Interleukin-1beta inhibits phospholipase C and insulin secretion at sites apart from KATP channel

Jacob Vadakekalam, Mary E. Rabaglia, and Stewart A. Metz

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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Although interleukin-1beta (IL-1beta ) 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-1beta (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-1beta . IL-1beta 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-1beta -treated islets were exposed to 100 µM carbachol (which activates PLC partially independent of extracellular Ca2+), the effects were still obliterated by IL-1beta . These data (together with the finding that IL-1beta inhibited Ca2+-induced insulin release) suggest that, in addition to its effects on ATP synthesis and thereby on the KATP channel, IL-1beta has at least two undescribed, distal effects to block both PLC as well as Ca2+-induced exocytosis. The latter correlated best with IL-1beta 's effect to impede phosphoinositide synthesis, since it also was reversed by guanosine or adenosine.

pancreatic islet; calcium; exocytosis; purine nucleotides

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IT IS WIDELY ACCEPTED that interleukin-1beta (IL-1beta ) contributes in a significant way to the functional, as well as the anatomic, insult to pancreatic beta -cells at the onset of insulin-dependent diabetes mellitus in humans. For example, exposure of rodent islets to IL-1beta (or exposure of human islets to IL-1beta 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-1beta on energetics in the pancreatic beta -cell (20). For example, IL-1beta , 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 beta -cells, we observed that IL-1beta 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-1beta 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 beta -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-1beta 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-1beta , 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-1beta . Our results support the hypothesis that IL-1beta has additional, previously unanticipated, effects to inhibit "directly" the activation of PLC in the pancreatic islet and reduce Ca2+-activated insulin secretion.

    METHODS
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Introduction
Methods
Results
Discussion
References

Materials. Human recombinant IL-1beta 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-alpha )quinoxalin-1-one (ODQ) was from Biomol (Plymouth Meeting, PA). The diluents for making stock solutions of these drugs were water for IL-1beta 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-1beta . The dose of IL-1beta was chosen on the basis of our previous observation that exposure to 100 pM of IL-1beta 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-1beta 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-1beta but in the presence of myo-[3H]inositol and were then labeled for an additional 20 h (in the presence or absence of IL-1beta ) in myo-[3H]inositol. These islets were then perifused in the absence of unlabeled inositol to preserve equilibrium labeling (25).

Static measurement of insulin release. This was carried out as previously described for IL-1beta 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-1beta 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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of IL-1beta 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-1beta . 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-1beta -pretreated islets, there was a virtually complete absence of response during incubation with 16.7 mM glucose (Fig. 1).


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Fig. 1.   Effect of interleukin-1beta (IL-1beta ) on glucose-induced fractional efflux of [3H]inositol (FEI). Islets were labeled with [3H]inositol (20 h) in batches of 100. Control group (open circle ) and islets treated with IL-1beta (bullet ) were then perifused with 16.7 mM glucose starting at 0 min, after a 30-min preincubation at 3.3 mM glucose (n = 4). Results are shown as means ± SE. P value shown refers to statistical analysis of area under curves (AUCs) for FEI (above prestimulus basal rate) induced by 16.7 mM glucose in control group vs. IL-1beta -pretreated group.

During these studies, prelabeling of phosphoinositides was carried out for 20 h. We have previously shown that the labeling of PtdIns(4)P and PtdIns(4,5)P2 does reach isotopic steady state by 20 h; however, it requires 50 h to label PtdIns to steady state (32). Hence, to confirm these findings in islets labeled to full isotopic equilibrium, additional experiments were carried out using islets labeled for 50 h. IL-1beta exposure was only for the last 20 h (18 h of culture and 2 h of final labeling). Unlabeled inositol was excluded from the perifusion media to avoid disturbing isotopic equilibrium. Under these conditions, IL-1beta again completely inhibited the glucose-induced FEI (Fig. 2).


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Fig. 2.   Effect of IL-1beta on glucose-induced FEI in islets labeled to complete isotopic equilibrium. Islets were labeled with [3H]inositol (50 h) in batches of 100. Control group (open circle ) and islets treated with IL-1beta (IL-1beta present only for last 20 h; bullet ) were perifused with 16.7 mM glucose starting at 0 min, after a 30-min preincubation at 3.3 mM glucose (n = 3). Results are shown as means ± SE. P value shown refers to statistical analysis of AUCs for FEI (above prestimulus basal rate) induced by 16.7 mM glucose in control group vs. IL-1beta -pretreated group

Effect of IL-1beta on inositol efflux induced by glyburide or high K+. Inhibition of glucose-induced PLC activity by IL-1beta 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-1beta pretreatment. In static incubations, IL-1beta also inhibited the insulin secretion induced by 10 µM glyburide (Fig. 4). Thus, in IL-1beta -pretreated islets, glyburide-induced (i.e., Ca2+-stimulated) PLC activation and insulin secretion were absent.


