1 Yale University School of Nursing, New Haven, Connecticut 06536-0740; and 2 Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118
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ABSTRACT |
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The insulin secretory responses of rat islets to glucose (15 mM), 12-O-tetradecanoylphorbol
13-acetate (TPA; 500 nM), and potassium (30 mM) were determined from
perifused islets cultured for 22-24 h in CMRL-1066 medium (control
cultured) or islets cultured in the additional presence of 500 nM TPA.
Islet content of protein kinase C (PKC
) and serine and threonine
phosphoprotein patterns were also monitored after the culture period.
Compared with freshly isolated islets, culturing alone had no adverse
effect on the capacity of TPA or 30 mM potassium to stimulate secretion
or on the islet content of PKC
. In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKC
by
>95% and abolished the capacity of the phorbol ester to stimulate secretion during a subsequent dynamic perifusion. Culturing in TPA
slightly improved the insulin secretory response to 15 mM glucose
compared with control-cultured islets; however, sustained rates of 15 mM glucose-induced secretion from these islets were significantly less
than the responses of freshly isolated islets. Islets cultured in TPA
responded to 30 mM potassium with a markedly amplified insulin
secretory response that was abolished by nitrendipine. Enhanced
phosphorylation of several islet proteins was also observed in
TPA-cultured islets compared with control-cultured islets. These
findings demonstrate that culturing alone impairs glucose-induced secretion, a response that is improved but still subnormal compared with freshly isolated islet responses, if TPA is included in the culture medium. Sustained phosphorylation of several islet proteins in
TPA-cultured islets may account, at least in part, for augmented calcium-dependent secretion.
insulin release; phorbol ester; 12-O-tetradecanoylphorbol 13-acetate; downregulation; protein kinase C
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INTRODUCTION |
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CHRONIC EXPOSURE TO THE phorbol ester
12-O-tetradecanoylphorbol 13-acetate
(TPA) reduces the cellular levels of the enzyme protein kinase C (PKC
) (25). The mechanism underlying this effect is thought to be
enhanced degradation of the enzyme. Two reports documenting the effect
of this pretreatment protocol on rat pancreatic islet responsiveness to
glucose have been published (21, 28). Both studies have concluded that,
although 18-24 h of exposure to TPA abolished the normal
insulinotropic effect of TPA (indicating that TPA pretreatment reduced
or abolished the PKC
component in
-cells responsible for its
insulinotropic action), the insulin stimulatory effect of glucose is
not only maintained from these cultured islets but is slightly enhanced compared with the same response from control-cultured islets.
The findings made with islets chronically exposed to TPA have been used
as evidence against the involvement of PKC activation in
glucose-stimulated secretion (21). However, the stimulatory effects of
TPA last for many hours (11), and it has yet to be established whether
the sustained phosphorylation of PKC
-target proteins contributes to
release from PKC
-depleted islets. Furthermore, findings made with
freshly isolated rat islets displaying insulin secretory response
characteristics comparable to those observed with the perfused rat
pancreas (15, 16, 32, 47, 49) have supported a role for PKC
activation in the physiological regulation of glucose-induced
secretion. For example, 20 mM glucose stimulation of freshly isolated
islets or the perfused pancreas in vivo results in the translocation of
PKC
from a predominantly cytosolic location to a membrane
compartment (13, 14). Translocation to the cell membrane is often used
as a surrogate marker for the activation of PKC
(22, 25). Glucose
(20 mM) stimulation of freshly isolated islets also increases the
phosphorylation of the myristoylated alanine-rich C kinase substrate
(MARCKS) protein (8), an established intracellular target for activated
PKC
(6). Glucose (20-22 mM) increases the accumulation of
phosphoinositide-derived inositol phosphates (5, 39), and, from a
quantitative perspective, the magnitude of this effect is comparable to
cholinergic stimulation of the
-cell (5, 23). In addition, high
glucose shares many of the characteristics of several agonists whose
effects on the
-cell are generally attributed to phospholipase C
(PLC)/PKC
activation (16, 29, 34, 40, 43, 44, 48).
We decided to reinvestigate the effects of culturing islets in the
presence or absence of TPA on the secretory responses of the -cell.
