Section of Endocrinology and Medical Service, Middleton Veterans Affairs Medical Center, Madison 53705; and Division of Endocrinology and Metabolism and Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53792
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ABSTRACT |
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Although interleukin-1 (IL-1
) reduces
pancreatic islet content of ATP and GTP, the distal events that mediate
its inhibitory effects on insulin secretion remain poorly understood.
Herein, the activation of phospholipase C (PLC) was quantified during islet perifusions. An 18-h exposure to IL-1
(100 pM) totally vitiated activation of PLC induced by glucose, an effect that requires
ATP and GTP and closure of the ATP-dependent
K+
(KATP) channel. Surprisingly,
however, when islets were depolarized directly using either of two
agonists, glyburide (which does not act via generation of purine
nucleotides) or 40 mM K+ (which
acts distal to KATP channel), PLC
and insulin secretion were again obliterated by IL-1
. IL-1
also
reduced the labeling of phosphoinositide substrates; however, this
effect was insufficient to explain the inhibition of PLC, since the
effects on substrate labeling, but not on PLC, were prevented by
coprovision of guanosine or adenosine. Furthermore, when
IL-1
-treated islets were exposed to 100 µM carbachol (which
activates PLC partially independent of extracellular
Ca2+), the effects were still
obliterated by IL-1
. These data (together with the finding that
IL-1
inhibited Ca2+-induced
insulin release) suggest that, in addition to its effects on ATP
synthesis and thereby on the KATP
channel, IL-1
has at least two undescribed, distal effects to block
both PLC as well as Ca2+-induced
exocytosis. The latter correlated best with IL-1
's effect to impede
phosphoinositide synthesis, since it also was reversed by guanosine or
adenosine.
pancreatic islet; calcium; exocytosis; purine nucleotides
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INTRODUCTION |
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IT IS WIDELY ACCEPTED that interleukin-1 (IL-1
)
contributes in a significant way to the functional, as well as the
anatomic, insult to pancreatic
-cells at the onset of
insulin-dependent diabetes mellitus in humans. For example, exposure of
rodent islets to IL-1
(or exposure of human islets to IL-1
in the
concomitant presence of other cytokines) leads to inhibition of
glucose-induced insulin secretion (4, 16) followed, after longer
periods, by the onset of programmed cell death (apoptosis; Ref. 12). It
has frequently been assumed that many or all of these effects are
attributable to deleterious effects of IL-1
on energetics in the
pancreatic
-cell (20). For example, IL-1
, acting via the release
of nitric oxide (NO), can interfere with the activation of
mitochondrial (and possibly cytosolic) enzymes involved in glucose
metabolism and electron transport (20). The result of this would be to
impede the generation of high-energy phosphate-containing molecules
required for exocytosis. Indeed, in a recent comprehensive study of ATP
and GTP synthesis in rodent pancreatic islets and transformed
-cells, we observed that IL-1
preexposure has profound effects
not only on ATP metabolism but also on that of GTP (16). The latter
might also be relevant, since we have recently identified a requisite
role for GTP in glucose-induced activation of phospholipase C (PLC) and
glucose-induced insulin secretion (18, 25).
It might be assumed that inhibition by IL-1 of ATP synthesis,
and/or of the glucose-induced rise in the ATP-to-ADP ratio, would impede the closure of the ATP-dependent
K+ channel
(KATP channel) in the
-cell,
thereby blocking cell depolarization and the consequent
Ca2+ influx (cf. Ref. 27). The
latter should in turn impede the activation of PLC in islets, where PLC
is Ca2+ stimulated (25 and
references therein). Inhibition of PLC may in turn mediate, at least in
part, the reduction of glucose-induced insulin secretion via blockade
of the generation of signaling molecules such as inositol phosphates
and/or diacylglycerol.