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Fig. 3.   Effect of IL-1beta on glyburide-induced FEI. Islets were labeled with [3H]inositol (20 h) in batches of 100. Control group (open circle ) and islets treated with IL-1beta (bullet ) were perifused with 10 µM glyburide starting at 0 min, after a 30-min preincubation at 3.3 mM glucose (n = 5). Results are shown as means ± SE. P value shown refers to statistical analysis of AUCs for FEI (above prestimulus basal rate) induced by 10 µM glyburide in control group vs. IL-1beta -pretreated group.


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Fig. 4.   Effect of guanosine to prevent inhibition by IL-1beta of glyburide (10 µM)-induced insulin secretion. Ten islets/tube were treated with glyburide, in static incubations of 45-min duration, after overnight culture in presence or absence of 100 pM IL-1beta  ± 500 µM guanosine (Guo; C = control). Incubations were then carried out at 3.3 mM glucose. Results are expressed as means ± SE for no. of replicates in parentheses, derived by pooling data from 3 independent experiments. * P < 0.05 (or greater) comparing bars 5 and 6, bars 6 and 8, or bars 7 and 8.

To totally bypass the involvement of the KATP channel by another means, high K+ was next studied. K+ (40 mM) directly induces cell depolarization and subsequent Ca2+ influx via voltage-dependent Ca2+ channels. K+ (40 mM) induced an increase in FEI from a basal rate of 0.28 ± 0.02%/min to a peak rate of 0.97 ± 0.21%/min in control islets; the AUC above the prestimulus basal rate was 11.73 ± 2.07/45 min. Once again, IL-1beta pretreatment induced a nearly complete inhibition of the FEI (Fig. 5), reducing the AUC above the prestimulus basal rate to 1.92 ± 0.85/45 min. Insulin secretion induced by 40 mM K+ was inhibited by IL-1beta pretreatment (see below), confirming our previous observation (16).


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Fig. 5.   Effect of IL-1beta on 40 mM K+-induced FEI in presence or absence of adenosine or guanosine. Islets were labeled with [3H]inositol (20 h) in batches of 100. Control group (open circle ), islets treated with IL-1beta (bullet ), islets treated with IL-1beta  + adenosine (square ), and islets treated with IL-1beta  + guanosine (down-triangle) were perifused with 40 mM K+ starting at 0 min, after a 30-min preincubation at 3.3 mM glucose (n = 4). Results are shown as means ± SE. P value shown refers to statistical analysis of AUCs for FEI (above prestimulus basal rate) induced by 40 mM K+ in control group vs. IL-1beta -treated group.

Effect of IL-1beta on content of phospholipid substrates. One of the possible explanations for absence of PLC activation in IL-1beta -pretreated islets might be that IL-1beta 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-1beta effects on labeling, some islets were extracted immediately after the 20-h labeling period (in presence or absence of IL-1beta ) without additional experimental incubations. IL-1beta 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-1beta '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-1beta now failed to inhibit the labeling of the phosphoinositide, indicating that the inhibition of phosphoinositide labeling by IL-1beta is mediated, at least in part, via NO.

                              
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Table 1.   Effect of IL-1beta on labeling of phosphoinositides

                              
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Table 2.   Prevention of IL-1beta effect on phosphoinositide labeling by L-NAME

Effects of guanosine, adenosine, or ribose. We have previously shown that IL-1beta 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-1beta . Either adenosine or guanosine reversed the effect of IL-1beta 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-1beta 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-1beta : 71 ± 5, n = 6, P < 0.001; 40 mM K+ + IL-1beta  + 500 µM guanosine = 405 ± 43; n = 6). In fact, guanosine actually induced an overshoot in insulin secretion in IL-1beta -pretreated islets (only). In additional experiments (not shown), 500 µM adenosine was as effective as guanosine in preventing the inhibition by IL-1beta 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-1beta 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).