The subsequent insulin secretory responses not only to glucose and TPA
but also to potassium were measured and compared with the responses of
1-day control-cultured islets and with those of freshly isolated
perifused islets. Glucose utilization rates and immunoreactive PKC
,
PLC-
1, and PLC-
1 levels were also determined. Islet
phosphoprotein patterns after culture in the presence or absence of TPA
were examined as well.
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METHODS |
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The detailed methodologies employed to assess insulin output from collagenase-isolated islets have been previously described (45, 47). Male Sprague-Dawley rats purchased from Charles River Laboratories were used in all studies. All animals were treated in a manner that complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were fed ad libitum and weighed 300-450 g at the time of study. After Nembutal (pentobarbital sodium, 50 mg/kg; Abbott, North Chicago, IL)-induced anesthesia, islets were isolated by collagenase digestion and handpicked, using a glass loop pipette, under a stereomicroscope. They were free of exocrine contamination.
After isolation, groups of freshly isolated islets were loaded onto nylon filters and directly perifused in a Krebs-Ringer bicarbonate (KRB) buffer at a flow rate of 1 ml/min. Perifusate solutions were gassed with 95% O2-5% CO2 and maintained at 37°C. Perifusate samples were collected at appropriate times and analyzed for insulin content. Glucose utilization rates were monitored in some experiments by the rates of 3H2O formation from 5-[3H]glucose (see below).
Other groups of islets were studied after culturing for 22-24 h in CMRL-1066 with or without 500 nM TPA. TPA was dissolved in DMSO, and equivalent amounts of diluent were added to control-cultured islets. Fetal bovine serum (1 ml serum/9 ml CMRL-1066) was added to the CMRL-1066, and the final glucose concentration varied from 5 to 5.5 mM. Glutamine (to achieve a final concentration of 2 mM) and the antibiotics penicillin (50 U/ml) and streptomycin (50 µg/ml) were included. Groups of 16-20 islets were placed in Falcon culture dishes that contained 4 ml of warmed (to 37°C) and oxygenated tissue culture medium. They were incubated overnight at 37°C in a humidified 5% CO2-95% air atmosphere. After the culturing period and washing with 5 ml KRB buffer, they were then treated identically to the freshly isolated islets.
Glucose utilization rates. The usage of glucose was measured by determining the rate of 3H2O formation from 5-[3H]glucose using methods previously described (41, 42). Some groups of islets were studied immediately after isolation, whereas other groups of islets were studied after culture under control conditions or in the presence of TPA. These islets were washed with 5 ml of KRB and then incubated in 0.125 ml of 3 or 15 mM glucose supplemented with tracer 5-[3H]glucose. The 3H2O formed during the subsequent 1-h incubation was separated from the unused [3H]glucose as described previously (41, 42).
Western blot analyses.
Groups of freshly isolated islets or of islets cultured in the presence
or absence of TPA were pelleted by centrifugation and then suspended in
25-50 µl of homogenization buffer containing various protease
inhibitors, as described previously (23, 47). After sonication,
triplicate aliquots were analyzed for protein content according to the
Lowry method, using BSA as a standard. For the Western blots of
PLC-1 (20 µg), PLC-
1 (20 µg), PKC
(15 µg), or islet
phosphoproteins (20-23 µg), the protein sonicate from islets was
boiled for 90 s in 4× Laemmli sample buffer and separated by
SDS-PAGE using a 4% stacking gel and a 7% running gel run at 12 mA
and 16 mA, respectively. Gel-resolved proteins were electrotransferred
onto an Immobilon polyvinylidene difluoride membrane at 15 volts for 20 h. The Immobilon was stained with Ponceau S
solution for protein, washed, and blocked for 2 h in Tris-buffered
saline supplemented with 0.05% Tween 20 and 5% milk powder. (A
special blocking solution made by Zymed Laboratories was used when
blocking membranes for phosphoprotein analyses.) For PLC-
1
determinations, the membranes were incubated for 150 min with the
primary anti-PLC-
1 antibody (1.0 mg/ml dilution), washed, incubated
for 60 min with horseradish peroxidase (HRP)-conjugated IgG, and washed
again. For PLC-
1 determinations, the membranes were incubated for 60 min with the primary anti-PLC-
1 antibody (0.5 mg/ml dilution),
washed, incubated for 45 min with HRP-conjugated IgG, and washed again.
