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Department of Biochemistry and Molecular Biology (R.A.E., N.R.F., E.M.I., J.T.), University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107-2699; and Department of Pediatrics (M.L.), Washington University School of Medicine, St. Louis, Missouri 63110
Address all correspondence and requests for reprints to: Richard A. Easom, Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107-2699. E-mail: reasom{at}hsc.unt.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been hypothesized that the activation of Ca2+/calmodulin-dependent protein kinases (CaM kinases) function in the initiation of insulin release (8, 9, 10). Included in this group is the multifunctional CaM kinase II, which has been shown to be present (11, 12) and activated (13) by glucose in isolated rat islets. Despite numerous attempts to link this enzyme with insulin secretion via correlative studies or by the use of putative inhibitors of CaM kinase II, current evidence of a role of this kinase is ambiguous. Although glucose activation of CaM kinase II correlates with insulin secretion, at least with respect to glucose concentration (13), other experimental results suggest that these cellular events may be independent of each other. For example, exposure of islets to the muscarinic agonist, carbachol, in the presence of stimulatory concentrations of glucose, suppresses the activation of CaM kinase II while potentiating insulin secretion (14). Moreover, though several reports have documented that the putative inhibitor of CaM kinase II, KN-62 (15), inhibits glucose- and nutrient-induced insulin secretion from isolated islets (16, 17) or cultured ß-cells (18), other studies have shown that KN-62 does not inhibit Ca2+-induced insulin secretion from permeabilized ß-cells (18). Studies using intact cells are further complicated by the reported effect of KN-62 to inhibit Ca2+ channel activity (18, 19).
The suggestion that CaM kinase II activation and insulin secretion may be dissociated has major implications with respect to the role of this enzyme in the exocytotic process. This has prompted a direct analysis of the temporal correlation of glucose-induced kinase activation and insulin secretion in conditions where both could be measured simultaneously. Such conditions have been achieved using a perifusion technique in which isolated rat islets are immobilized on a hydrophilic membrane to allow their recovery at any given time. The analysis of the insulin content of the perifusate has thus permitted the direct measurement of the rate of insulin secretion at the moment of kinase assay. This analysis has provided convincing data for the association of CaM kinase II activation with the initiation of insulin secretion but suggests that kinase deactivation lags the cessation of secretion on the removal of stimulus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]ATP was purchased from NEN Research
Chemicals (DuPont, Boston, MA). Autocamtide-2, sequence KKALRRQETVDAL
(20), was synthesized by Bio-Synthesis, Inc. (Lewisville, TX). All
other chemicals were of the finest reagent grade available.
Isolation of islets
Pancreatic islets were isolated from male rats (250–350g) by
collagenase P digestion and subsequent enrichment by centrifugation on
a Ficoll gradient as described previously (14). After isolation, islets
were cultured for at least 1 h in CMRL-1066 containing 5.5
mM glucose and supplemented with 2 mM
L-glutamine, 10% heat-inactivated FBS at 24 C under an
atmosphere of 95% air/5% CO2. Animal maintenance and
surgical procedures were conducted in accordance with an Institutional
Animal Care and Use Committee-approved protocol (University of North
Texas Health Science Center at Fort Worth).
Insulin secretion models
Static secretion.
For static secretion experiments, islets
(160–200 per tube) were counted into polypropylene microcentrifuge
tubes and preincubated for 15 min at 37 C in Krebs-Ringer-Bicarbonate
(KRB) basal medium (25 mM HEPES, pH 7.4, 115 mM
NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5
mM CaCl2, 1 mM MgCl2)
containing 3 mM glucose and 0.1% BSA (500 µl) under an
atmosphere of oxygen/CO2 (95/5%). The medium was then
replaced with fresh KRB basal medium (3 mM glucose) or KRB
medium supplemented with 17 mM glucose, and the incubation
continued for the indicated times. Incubations were terminated by brief
centrifugation (7000 x g, 5 sec) and supernatants
recovered for the analysis of insulin content by double-antibody RIA
(21). The pelleted islets were washed once in 500 ml ice-cold
homogenization buffer (20 mM Tris-HCl, pH 7.5, 0.5
mM EGTA, 1.0 mM EDTA, 2.0 mM
dithiothreitol, 10 mM sodium pyrophosphate, 0.4
mM ammonium molybdate) and homogenized by sonication (10
pulses, setting 3, 30% duty cycle) in 50 ml homogenization buffer
supplemented with 100 µg/ml leupeptin. The resulting homogenate was
used for the assay of CaM kinase II activation.