However, we recently reported that IL-1 inhibits the secretory
response to a depolarizing concentration of
K+ during static islet incubations
(16). Because high K+ stimulates
insulin secretion by directly provoking
Ca2+ influx in a fashion that is
independent of closure of KATP
channels or the activation of PLC, this finding implied additional
distal mechanism(s) of action of IL-1
, possibly at the site of
Ca2+ influx and/or the
subsequent effects of Ca2+. We
therefore systematically studied PLC activation in perifused normal rat
pancreatic islets after exposure to IL-1
. Our results support the
hypothesis that IL-1
has additional, previously unanticipated, effects to inhibit "directly" the activation of PLC in the
pancreatic islet and reduce
Ca2+-activated insulin secretion.
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METHODS |
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Materials.
Human recombinant IL-1 was purchased from Boehringer Mannheim
(Indianapolis, IN).
NG-nitro-L-arginine
methyl ester (L-NAME),
guanosine, indomethacin, adenosine, carbamylcholine chloride
(carbachol), and glyburide were purchased from Sigma Chemical (St.
Louis, MO).
1H-(1,2,4)oxadiazolo(4,3-
)quinoxalin-1-one (ODQ) was from Biomol (Plymouth Meeting, PA). The diluents for making
stock solutions of these drugs were water for IL-1
and L-NAME, dimethyl sulfoxide for
guanosine, adenosine, ODQ, and glyburide, and ethanol for indomethacin.
Control groups always contained an equal amount of the diluent as the
experimental groups. Myo-[2-3H]inositol
(20.5 Ci/mmol),
[inositol-2-3H(N)]phosphatidylinositol
(PtdIns; 10 Ci/mmol),
[inositol-2-3H(N)]phosphatidylinositol
4-phosphate [PtdIns(4)P; 7.3 Ci/mmol], and
[inositol-2-3H(N)]phosphatidylinositol
4,5-bisphosphate
[PtdIns(4,5)P2; 6 Ci/mmol] were purchased from Du Pont NEN Research Products
(Boston, MA). RPMI-1640 medium was purchased from GIBCO (Grand Island,
NY).
Isolation and treatment of pancreatic islets.
Intact pancreatic islets were isolated from adult male Sprague-Dawley
rats using collagenase digestion and separation from acinar tissue and
debris on Ficoll gradients, as previously described (25). To further
exclude contamination by exocrine tissue, islets were hand picked
(×2) under stereomicroscopic observation. The islets were
cultured for 18 h (unless otherwise indicated) in batches of 100 in
RPMI-1640 medium containing 10% fetal calf serum, 11.1 mM glucose, 100 U/ml of penicillin, 100 µg/ml of streptomycin, 5 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, and myo-[3H]inositol
(25 µCi/ml) in the presence or absence of 100 pM IL-1. The dose of
IL-1
was chosen on the basis of our previous observation that
exposure to 100 pM of IL-1
for 18 h exerted the maximal inhibition
of subsequent glucose-induced insulin secretion in cultured rat islets
without altering basal insulin secretion, insulin content, or protein
content (16). After the culture period, the islets were labeled for an
additional 2 h in Krebs-Ringer bicarbonate buffer (pH 7.4), gassed with
95% O2-5%
CO2 and containing 0.2% bovine
serum albumin (BSA), 4.4 mM glucose,
myo-[3H]inositol
(25 µCi/ml) and the continued presence or absence of IL-1
after a
previously described protocol (25). When used, 500 µM adenosine,
guanosine, or ribose was present throughout the 18 h and the following
2 h of labeling. When islets were exposed to
L-NAME, the medium was arginine
free (made from RPMI-1640 Select-amine kit, adding all amino acids
except arginine), since this combination has been shown to block NO
production totally (16, 23).