For PKC
determinations, the membranes were incubated for 45 min with
the primary anti-PKC
antibody (0.5 mg/ml dilution), washed,
incubated for 30 min with HRP-conjugated IgG, and washed again. For
phosphoprotein analyses, the membranes were incubated for 120 min with
the primary anti-phosphothreonine or anti-phosphoserine antibody (2 mg/ml dilution), washed, incubated for 75 min with HRP-conjugated IgG,
and washed again. The antigen-antibody complexes were visualized using
the enhanced chemiluminescence system (ECL; Amersham) and quantitated
densitometrically using the Visage 2000. Samples to be compared were
always run in parallel, and the optical densities of the experimental
islet samples are given as the percentage of the control islet samples.
Reagents.
Hanks' solution was used for the islet isolation. The perifusion
medium (KRB) consisted of (in mM) 115 NaCl, 5 KCl, 2.2 CaCl2, 1 MgCl2, and 24 NaHCO3, with 0.17 g/dl BSA. Other
compounds were added as indicated, and the solution was gassed with a
mixture of 95% O2-5%
CO2. The
125I-labeled insulin,
5-[3H]glucose, and
3H2O
were purchased from DuPont NEN (Boston, MA). BSA (RIA grade), glucose,
TPA, and glutamine, as well as the salts used to make the Hanks'
solution and perifusion medium, were purchased from Sigma (St. Louis,
MO). Culture medium (CMRL-1066), fetal bovine serum, and the
antibiotics penicillin and streptomycin were purchased from GIBCO
(Grand Island, NY). Glutamine was added to the CMRL-1066 to achieve a
final concentration of 2.0 mM. The nylon sheets (no. 3-48/33) cut into
small disks and used to support the islets during the perifusions and
glucose usage analyses were purchased from Tetko (Briarcliff Manor,
NY). Rat insulin standard (lot no. 615-ZS-157) was the generous gift of
Dr. Gerald Gold (Eli Lilly, Indianapolis, IN). Collagenase (type P) was
obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN).
Antibodies to PLC-1 were purchased from Upstate Biotechnology (Lake
Placid, NY). Antibodies to PLC-
1, PKC
, and the goat anti-rabbit
and goat anti-mouse IgG-HRP antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-phosphoserine and
anti-phosphothreonine antibodies were purchased from Zymed Laboratories
(South San Francisco, CA). ECL reagents and films were from Amersham.
Statistics. Statistical significance was determined using Student's t-test for unpaired data or ANOVA in conjunction with the Newman-Keuls test for unpaired data. A P < 0.05 was taken as significant. Values presented are means ± SE of at least three observations.
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RESULTS |
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Protein, insulin, and PKC contents of freshly
isolated or cultured islets.
In the initial series of experiments, we compared islet protein and
insulin contents in freshly isolated vs. cultured islets. The protein
content of fresh islets was 0.81 ± 0.05 µg/islet
(n = 6). Similar protein contents were
observed in control-cultured islets (0.76 ± 0.07 µg/islet) and islets cultured in the additional presence
of 500 nM TPA (0.72 ± 0.12 µg/islet;
n = 6 for each condition). The insulin
contents of fresh islets, control-cultured islets, and islets cultured
in TPA were 0.268 ± 0.031, 0.232 ± 0.017, and 0.165 ± 0.018 µg/islet, respectively (n = 6 for
each condition). The insulin content of islets cultured in 500 nM TPA
was significantly less than the contents of the other two groups.
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TPA-induced insulin secretion from freshly isolated and cultured islets. Insulin secretion to 500 nM TPA stimulation during a dynamic perifusion was abolished from islets previously cultured with 500 nM TPA for 1 day (Fig. 3). In contrast, both freshly isolated islets and control-cultured islets exhibited similar responses to TPA (Fig. 3). As reported previously (27, 38), a slowly rising phase of release followed sustained exposure to the phorbol ester. In quantitative terms the response achieved 35-40 min after the onset of stimulation with TPA (in the simultaneous presence of 3 mM glucose) was only ~15-20% of the second phase response to 15 mM glucose stimulation from freshly isolated islets (Fig. 4).
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Glucose-induced insulin secretory responses of freshly isolated and
cultured islets.
