitated
ose ccl
exert
a direct
transport
(3),
1 types
microfilamentous In
(7-9)
-
cell
lines,
similar The sin-D compare
on
into
effect
on to
system
and
it
of
shown
effect
share
been
glucose-induced does
not
disruption
of
concentrations almost
a common
by hexose
D-
hexosehas
been
of
hex-
inhibition transport
shown
to
in
disrupt in
transport
in
microfilamentous
other
the
release
hexose
the
complete
hexoses,
insulin
inhibit
five
However,
Cytochalasin-B
on
has
(1).
high
membrane
inhibitory
potentiate
produces
to
for
protective
(2).
beta-cell
alloxan
provides
The
cytochalasin-B
islets certain
system
(10). the
present
glucose-induced
cytochalasin-B
D-mannose
beta-cell
the
its
with
alloxan. been
the
cytochalasin-D
cytochalasin-6
both
of
to release
alloxan or
entry
addition,
contrast,
purpose
to
have
In
although to
islets
effect
similar
(4-6).
islets insulin
3-O-methyl-D-glucose
and
to
pancreatic
glucose-induced isolated
glucose
shown
isolated
study insulin
and
0 as
Copyright o 1975 by Academic Press, Inc. All rights of reproductiorz irz arzy fbrm reserved.
was
to
release
protective
determine and
agents
1089
the
effect
hexose
transport,
against
alloxan
of
cytochalaand
inhibition
to of
Vol. 66, No. 4, 1975
glucose-induced
BIOCHEMICAL
insulin
release
in
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
isolated
MATERIAL
islets.
AND
METHODS
Perifusion of isolated Islets: Islets were isolated from the pancreas of male Wistar rats (2OO-3OO gm) using the collagenase technique described previously (II). The perifusion system utilized-was identical to that described in studies on alloxan inhibition of insulin release, b vitro (1). lnsul in was determined by the radioimmunoassay of Wright et al. musing crystalline pork insulin as the standard and []25] I labeled pork insulin (Cambridge Nuclear Pharmaceutical Co.) as the tracer. Exposure to A 1 loxan : was added to previously to the perifusion of
MO.) prior
Alioxan monohydrate warmed and gassed the islets to yield
(Sigma perifusion a final
Chemical Co., St. Louis, medium immediately concentration of 20 mg/lOOml.
Transport Studies : Hexose transport in isolated islets was determined with a double-labeled isotope procedure described previously (3). This non-wash procedure is a modification of the original technique described by Hellman et al. (13). [3H]Sucrose, which does not penetr te the beta-ccl 1 membrane, w s used as an extracellylar marker in determining &] O-glu[ 18 CID-glucose uptake (14). cose-U and [ HIsucrose were supplied by New England Nuclear. insulin Static Incubation of Islets: isolated islets was determined in the following transfered into incubation vials (11 mm IO x Krebs-bicarbonate media. The vials were then which were stoppered, and maintained at 37°C and at pH 7.4 by continuous gassing with 95% media changes were made using a constriction scope.
release following static incubation Twenty-five islets were manner. 20 mm height) containing 200 ul of transfered to scintillation vials in a Oubnoff shaking water bath 02 and 5% CO2. All subsequent pipette and a dissecting micro-
Cytochalasin B and 0: Cytochalasin B and 0 were obtained from Aldrich Chemical Co., Mi Iwaukee, Wise. The cytochalasins were dissolved in dimethylsulfoxide (Fisher Scientific Co.) to provide stock solutions which when added to the perifusion media at 0.4% dimethylsulfoxide provided final cytochalasin conOimethylsulfoxide at 0.4% had no effect on either centrations of l-30 ug/ml. hexose transport, glucose-induced insulin release or protection against alloxan. Cytochalasin-B and 0 did not alter the rate of decomposition of alloxan as determined by the procedure of Patterson et al. (15). RESULTS Glucose-Induced on
the
pattern
of
isolated
Insulin of
islets
and
experimental
ml)
for
to
glucose
Release:
glucose-induced is
At
50 minutes. (3.0
imental
chamber
exposed
to
glucose
insulin
shown
chambers
mg/ml) resulted (3.0
in were
the and
The
end
Figure
1.
perifused of
this
mg/ml)
an
cytochalasin-0
as
In
study,
this media
pretreatment
enhancement alone
of
release
with
cytochalasin-0 in
effect
(15
ug/ml)
of
insulin
(control
1090
chambers
(15
determined
by
the
perifusion
in
the
control
islets
containing period, for
glucose
(0.3
exposure
60
release
18.7
of
minutes
in
compared + 2.9
ug/ml)
mg/
islets the to
mU insulin/
experislets
of
Vol.