Perifusion experiments. After completion of the final 2-h labeling period, groups of 100 islets were transferred into each of four or six perifusion chambers and were perifused using a previously described procedure (25). The islets were first perifused (at 1 ml/min) for 30 min at 3.3 mM glucose to establish a stable basal rate of inositol efflux. The ensuing incubation period was for 45 min. The standard perifusion medium consisted of Krebs-Ringer bicarbonate buffer containing 0.2% BSA, 1 mM unlabeled myo-inositol (to prevent reincorporation of released [3H]inositol), and the agonists as indicated in RESULTS and gassed with 95% O2-5% CO2. In experiments where K+ was the agonist, an appropriate amount of sodium chloride was removed from the medium to keep the osmolarity constant. The samples of the effluent were collected every 2 min using a Gilson fractionator (Middleton, WI). Data were corrected for the dead space of 4 ml. The perifusate was analyzed for the content of [3H]inositol using previously described methods (25).
In some experiments, where indicated, islets were cultured for 30 h in the absence of IL-1Static measurement of insulin release.
This was carried out as previously described for IL-1 studies (16).
In brief, insulin secretion was assessed in groups of 10 islets; a
preincubation period of 45 min was followed by a 45-min incubation
period. Unlabeled inositol was not included in the medium for these
secretory studies. Insulin in the medium was measured by
radioimmunoassay (16).
Islet lipid extraction.
At the end of the perifusion incubation periods, the islets on their
polyethylene filters were transferred to polypropylene centrifuge
tubes. Absolute methanol (1 ml), 2 ml of chloroform, and 10 µl of
concentrated hydrochloric acid were added. Islets were sonified for 10 s using a Branson 450 sonifier (Danbury, CO) and were left overnight at
4°C. The next day, phase separation was induced by addition of 750 µl of 500 mM KCl-50 mM EDTA mix followed by centrifugation at 1,200 rpm for 6 min. The upper (aqueous) and lower (organic) phases were
separated, and 100-µl aliquots of the organic phase were counted for
[3H]inositol. In some
experiments, the islets were extracted immediately after the labeling
to study the effect of IL-1 on phosphoinositide labeling.
Thin-layer chromatography.
SL60 silica gels, 250 µm thick (Whatman, Clifton, NJ), were treated
with oxalate by running the plates overnight in 1.2% potassium oxalate, dissolved in methanol and water (2:3). The plates were preactivated by heating at 110°C for 30 min before loading the samples. The organic phase of the islet extract was dried down on ice
under argon gas and was resuspended in 20 µl of chloroform-methanol (2:1) ×2 and was loaded onto the plates. The plates were then developed in a solvent system consisting of
methanol-chloroform-ammonium hydroxide-water (100:70:15:25). The
relevant areas [PtdIns, PtdIns(4)P, and
PtdIns(4,5)P2] of the plates
were subsequently scraped after autoradiographic exposure for 48 h at
70°C. The scrapings were counted for
3H content using 4 ml of
scintillation fluid (Packard, Meriden, CT) with 1 ml of methanol.
Data presentation and statistical analysis. Efflux of [3H]inositol is expressed as fractional efflux (FEI) per minute per 100 islets, calculated using the disintegrations per minute (dpm) in each perifusate sample as the numerator and the total [3H]phosphoinositide content as the denominator. The latter was determined by adding the total dpm in each perifusate sample to the 3H-containing phospholipid fraction of the islets, obtained by phospholipid extraction of the islets at the end of the experimental incubation. At each time point, the dpm released during the previous 2 min were subtracted from this total. The incremental responses for inositol release were calculated by subtracting the mean of the last four basal values during the preincubation period from each of the values during the incubation periods. Areas under the curve (AUC) were calculated using the trapezoid rule; AUC for inositol efflux is expressed as percentage of [3H]inositol-containing phospholipids per 45 min per 100 islets. Insulin release (in static incubations) is expressed as microunits per 10 islets per 45 min. The data are expressed as means ± SE, with n representing the number of experiments except as otherwise indicated. Statistical analyses of the results in perifusion experiments were carried out on AUC; analyses were done by paired or nonpaired t-test as appropriate. When comparisons involving more than two groups were made, one-way analysis of variance (ANOVA) was first carried out using Student-Newman-Keuls method. P < 0.05 was considered as significant.