In agreement with previous studies (21, 28), insulin secretory
responses to 15 mM glucose from islets cultured in TPA were greater
than the responses from islets cultured in its absence (Fig. 4). Both
first and second phase responses were increased. For example, peak
first phase release rates from islets cultured in TPA averaged 554 ± 56 pg · islet1 · min
1.
Release rates measured 35-40 min after the onset of stimulation averaged 331 ± 65 pg · islet
1 · min
1. The comparable
responses from controlcultured islets were 167 ± 35 and 235 ± 49 pg · islet
1 · min
1.
Potassium-induced insulin secretion from freshly isolated and
cultured islets.
Because TPA sensitizes islets to calcium, the possible contribution of
this cation to the augmentation of secretion observed from TPA-cultured
islets was examined by determining islet responses to depolarizing
levels of potassium. Potassium depolarizes -cells, an event that
opens voltage-regulated calcium channels and results in a
calcium-dependent insulin secretory response (18). Freshly isolated
islets responded to 30 mM potassium stimulation (in the simultaneous
presence of 3 mM glucose) with an acute insulin secretory response that
waned as the perifusion progressed (Fig.
5). For example, 2-3 min after the
onset of potassium stimulation, release increased from prestimulatory
rates of 42 ± 9 to 415 ± 71 pg · islet
1 · min
1.
Similar peak insulin secretory responses (500 ± 119 pg · islet
1 · min
1)
were obtained from 1-day control-cultured islets (Fig. 5). The secretory response was more sustained however. In contrast to control-cultured islets, significantly amplified secretory responses to
potassium were observed from TPA-cultured islets (Fig. 5). In
particular, the magnitude of the initial response was striking. It
averaged 1,905 ± 319 pg · islet
1 · min
1
at its apex and slowly fell as the perifusion progressed. Even after 30 min of stimulation with potassium, the secretory response from
TPA-cultured islets (585 ± 173 pg · islet
1 · min
1)
was still greater than that seen from control-cultured islets (264 ± 59 pg · islet
1 · min
1).
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Effects of nitrendipine on potassium- and glucose-induced secretion. Additional studies were conducted with a calcium channel blocker. In these studies, islets were stimulated with 30 mM potassium in the presence of nitrendipine. At a level of 5 µM, nitrendipine abolished potassium-induced secretion from freshly isolated islets or from 1-day control-cultured islets (results not shown). This level of nitrendipine abolished the amplified insulin secretory response to high potassium from TPA-cultured islets (Fig. 6).
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Glucose usage rates in freshly isolated or cultured islets. Rates of glucose usage at two different glucose levels were comparable for freshly isolated islets and for islets after a 1-day culturing period (Table 1). The presence of TPA had no obvious adverse effect on glucose usage by islets.
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Phosphoprotein analysis of cultured islets: effects of TPA.
Because TPA phosphorylates a large number of protein substrates on
serine or threonine residues (25) and because its effects are sustained
for many hours (11), we considered the possibility that, despite the
dramatic decline in immunoreactive PKC content, the sustained
phosphorylation of proteins may be involved in the amplified insulin
secretory responses to potassium and in the modest increase (compared
with control-cultured islets) when these islets are stimulated by 15 mM
glucose. To address this possibility, islets were cultured in the
presence or absence of 500 nM TPA, harvested, sonicated, and then
analyzed for phosphoprotein content using antibodies that detect
phosphoserine or phosphothreonine residues on islet proteins.
Densitometric analyses revealed a 119 ± 18% increase in the
phosphoserine content of one islet protein (~195 kDa) in sonicates
from TPA-cultured islets compared with those from islets cultured in
the absence of the phorbol ester (Fig. 7).
Four other proteins (~120 kDa, 104 ± 12% increase; ~140 kDa,
110 ± 19% increase; ~185 kDa, 220 ± 32% increase; ~225
kDa, 773 ± 93% increase) in TPA-cultured islets also displayed
increased phosphothreonine labeling.
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PLC isozyme alterations in freshly isolated or cultured islets.
A possible explanation for the reduction in glucose-stimulated insulin
secretion (Fig. 4) and glucose-induced inositol phosphate accumulation
in cultured islets (46) compared with freshly isolated islets is that
islet content of PLC is adversely affected. As shown in Fig.
8, culturing for 22-24 h reduced the
expression of both PLC-1 and PLC-
1. Compared with freshly studied
islets, both isozymes were reduced ~50%.