This restorative effect of guanosine or adenosine on IL-1beta -inhibited phosphoinositide synthesis might be related to the effects on ATP and GTP content. Alternatively, however, adenosine or guanosine can be metabolized to yield ribose 1-phosphate (along with adenine and guanine) by purine nucleoside phosphorylase. Ribose 1-phosphate, in turn, can be converted to phosphoribosylpyrophosphate (PRPP) for nucleotide synthesis or to glycerol phosphate for de novo synthesis of phospholipids. To separate these alternate possibilities, ribose alone was provided with IL-1beta . When ribose (500 µM) was coprovided with IL-1beta , there was a significant (35%) restoration of PtdIns, but no significant restorative effect on IL-1beta inhibition of the labeling of PtdIns(4)P or PtdIns(4,5)P2 (Table 1). Additionally, provision of either adenosine or guanosine or ribose significantly prevented the inhibition of phospholipid synthesis induced by IL-1beta (as assessed by total dpm in organic phase of islet extracts at end of the incubation period; additional data, not shown). Concomitantly, the inhibition by IL-1beta of glyburide-induced insulin release was prevented by ribose, which by itself did not alter insulin release at 3.3 mM glucose (additional data not shown).

Effect of carbachol on PLC activation in IL-1beta -treated islets. The findings of a blockade by IL-1beta 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-1beta affected PLC activation by affecting Ca2+ influx, then the direct receptor-mediated effects of carbachol might be resistant to inhibition by IL-1beta . 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-1beta pretreatment completely abolished this response as well (Fig. 6).


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Fig. 6.   Effect of IL-1beta on carbachol-induced FEI. Control (open circle ) and IL-1beta treated (bullet ) islets were perifused with 100 µM carbachol, starting at 0 min after a 30-min preincubation at 3.3 mM glucose. Results are shown as means ± SE; n = 4. P value shown refers to statistical analysis of AUCs for FEI (above prestimulus basal rate) induced by 2 groups.

Possible cellular mediators of inhibition of PLC by IL-1beta . IL-1beta is known to generate large increases in prostaglandins (10) or guanosine 3',5'-cyclic monophosphate (cGMP) (5) in islets or beta -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-1beta -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].

    DISCUSSION
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Abstract
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Methods
Results
Discussion
References

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-1beta 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-1beta 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-1beta , 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-1beta 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-1beta 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-1beta be attributed to decrements in GTP (32) or ATP, such as those known to be induced by IL-1beta (16)? In previous studies, when the purine precursors adenine or guanine were provided to pancreatic islets pretreated with IL-1beta (16), they were unable to restore ATP or GTP levels, either because IL-1beta blocked the "salvage" of these purine bases to their phosphorylated derivatives or, more likely, because IL-1beta (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-1beta 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-1beta . 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-1beta (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-1beta (27), no inhibition of phosphoinositide synthesis by IL-1beta 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-1alpha for several hours only; it was observed that IL-1alpha 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-1alpha 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-1beta . IL-1beta 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 beta -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 beta -cell to activation by Ca2+ (25).

We felt, however, that it was important to investigate other potential mechanisms of action of IL-1beta more formally. We observed that IL-1beta (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-1beta 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 beta -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-1beta must directly impede depolarization of the beta -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-1beta 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-1beta . Indeed, IL-1beta (or other agents generating NO) may retard Ca2+ entry via voltage-dependent Ca2+ channels in beta -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-1beta effects on PLC or secretion. Furthermore, even if inhibition of Ca2+ entry did contribute to IL-1beta '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-1beta 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-1beta . A unitary hypothesis might be that IL-1beta 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-1beta 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-1beta of adenosine or guanosine, failed to reverse IL-1beta 's effect on glucose-induced insulin secretion (16). In contrast, in the current studies, guanosine totally reversed the effect of IL-1beta on 40 mM K+- or glyburide (i.e., Ca2+)-induced secretion. Thus IL-1beta 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-1beta 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-1beta cannot provide the full explanation for its effect on agonist-induced PLC. It is possible that IL-1beta 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-1beta . However, as indicated above, the studies with carbachol would seem to exclude a blockade of Ca2+ influx as the (sole) mechanism of IL-1beta 's inhibition of PLC. On the basis of our previous data, the inhibition of the GTP-to-GDP ratio induced by IL-1beta also cannot explain the total inhibition of carbachol effect on PLC (25). The generation of NO by exposure of cells to IL-1beta 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-1beta treatment (11); PGE2 has been reported to inhibit glucose- or carbachol-induced PLC activation (14). However, the effects of IL-1beta 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-1beta (A. Kowluru and S. Metz; unpublished data). An attractive explanation may lie in the fact that a prior exposure to IL-1beta (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-1beta in mitochondrial energetics do not appear to adequately explain its effects on PLC activation.