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RESULTS |
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Effect of IL-1 on glucose-induced phosphoinositide
hydrolysis.
During the 30-min preincubation period (at 3.3 mM glucose), the FEI
reached a steady-state level in all experiments and was not influenced
by IL-1
. Perifusion of control islets with 16.7 mM glucose induced
an increase in FEI from a basal rate of 0.28 ± 0.03%/min to a peak
rate of 0.95 ± 0.44%/min (Fig. 1); AUC
above the prestimulus basal rate was 12.9 ± 1.94%/45 min. In
IL-1
-pretreated islets, there was a virtually complete absence of
response during incubation with 16.7 mM glucose (Fig. 1).
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Effect of IL-1 on inositol efflux induced by
glyburide or high
K+.
Inhibition of glucose-induced PLC activity by IL-1
is consistent
with the dramatic fall in ATP induced by the latter (16), leading to a
failure to close the KATP channel.
The latter effect would impede depolarization of the islets, preventing
the Ca2+ influx required for PLC
activation (cf. Ref. 27). To study the effect of directly closing
KATP channels and bypassing ATP synthesis steps, a sulfonylurea, which directly closes the
KATP channel, was next studied. In
control islets, 10 µM glyburide induced an increase in FEI from a
basal rate of 0.30 ± 0.01%/min to a peak rate of 0.66 ± 0.05%/min (Fig. 3); unexpectedly, however, this response was abolished by IL-1
pretreatment. In static
incubations, IL-1
also inhibited the insulin secretion induced by 10 µM glyburide (Fig. 4). Thus, in
IL-1
-pretreated islets, glyburide-induced (i.e.,
Ca2+-stimulated) PLC activation
and insulin secretion were absent.
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Effect of IL-1 on content of phospholipid
substrates.
One of the possible explanations for absence of PLC activation in
IL-1
-pretreated islets might be that IL-1
affected
phosphoinositide labeling (26), thereby reducing the substrates needed
for all agonists to activate PLC. Phosphoinositides may also have a
direct role in exocytosis (see
DISCUSSION). To assess IL-1
effects on labeling, some islets were extracted immediately after the
20-h labeling period (in presence or absence of IL-1
) without
additional experimental incubations. IL-1
reduced the labeling of
PtdIns(4,5)P2 by 67%, PtdIns(4)P
by 55%, and PtdIns by 53% (P < 0.05, by ANOVA; Table 1). To determine
whether IL-1
's inhibitory effect on labeling is mediated via NO, in
some experiments, islets were labeled in arginine-free media (18 h of
culture + 2 h of 2nd-day labeling) in the presence of 1 mM
L-NAME. The phosphoinositide
contents of the islets studied without any experimental incubation are summarized in the Table 2. In the presence
of L-NAME, IL-1
now failed to
inhibit the labeling of the phosphoinositide, indicating that the
inhibition of phosphoinositide labeling by IL-1
is mediated, at
least in part, via NO.
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Effects of guanosine, adenosine, or ribose.