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Restoration of glucose-induced secretion by PKC activation in control-cultured islets. Based on the observations that PLC isozyme expression (Fig. 8) and activation (46) are impaired as a result of a 22- to 24-h culturing period, a possible explanation for the decline in insulin secretory responsiveness to 15 mM glucose from control-cultured islets is that they are unable to generate the necessary signals, particularly perhaps diacylglycerol, to activate PKC. We tested this hypothesis by stimulating control-cultured islets with 15 mM glucose plus the exogenous PKC activator TPA (500 nM). Insulin secretion from these islets was brisk and biphasic (Fig. 9), suggesting that the secretory responsiveness of these islets can be amplified by the concomitant activation of PKC. Release rates from these cultured islets were, however, slightly reduced compared with the responses of freshly isolated islets.
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DISCUSSION |
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At the outset, several comments are warranted concerning the approaches
used in these experiments and their potential impact on our results and
their interpretation. Rat islets were used in all experiments. We are
aware of several publications on mouse islets using a similar approach
to assess the contribution of PKC to glucose-stimulated secretion (1,
36). However, mouse islets differ in several significant respects from
both rat and human islets in their responses to glucose. Mouse islets
neither exhibit a rising second phase response to the hexose (3, 24, 26, 47) nor develop time-dependent potentiation when briefly primed by
glucose (4, 44). Both of these time-dependent phenomena, which appear
to involve an increase in signaling via the PLC/PKC transduction system
(37), are well characterized in both rat and human islets (9, 12, 16,
34, 40). We focused on PKC in these experiments because it is
calcium dependent, it translocates in response to stimulatory glucose
and TPA (11, 13, 14), and its islet content can be significantly
reduced by sustained exposure to phorbol esters (25). The possibility that other isozymes of PKC or other signal transduction systems (cAMP,
calmodulin, or other kinases effectors) involved in stimulus-response coupling contribute to the responses observed cannot be excluded.
Two separate but interrelated issues are addressed in the present
series of experiments: the effect of culturing alone on islet responses
and the nature and specificity of the alterations produced in islets as
a result of culturing in the additional presence of TPA. An underlying
assumption made when islets are cultured in TPA is that PKC
depletion is the only pertinent (to insulin secretion) alteration
produced. As suggested by others (33), this is not necessarily the
case, and our findings reinforce this concept. Moreover, these
additional cellular changes may explain why glucose stimulation is
still able to induce insulin release despite the dramatic reductions in
PKC
immunoreactivity or activation, monitored in this report by the
abolition of its insulin stimulatory effect or by Western blot
measurements using highly specific antibodies directed against PKC
.
Our conclusion as to the involvement of PKC
activation in the
physiological regulation of glucose-induced insulin secretion differs
from the earlier study by Hii and co-workers (21) when one takes into account three other parameters of the
-cell: the decline in islet responsiveness to glucose stimulation from cultured islets compared with freshly isolated islets, the islet responses to potassium, and the
sustained phosphorylation of islet proteins in TPA-cultured islets.
Hii and co-workers (21) were the first to report that the depletion of
PKC (monitored by failure of the these islets to respond to TPA in
terms of insulin secretion and phosphorylation of histone) by chronic
20- to 24-h exposure to TPA (0.1-1.0 µM) left 20 mM
glucose-induced insulin release from rat islets intact. In fact, 20 mM
glucose-induced insulin secretion was slightly augmented from
PKC-depleted islets compared with control-cultured islets, a finding
also made herein. However, in their study (21), insulin release rates
to 20 mM glucose stimulation from control-cultured islets less than
doubled (from 0.44 to 0.75 ng · h1 · islet
1)
compared with the responses observed with 5.6 mM glucose. This marked
deviation of the response of their control-cultured islets to 20 mM
glucose from the physiological secretory responses of freshly isolated
islets or of the perfused pancreas preparation (7, 15, 16, 19, 39) was
not commented on in the report (21). Shortly after, another report (28)
on experiments using cultured islets (in RPMI-1640 medium) exposed to
TPA for 18 h was published. In these studies, 16.7 mM glucose-induced
insulin secretion from PKC-depleted islets (assessed by the failure of TPA to induce insulin secretion) was again greater than that seen from
control-cultured islets, a finding reported herein as well. Also
included in this report were insulin release rates from freshly isolated islets. The decline in insulin release rates from
control-cultured islets compared with freshly isolated islets,
regardless of whether or not they were previously exposed to TPA during
the culture period, was ~70% in this report (28) and is comparable
to the findings reported here.