66, No.
BIOCHEMICAL
4, 1975
AND
BIOPHYSICAL
GLUCOSE 03
1
0
IO
30
40
50 60 MINUTES
70
of
Cytochalasin-D
on
90
100
110
with glucose (0.3 mg/ml) for 50 minfor 60 minutes. The experimental 0.3 mq/ml) for 50 minutes. then lasin-I? (1s ug/mi) for 60 minutes.
I
(
I
Table Effect
80
1
(-) were perfused to qlucose (3.0 mq/ml) perfused with glucose (3.0 mg/ml) and cytocha
mm
1
C”L
Figure The control chambers exposed utes, then ) were chambers ( exposed to glucose N=4.
COMMUNICATIONS
mp/ml
I
‘20
RESEARCH
Glucose
(3
Cytochalasin-D uq/ml
0 mg/ml)
Induced
% increase insulin
in
Release
of control release
10
+ 114 (8) + 90 (8)
:z
+ +
5
lnsul
89 go
(lo) (IO)
Islets were both preincubated 30 minutes with glucose (0.3 mg/ml) and incubated 30 minutes with glucose (3.0 mg/ml) in the presence and absence of cytochalasin-D The number of individual observations are in parentheses and the S.E.M. in all cases was less than 10% of the mean.
100
islets
as
experimental of
1 and
compared
29.1
chambers). 3 ug/ml
in
glucose-induced of
concentrations
the
5-30
Transport: on
D-glucose
3.5
mU
of
perifusion
insulin/l00
islets
release.
With
(Table The
transport
islets,
static
no
was
.005, at
of observed
in
the
concentrations
potentiating
incubation
release
p<
cytochalasin-D
exerted
insulin
ug/ml
to
system
glucose-induced of
D-Glucose
f
Exposure
insulin
potentiation
determined
to
effect isolated at
on islets,
cytochalasin-D
1).
effect in
of isolated
1091
cytochalasin-D islets
at (Table
3 and 2).
15 ug/ml In
this
was study,
Vol.
66, No. 4,1975
cytochalasin-D
was
as
during
the
at
either
3 or
to
had
mg/ml)
for
minutes
while
still
were
perfused
in
a similar
mu
28.1
+ 2.3
insulin/100
tions sure exposure substantial
islets of
to
the
for
the
alloxan
protection
Effect
against
of
and the
Cytochalasin
Expe ri men ta 1 Condition
in
the
these
effect
Table
2
D and
B on
pmoles
of
D-glucose
I-
(control
22.6
2
1.7
condipre-expo-
simultaneous
minutes alloxan
also (control
provided chamber
Transport
D-glucose/islet/min.
control
23.6
+
1.1
(12)
cy to-D 3 ug/ml
24.3
2
1.2
(12)
control
20.1
+ 2.7
(16)
cyto-D 15 ug/ml
20.6
+
(16)
islets were both preincubated and incubated for one minutes The final of cytocha las i n-D. [lk,llabeled D-glucose (sp. 2.0 mCi/mm). Mean sp. act. in parentheses.
alloxan
minute the
five
chambers Cytocha
experiments,
forty-five
for
inhibitory
of
for
control
of
conditions,
cytochalasin-B
alloxan
chamber
series
and
alloxan.
experimental
of
experimental
to
effect
of
subsequent
ug/ml)
The
the
a second
the
exposed
of
ability
on
(15
omission
against
Under
well
cytochalasin-D
alloxan
Islets
then
the
omission
as
The
cytochalasin-8.
for
In
of
2.
islets/hour;
cytochalasin-8. to
Fig.
and
protection
p > .2).
to
cytochalasin-B
of
minutes
Alloxan:
effect
containing
20
islets
D Against
in
except
complete
except
islets
shown
COMMUNICATIONS
transport.
inhibitory
presence
manner
of of
D-glucose
minutes,
mU insulin/100
identical
period
B and
forty-five
islets/hour,
were of
media
in
almost
is
RESEARCH
Exposure
on
the
release
five
provided
effect
against
with
BIOPHYSICAL
period.
Cytochalasin
protect
perifused
(1.0
no
of
insulin were
chamber
a preincubation
incubation
Actions
glucose-fnduced
asin-
during
present
15 ug/ml
cytochalasin-B
D-glucose
AND
one-minute
Protective
chambers
BIOCHEMICAL
1.1
for 20 minutes at 20°C in a glucose-free medium with glucose (3 mg/ml) in the presence and absence incubation medium contained 3 mg/ml uniformly act. 0.6 mCi/mm) and 1.8 mg/ml (6,6’ - [3HI-sucrose, The number of individual observations are f S.E.M.