We have previously shown that IL-1 pretreatment leads to reduction
of ATP and GTP as well as the ATP-to-ADP and GTP-to-GDP ratios in
islets (16). To determine whether the inhibition of labeling of
phosphoinositides is secondary to a decline in purine nucleotide
content, 500 µM adenosine (which partially restores ATP content but
not ATP-to-ADP ratio; Ref. 16) or 500 µM guanosine (which restores
GTP but not GTP-to-GDP ratio; Ref. 16) was concurrently provided with
IL-1
. Either adenosine or guanosine reversed the effect of IL-1
on labeling of phosphoinositides to a substantial, though incomplete,
degree [PtdIns(4,5)P2 by
82%, PtdIns(4)P by 46%, and PtdIns by 39%; Table 1]; neither
had any effect on phosphoinositide content in control islets (data not
shown). Coprovision of guanosine was also able to reverse the effect of
IL-1
on insulin secretion stimulated by glyburide (Fig. 4) or 40 mM
K+ (basal = 51 ± 9, n = 6; 40 mM
K+ = 320 ± 28, n = 5; 40 mM
K+ + IL-1
: 71 ± 5, n = 6, P < 0.001; 40 mM
K+ + IL-1
+ 500 µM guanosine = 405 ± 43; n = 6). In
fact, guanosine actually induced an overshoot in insulin secretion in
IL-1
-pretreated islets (only). In additional experiments (not
shown), 500 µM adenosine was as effective as guanosine in preventing
the inhibition by IL-1
of glyburide-induced insulin release, though
having no direct stimulatory effects of its own. However, neither
adenosine nor guanosine reversed the inhibition of PLC
activation (Fig. 5). It is thus unlikely that IL-1
exerted most of
its effect on FEI by inhibiting the synthesis of phosphoinositides;
however, the latter effect (especially on PtdIns) may be involved in
its antisecretory actions (see
DISCUSSION).
Effect of carbachol on PLC activation in
IL-1-treated islets.
The findings of a blockade by IL-1
of PLC activation in response to
either glyburide or 40 mM K+ could
indicate a defect either in Ca2+
influx (via voltage-dependent calcium channels) or distal
to Ca2+ influx. To analyze this
defect further, muscarinic cholinergic receptor agonist
(carbachol)-induced PLC activation was next studied. Carbachol induces
a direct receptor-mediated stimulation of PLC, a significant proportion
of which is independent of extracellular Ca2+ (8, 25). We reasoned that if
IL-1
affected PLC activation by affecting
Ca2+ influx, then the direct
receptor-mediated effects of carbachol might be resistant to inhibition
by IL-1
. Carbachol (100 µM) induced an increase in FEI from a
basal rate of 0.37 ± 0.03%/min to a peak rate of 3.03 ± 0.17%/min in control islets (Fig. 6). IL-1
pretreatment completely abolished this response as well (Fig.
6).
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Possible cellular mediators of inhibition of PLC by
IL-1.
IL-1
is known to generate large increases in prostaglandins (10) or
guanosine 3',5'-cyclic monophosphate (cGMP) (5) in islets
or
-cells in a NO-dependent fashion. Because either prostaglandin
E2
(PGE2) (14) or cGMP (10) may
inhibit PLC activation, we investigated the effects of 5-10 µM
indomethacin (an inhibitor of prostaglandin synthesis) or 10 µM ODQ
(a specific inhibitor of NO-activated guanylate cyclase; Ref. 21) on
PLC activation. Despite the inclusion of these inhibitors during the labeling, preincubation and incubation periods, absolutely no reversal
of IL-1
-induced inhibition of phosphoinositide synthesis, PLC
activation, or insulin release was observed, either in response to 16.7 mM glucose or 40 mM K+
[degrees of freedom (df) = 10 for glucose or K+
as agonist, in presence or absence of indomethacin; df = 2 for glucose
as agonist in presence or absence of ODQ; data not shown].
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DISCUSSION |
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In these studies, we have used FEI as our principal index of PLC activation, as employed by Zawalich et al. (28-30). We have previously refined and standardized this technique and have summarized the reasons it serves as a specific index of PLC activity when carried out as described above (25). PLC activation generates intracellular inositol phosphates, which are degraded to produce free inositol; in the presence of a saturating concentration of unlabeled inositol, the labeled inositol released cannot be reincorporated into phospholipids and subsequently effluxes into the medium. We have also previously documented that PtdIns(4)P and PtdIns(4, 5)P2 are labeled to isotopic equilibrium during an overnight labeling period, during which PtdIns is labeled to ~75% of isotopic steady state (25). Furthermore, when islets were labeled to full isotopic equilibrium (50 h of labeling), similar results were obtained. Thus we are confident that our FEI results represent quantitatively the true activation of PLC and not base exchange (or other) mechanisms.