Perifusion.
Islets (150–200 per chamber) were placed on
hydrophilic 13-mm polycarbonate cyclopore filters (Whatman, Fairfield,
NJ) in Swinnex chambers (Whatman) and perifused with KRB basal medium
(3 mM glucose) at 1 ml/min for 30 min at 37 C. The
perifusion was then continued with either the same medium or KRB medium
containing 17 mM glucose. The perifusate was collected in
1- to 5-ml fractions and analyzed for insulin or C-peptide content by
RIA (21, 22). At the indicated time, the perifusion was terminated, and
the islets were recovered from the filter by washing twice with
ice-cold homogenization buffer containing 1 mg/ml BSA. The islets were
further harvested by centrifugation and homogenized as described
above.
Assay of CaM kinase II activity
CaM kinase II activity was assayed using the determination of
32P incorporation into an exogenously added selective
peptide substrate, autocamtide-2, by a method described previously
(14). CaM kinase II activity was assayed in a reaction mixture
containing 50 mM piperazine diethanesulfonic acid, pH 7.0,
10 mM MgCl2, 0.1 mg/ml BSA (fraction V), 10
µM autocamtide-2, 20 µM ATP (specific
activity, 40 Ci/mmol), and either 0.5 mM
CaCl2/5 µg/ml calmodulin for Ca2+-stimulated
activity or 0.9 mM EGTA for Ca2+-independent
activity. Total reaction vol was 50 µl. The assay was initiated by
the addition of 10 µl of islet homogenate and continued for 30 sec at
30 C before termination by the addition of ice-cold trichloroacetic
acid (25 µl, 15%). After centrifugation (12,000 x
g, 1 min), 35 µl of the resulting supernatant was spotted
onto 3 cm by 0.5-cm strips of phosphocellulose paper (Whatman P81),
washed 5 times in 500 ml distilled H2O, and dried at 110 C
for 15 min. 32Pi incorporation into
autocamtide-2 was determined by Cerenkov radiation (Beckman
Instruments, Inc., Fullerton, CA). In the described experiments,
32Pi incorporation into autocamtide-2 in the
absence of Ca2+/calmodulin (autonomous CaM kinase II
activity) is expressed as percentage of incorporation in the presence
of these cofactors (Ca2+-dependent CaM kinase II activity).
Because autonomous activity is a property of autophosphorylated,
activated CaM kinase II (23), this ratio was used as a measure of
enzyme activation.
Statistical treatment of data
Data are presented as mean ± SE determined
from at least three independent observations, unless otherwise stated.
Where indicated, statistical significance was assessed by a Student’s
t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This pattern of secretion was similar to that
reported previously (24, 25). To correlate CaM kinase II activation
with biphasic hormone release, islets were recovered from the
perifusion chambers at various time points subsequent to the
introduction of stimulatory glucose concentrations (17 mM)
and the amount of CaM kinase II in an autonomous, autophosphorylated
form determined. As demonstrated in Fig. 1B, glucose (17
mM) induced the activation of CaM kinase II with a temporal
profile that was very similar to insulin secretion from the same
islets. Before stimulation, the ratio of autonomous to
Ca2+-dependent CaM kinase II activity was determined to be
16.14 ± 2.89% of CaM kinase II, and this ratio did not change
significantly throughout the period of incubation in the presence of
basal (3 mM) glucose. On exposure to stimulatory
concentrations of glucose (17 mM), the activation state of
CaM kinase II increased rapidly to 31.89 ± 2.86% by 3 min and
then onto 39.42 ± 4.52% at 30 min. A closer analysis suggested
that enzyme activation also was biphasic, although the observed nadir
in activation state was not statistically significant from the peak
activation at 3 min. Nevertheless, Fig. 1C demonstrates that the
temporal profiles of insulin secretion and CaM kinase II activation
were, for the most part, superimposable, suggesting that the two
processes are closely related.