Compared with freshly isolated islets, significant reductions in the
expression of several major PLC isozymes and reduced PLC-mediated
hydrolysis of islet phosphoinositide pools (46) characterize cultured
rat islets. Altered information flow in the PLC/PKC signaling system in
cultured -cells may explain, at least in part, the reduction in
glucose-induced insulin secretion from cultured islets compared with
freshly isolated islets or the perfused pancreas preparation. The loss
of PLC during the culture period would result in the failure of
glucose-stimulated islets to generate amounts of
phosphoinositide-derived signals necessary to support secretion to the
same quantitative extent as is seen from freshly isolated islets. This
defect in cultured islets would also be accompanied by the failure of
glucose stimulation to activate PKC, since lipid factors, particularly
diacylglycerol (2), would not be generated in the appropriate amounts.
The observations that protein content, glucose usage rates, PKC
content, and TPA-induced insulin secretion were comparable from freshly isolated and control-cultured islets suggest that culturing has not
induced a generalized and nonspecific reduction in protein synthesis or
in the capacity of the islet to be stimulated. Of particular
significance is the observation that the addition of the pharmacologic
PKC activator TPA to control-cultured islets exerted a significant
restorative effect on 15 mM glucose-induced insulin secretion. This
finding also reinforces the concept that cultured islets do retain a
high degree of secretory responsiveness and that TPA provides, perhaps,
a signal missing from these cultured islets and one that contributes to
the evocation of a large and sustained insulin secretory response.
In freshly isolated islets or in control-cultured islets, depolarizing
levels of potassium (in the presence of 3 mM glucose) initiate
comparable rates of insulin secretion. This response is characterized
by a large spike and a subsequent decline in hormone output. When
PKC-depleted islets are similarly treated, the response to potassium
is dramatically enhanced. Both the initial and sustained phases of
release are amplified but still susceptible to complete inhibition by
the calcium channel antagonist nitrendipine (5 µM). Because
potassium-induced insulin release is dependent on calcium influx into
the
-cell (20), this suggests that
-cell calcium sensitivity has
been enhanced by culturing in TPA and that this alteration is sustained
despite dramatic reductions in immunoreactive PKC
content. This
finding is also in accord with the established acute effect
of TPA to increase the sensitivity of the
-cell to calcium (17, 35),
an effect that is sustained for hours (11).
Islets cultured in the presence of TPA are characterized by an increase
in the phosphorylation state of serine and threonine residues on at
least several islet proteins. It was not the goal of these studies to
establish which of the approximately one hundred target substrates (25)
for PKC remain in a highly phosphorylated state even as PKC is being
depleted. Rather, we wanted to investigate the possibility that
prolonged treatment with TPA may exert long-term and sustained effects
on islet phosphoprotein patterns. Although their nature remains to be
determined, the sustained phosphorylation of PKC-dependent target
proteins, as documented in other cell types (30), may account for the
ability of potassium to evoke a markedly amplified secretory response
from PKC
-depleted islets and for the persistence of the small
enhancing effect of glucose on secretion.
In conclusion, the studies presented here emphasize the inherent
complexity of the islet stimulus-response coupling processes, the
concept that the prior history of the islet plays a key role in
determining the subsequent biochemical and secretory responses to
glucose, the importance of response elements distal to PKC activation
in the regulation of insulin secretion, and the physiological shortcomings of studies with cultured islets. Emphasis should now,
perhaps, be placed on identifying the factors necessary to maintain the
PLC isozymes in cultured islets and on the protein substrates that are
phosphorylated during long-term exposure to TPA and that may serve to
enhance the sensitivity of the -cell to calcium.
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ACKNOWLEDGEMENTS |
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These studies were supported by National Institutes of Health Grants DK-41230 and DK-50662.
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FOOTNOTES |
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Address for reprint requests: W. S. Zawalich, Yale University School of Nursing, 100 Church St. South, New Haven, CT 06536-0740.
Received 6 November 1997; accepted in final form 4 February 1998.
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