We observed that exposure to IL-1 obliterated the activation of PLC
induced by all agonists studied. There are extant data suggesting that
ATP and/or GTP may be involved in the synthesis of
phosphoinositide substrates (2, 22). Because IL-1
decreases ATP
and/or GTP content (as we have previously documented; Ref. 16),
then PLC substrate (rather than enzyme activity) might be altered, as
was observed. However, this effect cannot (fully) explain the apparent
inhibition of PLC activity by IL-1
, for two reasons. First, we have
expressed our data as fractional efflux of inositol, thereby correcting
the denominator for any alteration in the labeling of
phosphoinositides. In addition, the reduction by IL-1
in labeling of
phospholipid substrates was largely prevented by provision of adenosine
or guanosine, but this did not lead to any substantial restoration of
the PLC response. Thus we conclude that IL-1
inhibits PLC activity
but, in addition, has effects to impede the resynthesis of phospholipid
substrates, an effect that may potentiate the net inhibition of
phosphoinositide turnover.
Could the inhibition of PLC by IL-1 be attributed to decrements in
GTP (32) or ATP, such as those known to be induced by IL-1
(16)? In
previous studies, when the purine precursors adenine or guanine were
provided to pancreatic islets pretreated with IL-1
(16), they were
unable to restore ATP or GTP levels, either because IL-1
blocked the
"salvage" of these purine bases to their phosphorylated
derivatives or, more likely, because IL-1
(via its putative effects
on glucose metabolism) impeded the synthesis of the precursor PRPP,
which is needed to salvage adenine or guanine (16). Therefore, in the
current studies, we provided the nucleosides adenosine or guanosine, or
ribose, alone. The two nucleosides are degraded intracellularly (via
action of enzyme purine nucleoside phosphorylase) to their
respective bases and, in addition, to ribose 1-phosphate. Ribose
1-phosphate is the forecursor of PRPP and thus putatively might
circumvent any impedance by IL-1
of the formation of PRPP
precursors. We indeed found that adenosine, guanosine, or ribose
substantially corrected PtdIns synthesis; however, only guanosine or
adenosine corrected the inhibition of synthesis of
PtdIns(4,5)P2 induced by IL-1
.
The latter is therefore probably attributable to restoration of
cellular levels of purine nucleotides. For PtdIns, we speculate that
putative defect in glucose metabolism induced by IL-1
(cf.
references in Ref. 16) reduces the supply of 3-carbon triose needed as backbone for new PtdIns synthesis, a defect bypassed by providing ribose (either directly or in nucleosides). Interestingly, in a
previous study of IL-1
(27), no inhibition of phosphoinositide synthesis by IL-1
was found; however, labeling was carried out in
CMRL-1066, which contains substantial amounts of deoxynucleosides (19),
which would provide both ribose phosphate and nucleobases.
There are two previous studies also suggesting that IL-1 might inhibit
glucose-induced insulin secretion in part via a reduction of PLC
activation (27, 30). In one study, islets were labeled in the presence
of IL-1 for several hours only; it was observed that IL-1
acutely
stimulated, and then somewhat later inhibited, glucose-induced FEI
(30). These studies are somewhat difficult to interpret, since labeling
of substrate was not formally assessed and a very high concentration (5 nM) of IL-1
was used. In addition, 2 h of labeling is insufficient
to achieve isotopic equilibrium in inositol-containing substrates in
islets (25). In another study, a high concentration of IL-1 (2 nM) was
again used; the islets were labeled for 135 min in the absence of IL-1
followed by labeling in the presence of IL-1 for 18 h (27). In this
study, it was observed that the intracellular generation of inositol phosphates was impeded by IL-1; however, glucose alone was used as the
agonist. It would indeed be anticipated, based on current dogma, that
glucose-induced PLC activation should be inhibited by IL-1
. IL-1
leads to the generation of NO, which profoundly reduces both ATP and
GTP within the pancreatic islet (16). A reduction in ATP
(and/or ATP-to-ADP ratio) might be expected to impede the
closure of the KATP channel and
thereby impede Ca2+ influx, which
is necessary for PLC activation in the pancreatic
-cell.