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). Because of this disparity, it
was of interest to study the temporal profile of insulin secretion from
islets incubated in a static medium. As demonstrated in Fig. 2B, under
these conditions the absolute amount of insulin secreted increased
rapidly on the introduction of stimulatory concentrations of glucose
(17 mM at time zero), such that approximately 15 µU was
released in the first 3 min. The amounts of insulin accumulated between
subsequent time points was not, however, consistent. For example, only
a net 2 µU of insulin was secreted as the incubation was extended
from 15 min to 20 min (Fig. 2B). These differences are revealed in an
additional plot of the calculated rate of secretion between the time
increments (Fig. 2C). A maximal secretion rate was achieved during the
first 3 min, followed by a transient return of secretion rates to the
basal level at 6 min. This initial burst of secretion likely
corresponded to the first phase of secretion observed in islet
perifusion (see Fig. 1
). This was followed by a second burst of
secretion that peaked at 10 min. However, unlike the steady incline of
secretion rates observed from perifused islets, this phase was
characterized by a marked decline of secretion such that by 20 min, the
rate of secretion was not significantly different from that observed
under basal glucose concentrations. This observation suggests that
insulin secretion from statically incubated islets was biphasic but
that the second phase of secretion was prohibited beyond 10 min, even
in the presence of stimulatory concentrations of glucose.
Interestingly, this prohibition of secretion correlated with the
observed decline in CaM kinase II activation under similar conditions
(Fig. 2A), though the temporal pattern of kinase activation did not
include a clear second phase in these static secretion conditions.
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, this treatment caused
no inhibition of the second phase of insulin secretion, indirectly
determined by the measurement of secreted C-peptide, despite being
added at concentrations likely to be representative of hormone
accumulation in static incubations. The introduction of insulin tended
to potentiate C-peptide accumulation, although this was likely the
result of an observed incremental affect to slow the perifusion rate.
The activation of CaM kinase II by glucose was not influenced in these
conditions in perifused islets (Fig. 3B), suggesting that insulin
accumulation did not account for the observed suppression of kinase
activation in static islet incubations.
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-cells of the pancreatic islet
and is known to inhibit insulin secretion (26, 27), similar experiments
were performed to test the hypothesis that the accumulation of this
hormone accounted for the decline in insulin secretion and CaM kinase
II activation. As shown in Fig. 4A, the introduction of
1 µM SRIF, 15 min subsequent to the exposure of islets to
17 mM glucose, rapidly decreased insulin secretion,
reaching a maximal inhibition of approximately 70%. By contrast, SRIF
had no significant effect on glucose activation of CaM kinase II (Fig. 4B). In additional experiments, SRIF (0–1 µM)
dose-dependently inhibited the onset of insulin secretion but had no
significant effect on glucose activation of CaM kinase II determined at
3 min after the addition of glucose (data not shown). These results
suggest that neither insulin nor SRIF account, in toto, for
the observed suppression of insulin secretion or CaM kinase II
activation, although it is possible that SRIF contributed to the
suppression of insulin secretion.
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2-adrenergic agonist, epinephrine (Epi) (28), and so the
ability of this hormone to alter CaM kinase II activation also was
studied. As demonstrated in Fig. 5A, Epi totally
reversed glucose-induced insulin secretion, when added 10 min
subsequent to the initiation of secretion, by the addition of
stimulatory concentrations of glucose. As observed for SRIF, this
concentration of Epi only modestly decreased the activation status of
CaM kinase II (Fig. 5B, P > 0.05). These observations
suggest that insulin secretion and CaM kinase II, though showing
similarities with respect to the initiation of insulin secretion, can
be dissociated and that this dissociation may occur with respect to the
exocytotic site.
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, insulin secretion declined rapidly after the withdrawal of stimulatory
concentrations of glucose from the perifusion medium. By 3 min, a 50%
reversal of secretion was observed, and basal secretion rates were
achieved by 15 min. In contrast, a maximal level of CaM kinase II
activation (i.e. 50% autonomous) was maintained for at
least 5 min subsequent to the withdrawal of glucose. Only then was
enzyme deactivation observed such that greater than 90% reversal was
achieved by 30 min, a time significantly longer than that required for
the reversal of insulin secretion. These observations again suggest
that although insulin secretion and CaM kinase II may be similar in
activation profile, these parameters may be dissociated at the point of
exocytosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a general sense, the activation of CaM kinase II was found to
correlate closely with insulin secretion. By perifusion, the activation
of CaM kinase II coincided with the onset of hormone release, and for
the most part, the two time profiles were superimposable, even to the
extent that kinase activation seemed biphasic, although not in a manner
that could be statistically established. This close temporal
correlation suggests that these cellular events are associated and
constitutes evidence in support of a function of CaM kinase II in the
insulin secretion process. Furthermore, this temporal profile,
particularly with respect to sustained kinase activation that
correlated with the second phase of insulin secretion, was distinct
from the monophasic-like profile observed from static incubation of
islets, in which kinase activation was steadily repressed after 5 min
of stimulation (see Fig. 2 and 13 . It is interesting to note,
therefore, that both kinase activation and insulin secretion were
suppressed in static incubations after a 5- to 10-min period of
stimulation. Although there is no direct evidence for a cause-effect
relationship between these two parameters, it is tempting to speculate
that the activation of CaM kinase II is required for insulin secretion
and that the observed suppression of secretion is the result of
inhibition of this enzyme.