    ACKNOWLEDGEMENTS

The technical assistance of J. Stephens is gratefully acknowledged.

    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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ashcroft, S. J., L. C. Weerasinghe, and P. J. Randle. Interrelationship of islet metabolism, adenosine triphosphate content and insulin release. Biochem. J. 132: 223-231, 1973[Medline].

2.   Benistant, C., A. P. Thomas, and R. Rubin. Effect of guanine nucleotides on polyphosphoinositide synthesis in rat liver plasma membranes. Biochem. J. 271: 591-597, 1990[Medline].

3.   Clementi, E., C. Sciorati, M. Riccio, M. Miloso, J. Meldolesi, and G. Nistico. Nitric oxide action on growth factor-elicited signals. Phosphoinositide hydrolysis and [Ca2+]i responses are negatively modulated via a cGMP-dependent protein kinase pathway. J. Biol. Chem. 270: 22277-22282, 1995[Abstract/Free Full Text].

4.   Corbett, J. A., M. A. Sweetland, J. R. Lancaster, Jr., and M. L. McDaniel. A 1-hour pulse with IL-1beta induces formation of nitric oxide and inhibits insulin secretion by rat islets of Langerhans: evidence for a tyrosine kinase signaling mechanism. FASEB J. 7: 369-374, 1993[Abstract/Free Full Text].

5.   Corbett, J. A., J. L. Wang, J. H. Hughes, B. A. Wolf, M. A. Sweetland, J. R. Lancaster, Jr., and M. L. McDaniel. Nitric oxide and cyclic GMP formation induced by interleukin 1beta in islets of Langerhans: evidence for an effector role of nitric oxide in islet dysfunction. Biochem. J. 287: 229-235, 1992[Medline].

6.   Detimary, P., P. Gilon, M. Nenquin, and J. C. Henquin. Two sites of glucose control of insulin release with distinct dependence on the energy state in pancreatic beta -cells. Biochem. J. 297: 455-61, 1994[Medline].

7.   Eizirik, D. L., S. Sandler, N. Welsh, L. Juntti-Berggren, and P. O. Berggren. Interleukin-1beta -induced stimulation of insulin release in mouse pancreatic islets is related to diacylglycerol production and protein kinase C activation. Mol. Cell. Endocrinol. 111: 159-165, 1995[Medline].

8.   Gao, Z. Y., P. Gilon, and J. C. Henquin. The role of protein kinase-C in signal transduction through vasopressin and acetylcholine receptors in pancreatic beta -cells from normal mouse. Endocrinology 135: 191-199, 1994[Abstract].

9.   Gillis, K. D., W. M. Gee, A. Hammoud, M. L. McDaniel, L. C. Falke, and S. Misler. Effects of sulfonamides on a metabolite-regulated ATP-sensitive K+ channel in rat pancreatic beta -cells. Am. J. Physiol. 257 (Cell Physiol. 26): C1119-C1127, 1989[Abstract/Free Full Text].

10.   Hirata, M., K. P. Kohse, C.-H. Chang, T. Ikebe, and F. Murad. Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells. J. Biol. Chem. 265: 1268-1273, 1990[Abstract/Free Full Text].

11.   Hughes, J. H., R. A. Easom, B. A. Wolf, J. Turk, and M. L. McDaniel. Interleukin 1-induced prostaglandin E2 accumulation by isolated pancreatic islets. Diabetes 38: 1251-1257, 1989[Abstract].

12.   Kaneto, H., J. Fujii, H. G. Seo, K. Suzuki, T. Matsuoka, M. Nakamura, H. Tatsumi, Y. Yamasaki, T. Kamada, and N. Taniguchi. Apoptotic cell death triggered by nitric oxide in pancreatic beta -cells. Diabetes 44: 733-738, 1995[Abstract].

13.   Krippeit-Drews, P., K.-D. Kröncke, S. Welker, G. Zempel, M. Roenfeldt, H. P. T. Ammon, F. Lang, and G. Drews. The effects of nitric oxide on the membrane potential and ionic currents of mouse pancreatic beta  cells. Endocrinology 136: 5363-5369, 1995[Abstract].