Furthermore, our recent data indicate that a selective deficit in GTP
and/or GTP-to-GDP ratio will decrease the sensitivity of PLC in
the
-cell to activation by Ca2+
(25).
We felt, however, that it was important to investigate other potential
mechanisms of action of IL-1 more formally. We observed that IL-1
(at a much lower concentration than used by previous investigators) did
inhibit the effects of glucose on PLC activity and insulin secretion.
This inhibition was prevented by
L-NAME. However, to our
surprise, we also observed inhibition by IL-1
of PLC activity
stimulated by glyburide, a depolarizing concentration of
K+, or a maximally effective
concentration of the cholinergic agonist carbachol. Glyburide is known
to depolarize the
-cell by directly closing the
KATP channel pharmacologically in
a fashion independent of any increase in intracellular ATP (9); thus
these unexpected findings suggest that IL-1
must directly impede
depolarization of the
-cell, reduce
Ca2+ influx, or inhibit the
subsequent cellular effects of
Ca2+. Indeed, when depolarization
was directly stimulated by 40 mM K+, both PLC activation and
insulin secretion were again blocked. These findings support the
earlier finding (16) of a blockade by IL-1
of insulin secretion
induced by high concentrations of K+ (which does not depend on
activation of PLC) and suggest an apparent blockade of the sensitivity
of islet PLC to activation by Ca2+
influx.
An alternative explanation might be that the opening of
voltage-dependent Ca2+ channels
induced by either depolarizing maneuver was in some way blocked by
IL-1. Indeed, IL-1
(or other agents generating NO) may retard
Ca2+ entry via voltage-dependent
Ca2+ channels in
-cells (13) or
other cells, possibly dependent on cGMP generation (17). However,
selective pharmacological blockade of NO-activated guanylate cyclase by
ODQ (21) failed to prevent IL-1
effects on PLC or secretion.
Furthermore, even if inhibition of
Ca2+ entry did contribute to
IL-1
's inhibitory effects on PLC activation by glucose, 40 mM
K+, or glyburide, this formulation
cannot explain the findings with carbachol. Carbachol is known to
activate PLC by several mechanisms, one of which involves the
receptor-mediated activation of PLC in a fashion resistant to total
removal of extracellular Ca2+
(25). Therefore, if IL-1
was merely acting by impeding
Ca2+ influx, it would be expected
to reduce the effects of carbachol only incompletely; instead, complete
obliteration of carbachol-induced PLC activation was observed. Thus the
hypothesis must be refined to indicate that (at least 1 isoform of) PLC
is directly blocked by the effects of IL-1
. A unitary hypothesis
might be that IL-1
blunts the effects of
Ca2+ in the pancreatic islet, thus
blocking not only Ca2+-induced
insulin secretion but also the activation of PLC by all agonists
studied (since all isoforms of PLC require at least a permissive level
of intracellular Ca2+). Further
studies will be needed to examine this hypothesis directly.