1092
BIOCHEMICAL
Vol. 66, No. 4, 1975
GLUCOSE mg/ml 401 C& C+-B
I C”tO-B
1
AND BIOPHYSICAL RESEARCH COMMUNICATIONS *
1Or
Figure
5
I
2
The control chambers (-) were perfused with glucose (1 mg/ml) and cytochalasin(15 ug/ml) for 45 minutes, followed by cytochalasin-B alone for 5 minutes, and then exposed to glucose (5 mg/ml ) for 60 minutes. The experimental chambers (---) were perfused with glucose (1 mg/ml) and cytochalasin-B (15 ug/ml) for 45 minutes, followed by cytochalasin-B and alloxan (20 mg/lOO for 5 minutes, and then exposed to glucose (5 mg/ml) for 60 minutes. N=4.
21.9
+ 2.0
100
islets/hour, The
mU insulin/100
the I5
under
of
from
the
Omitting
cytochalasin-D
and
to
those
against
and
to
alloxan.
of
alloxan
as
19.9
3.5 mU insulin/
+
with
cytochalasin-D
in
3
exposing not
was
not
in
ug/ml)
present
for
during
protect
(control 2.2
and did
(I5
did
chambers
also
alloxan Islets
was
Figure
period
of
cytochalasin-B.
cytochalasin-D
shown
5 minutes
effect
for
Cytochalasin-D
pretreatment for
the
used
experimental
the
alloxan
not
protect
exposure,
islets/hour;
islets/hour).
to
preincubated
period
action
mU insulin/l00
alloxan(results
were
alloxan
exposure
islets
similar
chambers preceding
five-minute
4.3
cytochalasin-D
conditions
experimental minutes
chamber
p >.5).
ability
evaluated
experimental
islets/hour;
ml)
the
the
chamber,
27.9
+ mU insulin/100 the
provide
islets
to
protection
against
shown). DISCUSSION
The
present
enhancement
of
effective asin-B
studies glucose-induced
concentration potentiates
demonstrated
that
insulin for
glucose-induced
cytochalasin-D
release
cytochalasin-D insulin
1093
produces
from
isolated
ranged
from
release
at
5-30
rat ug/ml.
concentrations
significant islets,
The
Cytochalof
5-15
+
Vol. 66, No. 4, 1975
BIOCHEMICAL
0
20
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
,” .. ..----. 60 MINUTES
40
Figure
__..- r--L- -__... 80 100
3
were perfused with glucose (1 mg/ml) for 30 minutes, The control chambers (-) followed by cytochalasin-D (IS ug/ml) and glucose (1 mg/ml) for 1.5 minutes, cytochalasin-D alone for 5 minutes, and then exposed to glucose (5 mg/ml) for The experimental chambers (---) 60 minutes. were perfused with glucose (I mg/ ml) for 30 minutes, followed by cytochalasin-D (15 ug/ml) and glucose (I mg/ ml) for 15 minutes, then cytochalasin-D and alloxan (20 mg/lOO ml) for 5 minutes, and then exposed to glucose (5 mg/ml) for 60 minutes. N=4.
w/ml
(9).
Cytochalasin-8
located
beneath
similar
morphological
is
reported In
more
plasma
effective of
in
capacity
to
cytochalasin-D
at
3 and
respective and
Cytochalasin-B
has
including cytochalasin-B
and
cytochalasin-B
inhibits
hexose
shown
of
hexose
glucose-induced by
insulin cytochalasin
to
lines,
with
hexose
failed
to of
for
The D may
in
1094
to
present D-glucose both
release, that
to
ability
its
release. cell
lines, Since
(3).
appear
of
on
many
islets
unrelated
by
the
insulin
transport
would
(10).
represent
insulin
related
effect
glucose-induced
enhancement be
In
any
basis
characterized
transport.
pancreatic
it is
been
exert
a molar
cytochalasin-B
cytochalasin-D
glucose-induced transport,
on
than has
hexose in
system produces
and
changes
interfere
cytochalasin-B
B and
cell
cytochalasin-D
inhibit
microfilamentous
Cytochalasin-D
(IO),
ug/ml
the
cells.
visible
transport
hexose
release.
disrupt
of
levels
D potentiate
by
beta
concentrations
been
transport
to
a variety
I5
potentiating
inhibition
of
producing
limited
non-potentiating
release
in
lines
These
shown
membrane
cell
transport.
of
been
changes
a variety
relatively study,
the
has
and
only
the
inhibition to
glucose-induced
a primary
effect
both
enhance insulin
on
the
micro-
a
BIOCHEMICAL
Vol. 66, No. 4, 1975
filamentous
system
of
Cytochalasin-6 conditions the
of
action
protective with
site
action the
hexose
Previous does
flux tions to
tritiated
alloxan.