Interpretation of the observed dissociation of CaM kinase II activation and insulin secretion in the presence of SRIF or Epi, or after glucose withdrawal, are less straightforward. At a cursory level, the observed ability of Epi and SRIF to profoundly suppress glucose-induced insulin secretion by more than 95% and more than 70%, respectively, without any significant effect on kinase activation, could be taken as evidence that CaM kinase II has no functional role in the insulin secretory process. Indeed, it should be recognized that insulin secretion can be elicited by mechanisms independent of the elevation of intracellular Ca2+ (30). An alternative explanation, however, could be that the targets of these secretory antagonists are cellular components distal to the activation of CaM kinase II by Ca2+. This explanation is supported by a substantial amount of evidence to suggest that these hormones directly interfere with exocytosis through the modulation of G-proteins (Gi or possibly Go) (26, 30, 31). Other reported effects of these hormones to promote membrane repolarization and ultimately decrease cytosolic Ca2+ concentrations (32) may not be of sufficient magnitude to affect the activation state of CaM kinase II.
The rapid return of insulin secretion to baseline rates after the withdrawal of a glucose stimulus, indicates that this nutrient tightly controls the final stages of exocytosis (i.e. secretory granule fusion and content release into the extracellular medium). The observed delay in CaM kinase II deactivation (by 5–10 min) eliminates the involvement of autonomous kinase activity in these events but is consistent with a potential involvement of this enzyme to regulate upstream events that represent the ATP-dependent steps of insulin exocytosis (30), as previously proposed by Ammala et al. (19). The movement of secretory granules to preexocytotic sites is thought to involve both microtubule and microfilament components of the cytoskeleton (33, 34, 35, 36). It is of interest, therefore, that calmodulin-dependent kinases have been reported to be associated with this cellular structure (9) and mediate Ca2+-induced phosphorylation of cytoskeletal or vesicle-associated components, such as microtubule-associated protein-2 (37) and synapsin I (38, 39), in intact or permeabilized ß-cells. It is conceivable, therefore, that the activation of CaM kinase II regulates movement of nascent secretory granules to the exocytosis site via the modulation of microtubule dynamics. This process would be envisaged to be activated immediately on stimulation of the cell but, ideally, would be required further to continue past the termination of the stimulus to permit the restocking of pools of granules strategically stored in close proximity to the plasma membrane for immediate release on cell stimulation. The property of CaM kinase II to convert, on activation, to a Ca2+-independent, autonomous form that will continue to phosphorylate and modulate the function of substrates, despite the elimination of stimulatory second messengers (40, 41, 42), perfectly equips this enzyme for such a role.
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Footnotes |
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Received January 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adrenoceptor activation is paralleled by both a
repolarization and a reduction in cytoplasmic free Ca2+
concentration. J Biol Chem 263:1855–1860 subunit associated with the generation of
Ca2+-independent activity. Proc Natl Acad Sci USA 85:6337–6341This article has been cited by other articles:
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)
or KRB containing 17 mM glucose (•). Perifusates (1–5
ml) were collected for the determination of insulin content (A). At the
indicated times, islets were recovered from selected chambers,
homogenized, and assayed for CaM kinase II activation (B). C, Panels A
and B have been superimposed to illustrate the correlation of the two
parameters.
) or 17 mM (•,
) glucose
(Glc) for 15 min. The last 5 min of this period is represented by time
-5 to 0. The perifusion medium was then switched back to 3
mM and the incubation continued. During this period (0–30
min), insulin content of the perifusate (circles) and
activation state of CaM kinase II (triangles) were
analyzed at the indicated times.