As indicated above, GTP also has effects required both for the
activation of PLC by Ca2+ as well
as for insulin secretion. Although IL-1 does profoundly reduce the
GTP-to-GDP ratio, such an effect cannot explain the total obliteration
of K+- or carbachol-induced PLC
activation or of K+-induced
insulin release from normal islets (25). Could ATP depletion explain
all these results? In addition to its effects on the
KATP channel, ATP has more distal
effects on exocytosis, probably involving the margination of secretory
granules at the plasma membrane and/or priming for subsequent
fusion (15). Inhibition of this latter effect of ATP requires a greater
depletion of ATP than does opening of the
KATP channel (1, 6); it is
possible that a sufficient degree of ATP depletion was achieved in the current studies to bring about such an effect. However, in previous studies, correction of ATP content, achieved via coprovision with Il-1
of adenosine or guanosine, failed to reverse IL-1
's effect on glucose-induced insulin secretion (16). In contrast, in the current
studies, guanosine totally reversed the effect of IL-1
on 40 mM
K+- or glyburide (i.e.,
Ca2+)-induced secretion. Thus
IL-1
appear to inhibit secretion by (at least) two mechanisms,
blockade of PLC (the latter being required for glucose-induced
secretion) and blockade of the direct effect of
Ca2+ on exocytosis (which does not
require PLC). Because addition to IL-1
of guanosine, adenosine, or
ribose restored PtdIns content and
Ca2+-induced insulin release pari
passu but did not restore
Ca2+-induced PLC activity, these
findings suggest a direct participation of PtdIns (or other
phosphoinositides) in Ca2+-induced
exocytosis independent of its role as PLC substrate. Indeed, recent
studies (15) implicate intact phosphoinositides as direct cofactors in
the ATP-dependent priming of exocytosis. Although guanosine (partially)
restores the GTP content of IL-treated islets, this cannot directly
explain the effect on secretion, since even a more profound degree of
GTP depletion fails to inhibit K+-induced secretion from islets
(18).
As indicated above, the inhibition of phosphoinositides synthesis
induced by IL-1 cannot provide the full explanation for its effect
on agonist-induced PLC. It is possible that IL-1
impedes the rise in
cytosolic free Ca2+ levels in
response to agonists, which in turn plays a major role in the
activation of PLC in the pancreatic islet (cf. references in Ref. 25).
There are no extant studies to our knowledge measuring cytosolic free
Ca2+ levels in pancreatic islets
chronically exposed to IL-1
. However, as indicated above, the
studies with carbachol would seem to exclude a blockade of
Ca2+ influx as the (sole)
mechanism of IL-1
's inhibition of PLC. On the basis of our previous
data, the inhibition of the GTP-to-GDP ratio induced by IL-1
also
cannot explain the total inhibition of carbachol effect on PLC (25).
The generation of NO by exposure of cells to IL-1
can lead to a rise
in intracellular cGMP, which, at least in some cells, impedes PLC
activation (3, 10). However, ODQ (a potent and specific inhibitor of
NO-activated guanylate cyclase; Ref. 21) failed to prevent interleukin
effects. Another mechanism could be the increase in
PGE2 associated with IL-1
treatment (11); PGE2 has been
reported to inhibit glucose- or carbachol-induced PLC activation (14).
However, the effects of IL-1
on PLC activation or insulin release
were totally resistant to inhibition by blockade of prostaglandin
synthesis. In preliminary immunoblots, we have been unable to notice
any discernible abnormality in protein levels for the major isoforms of
PLC within the islet after exposure to IL-1
(A. Kowluru and S. Metz;
unpublished data). An attractive explanation may lie in the fact that a
prior exposure to IL-1
(presumably via a transient initial
activation of PLC) could generate an increased level of diacylglycerol
and thereby activate protein kinase C (7); in the pancreatic islet, as in some other cells, prior activation of PLC or protein kinase C may
heterologously "downregulate" the subsequent activation of PLC by
agonists such as carbachol or glucose (29). Alternately, it is possible
that NO inhibits PLC directly via
S-nitrosylation (24). Additional
studies will be needed to sort out these possibilities. Whatever the
molecular defect, the current studies suggest that previous theories as
to the role of IL-1
in mitochondrial energetics do not appear to
adequately explain its effects on PLC activation.
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ACKNOWLEDGEMENTS |
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The technical assistance of J. Stephens is gratefully acknowledged.
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FOOTNOTES |
---|
These studies were supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37312.
Address for reprint requests: S. A. Metz, Div. of Endocrinology and Metabolism, H4/554 Clinical Science Center, University of Wisconsin Medical School, 600 Highland Ave., Madison, WI 53792.
Received 14 March 1997; accepted in final form 7 July 1997.
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