alloxan
not
Thus, insulin
site
on
into
the
the ccl
subsequently
hexose of
in
islets
rat
I via
of the
exerts
involved
in
Supported
an
in
a direct
beta
interaction
concept
is
of
alteration
Zawalich,
et
the
per
alloxan the in
primary
inhibiting
I membrane,
transport
system
alloxan In
anaerobic
addition,
glycolytic
observed in
no
islets of
alteraexposed
action
factors: with
and/or
b)
without
on
to
of
glucose-induced
following proximity
ccl
interact
islets
site
the
se. in
involve
effect
also
(18)
al.
a
by
(16).
in
that
is
rat
that with
supported
which
indicate
which
inhibitory
insulin
to
[3H]5-D-glucose
may
hexose
appear
would
transport no
with
it
from
of
employed
transport,
derived
action
the
those
protection
transport,
exposure
system,
a gluco-receptor surface
to
provide
all
D-glucose
due
vitro
and
carrier
mechanism
release with
alloxan,
formation
to
under
inhibit
This
observed
observations
the
the
interacts
site
HZ0 These
is
to
unable
3-O-methyl-D-glucose
(17)
exposed
--in
(7,8).
al.
alloxan
3-O-methyl-D-glucose,
that or
not
et
of
similar
membrane. and
indicate
was
is
cell
Hellerstrzm
islets in
the
D-glucose
and
in
on
Orci
effect
D-glucose
cytochalasin-B
site.
toxic
does
of
carrier
by
conditions
inhibit
D-glucose
inhibit
Gunnarsson
with
of
studies
not
the
cytochalasin-D
does
transport
suggested
cytochalasin-D
cytochalasin-B
hexose
as
against
alloxan
Since
protective
cell
However,
effect
cytochalasin-B. whereas
beta
protected
employed.
against
the
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a)
a hexose alloxan
damaging
insulin
release
transport is
it, via
alloxan
transported and
an
then
intra-cellula
release.
part
by
NIH
grants
AM
01226, AM
06181
and
AM 03373
REFERENCES 1.
2. 3.
Tomita,
T.,
Lacy,
P.E.,
Matschinsky,
F.M.
and
McDaniel,
M.L.
(1974)
23, 517-524. He1 lman, McDaniel, Diabetologia
B., Sehlin, H. and T;iljedal, M.L., King, S., Anderson, 10, 303-308.
I. S.,
1095
(1973) Fink,
J.
Pfliigers Arch. and Lacy, P.E.
340, (1974)
Diabetes
51-58.
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4. 5.
Kletzien, 2964-2966. Estensen,
R.F., R.D.
BIOCHEMICAL
Perdue, and
J.F.
Plegemann,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and P.
Springer, (1972)
A. Proc.
(1972) Nat.
J. Acad.
Biol.
Chem.
Sci.
247,
(Wash.)
69,
1430-1434. 6. i: 9. IO. 11.
12.
Mizel, Orci Malaisse, 594-604. Lacy, Miranda, Biol. Lacy, Wright,
S.B. and Gabbay, L., W.J.,
Wilson, K.H. Hager,
L.J. (1972) and Malaisse, D. and Orci,
J.
Biol. Chem. 247, W.J. (1972) Science L. (1972) Diabetes
4102-4105.
175,
1128-1130.
(suppl.
Klein, N.J. and Fink, C.J. (1973) Endocrinology P. E., A., Godman, G., Deitch, A. and Tanenbaum, S. (1974) 61, 481-500. P-E. and Kostianovsky, M. (1967) Diabetes 16, 35-39. and Sussman, K.E. P.H., Makuiu, D.R., Vichick, D.
2) 92, J.
(1971)
21, 1458-1468. Cell
Diabetes
20, 33-45. 13. 14. 15. 16. 17. 18.
Sehlin, S. and T;iljedal, 1.8. (1971) Biochim. Biophys. Acta Hellman, B., 241, 147-154. Bloom, G.D., Hellman, B., Idahl, L., Lernmark, A., Sehlin, J. and Tgljedal, I. (1972) Biochem. J. 129, 241-254. Patterson, J-W., LazarMnl, A. and Levey, S. (1949) J. Bioi. Chem. 177, 187-196. McDaniel, M.L., Anderson, S., Fink, C., Roth, C. and Lacy, P.E. (1975) Endocrinology 97, 68-75. Gunnarsson, R. and HellerstrEm, C. (1973) Horm. Metab. Res. 5, 404-409. Zawalich, W.S., Karl, R.C., Ferrendelli, J.A. and Matschinsky, F.M. (1975) Diabetes 24, (suppl. 2) 405.
1096