14.   Laychock, S. G. Prostaglandin E2 inhibits phosphoinositide metabolism in isolated pancreatic islets. Biochem. J. 260: 291-294, 1989[Medline].

15.   Martin, T. F. J., J. C. Hay, A. Banerjee, V. A. Barry, K. Ann, H.-C. Yom, B. W. Porter, and J. A. Kowalchyk. Late ATP-dependent and Ca++-activated steps of dense core granule exocytosis. In: Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor, NY: Cold Spring Harbor Lab. Press, 1995, vol. LX, p. 197-204.

16.   Meredith, M., M. E. Rabaglia, J. A. Corbett, and S. Metz. Dual functional effects of interleukin-1beta on purine nucleotides and insulin secretion in rat islets and INS-1 cells. Diabetes 45: 1783-91, 1996[Abstract].

17.   Méry, P.-F., C. Pavoine, L. Belhassen, F. Pecker, and R. Fischmeister. Nitric oxide regulates cardiac Ca2+ current involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation. J. Biol. Chem. 268: 26286-26295, 1993[Abstract/Free Full Text].

18.   Metz, S. A., M. E. Rabaglia, and T. J. Pintar. Selective inhibitors of GTP synthesis impede exocytotic insulin release from intact rat islets. J. Biol. Chem. 267: 12517-12527, 1992[Abstract/Free Full Text].

19.   Parker, R. C., L. N. Castor, and E. A. McCuloch. Altered cell strains in continuous culture: a general survey. Ann. NY Acad. Sci. 5: 303-313, 1957.

20.   Sandler, S., K. Bendtzen, L. A. Borg, D. L. Eizirik, E. Strandell, and N. Welsh. Studies on the mechanisms causing inhibition of insulin secretion in rat pancreatic islets exposed to human interleukin-1beta indicate a perturbation in the mitochondrial function. Endocrinology 124: 1492-1501, 1989[Abstract].

21.   Schrammel, A., S. Behrends, K. Schmidt, D. Koesling, and B. Mayer. Characterization of 1H-[1,2,4]oxadiazolo[4,3-alpha ]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol. Pharmacol. 50: 1-5, 1996[Abstract].

22.   Smith, C. D., and K. J. Chang. Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins. J. Biol. Chem. 264: 3206-3210, 1989[Abstract/Free Full Text].

23.   Southern, C., D. Schulster, and I. C. Green. Inhibition of insulin secretion by interleukin-1 beta and tumour necrosis factor-alpha via an L-arginine-dependent nitric oxide generating mechanism. FEBS Lett. 276: 42-44, 1990[Medline].

24.   Stamler, J. S., D. I. Simon, J. A. Osborne, M. E. Mullins, O. Jaraki, T. Michel, D. J. Singel, and J. Loscalzo. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc. Natl. Acad. Sci. USA 89: 444-448, 1992[Abstract].

25.   Vadakekalam, J., M. E. Rabaglia, Q.-H. Chen, and S. A. Metz. Glucose-induced phosphoinositide hydrolysis: a requirement for GTP in calcium-induced phospholipase C activation in pancreatic islets. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E85-E95, 1996[Abstract/Free Full Text].

26.   Vara, E., J. Arias-Diaz, C. Garcia, and J. L. Balibrea. Cytokine-induced inhibition of lipid synthesis and hormone secretion by isolated human islets. Pancreas 9: 316-323, 1994[Medline].

27.   Wolf, B. A., J. H. Hughes, J. Florholmen, J. Turk, and M. L. McDaniel. Interleukin-1 inhibits glucose-induced Ca2+ uptake by islets of Langerhans. FEBS Lett. 248: 35-38, 1989[Medline].

28.   Zawalich, W. Multiple effects of increases in phosphoinositide hydrolysis on islets and their relationship to changing patterns of insulin secretion. Diabetes Res. 13: 101-111, 1990[Medline].

29.   Zawalich, W. S., K. C. Zawalich, and G. G. Kelley. Time-dependent effects of cholinergic stimulation on beta cell responsiveness. Pflügers Arch. 432: 589-596, 1996[Medline].

30.   Zawalich, W. S., K. C. Zawalich, and H. Rasmussen. Interleukin-1 alpha exerts glucose-dependent stimulatory and inhibitory effects on islet cell phosphoinositide hydrolysis and insulin secretion. Endocrinology 124: 2350-2357, 1989[Abstract].


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