Chronic exposure to TPA depletes PKCalpha and augments Ca-dependent insulin secretion from cultured rat islets

Walter S. Zawalich1, Marc Bonnet-Eymard1, Kathleen C. Zawalich1, and Gordon C. Yaney2

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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha  (PKCalpha ) 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 PKCalpha . In agreement with previous studies, culturing in TPA reduced the islet content of immunoreactive PKCalpha 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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CHRONIC EXPOSURE TO THE phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) reduces the cellular levels of the enzyme protein kinase C alpha  (PKCalpha ) (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 PKCalpha component in beta -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 PKCalpha 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 PKCalpha -target proteins contributes to release from PKCalpha -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 PKCalpha 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 PKCalpha 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 PKCalpha (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 PKCalpha (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 beta -cell (5, 23). In addition, high glucose shares many of the characteristics of several agonists whose effects on the beta -cell are generally attributed to phospholipase C (PLC)/PKCalpha 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 beta -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 PKCalpha , PLC-delta 1, and PLC-beta 1 levels were also determined. Islet phosphoprotein patterns after culture in the presence or absence of TPA were examined as well.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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-delta 1 (20 µg), PLC-beta 1 (20 µg), PKCalpha (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-delta 1 determinations, the membranes were incubated for 150 min with the primary anti-PLC-delta 1 antibody (1.0 mg/ml dilution), washed, incubated for 60 min with horseradish peroxidase (HRP)-conjugated IgG, and washed again. For PLC-beta 1 determinations, the membranes were incubated for 60 min with the primary anti-PLC-beta 1 antibody (0.5 mg/ml dilution), washed, incubated for 45 min with HRP-conjugated IgG, and washed again. For PKCalpha determinations, the membranes were incubated for 45 min with the primary anti-PKCalpha 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-delta 1 were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies to PLC-beta 1, PKCalpha , 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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Protein, insulin, and PKCalpha 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.

Western blot data and their analyses are given in Figs. 1 and 2. As a necessary control, we first established the linearity between the amount of islet protein analyzed and the optical density of the PKCalpha Western blot. A strong positive correlation was evident (Fig. 1). As shown in Fig. 2, A and B, culturing for 22-24 h in CMRL-1066 had no obvious deleterious effect, compared with fresh islets, on the optical density of the PKCalpha Western blot. However, and in agreement with studies in other tissues (22), the content of immunoreactive PKCalpha was dramatically reduced in 1-day cultured islets when TPA (500 nM) was included in the culture medium (Fig. 2, C and D). The optical density of the PKCalpha Western blot band was <5% of the value obtained from control-cultured islets.


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Fig. 1.   Quantitation of immunoreactive protein kinase C alpha  (PKCalpha ) in islets. A: different amounts of rat islet protein (6-18 µg) were used for Western blot analyses. Sample of brain (Br) homogenate is also shown. B: optical density (OD) measured using a Visage 2000 is plotted against amount of islet protein analyzed.


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Fig. 2.   Effects of culturing alone or with 12-O-tetradecanoylphorbol 13-acetate (TPA) on PKCalpha content of isolated islets. A and B: groups of islets were analyzed for PKCalpha content immediately after isolation or after 22-24 h of culture in CMRL-1066 medium. Fifteen micrograms of protein from fresh or cultured (Cult) islet sonicates were analyzed as described in METHODS. A: 2 separate blots from different islet preparations and sample of brain run in parallel. FIa and FIb, separate groups of fresh islets; CIa and CIb, separate groups of cultured islets. B: analysis of 10 paired experiments. C and D: groups of islets were cultured for 22-24 h in CMRL-1066 with (+) or without (-) further addition of 500 nM TPA. Islets were then prepared for PKCalpha determinations as indicated in METHODS. C: Western blots from 2 separate islet preparations. D: quantitative densitometry of bands in C. An analysis of 3 paired experiments revealed a 97.5% reduction in PKCalpha content in islets cultured in presence of 500 nM TPA compared with islets cultured in its absence.

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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Fig. 3.   TPA-induced insulin secretion from freshly isolated, control-cultured, and TPA-cultured islets. Three groups of islets were studied: 1st group was studied immediately after collagenase isolation (black-triangle), 2nd group was studied after 22-24 h of culture in CMRL-1066 (open circle ), and 3rd group was studied after 22-24 h of culture in CMRL-1066 + 500 nM TPA (bullet ). All islets were perifused for 30 min in a Krebs-Ringer bicarbonate (KRB) buffer supplemented with 3 mM glucose (G3). After this, they were stimulated for 40 min with 500 nM TPA in continued presence of 3 mM glucose. Data are means ± SE for at least 3 experiments. These data and those in Figs. 4-6 and 9 have not been corrected for dead space in perifusion apparatus (2.5 ml or 2.5 min with a flow rate of 1 ml/min).


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Fig. 4.   Glucose-induced insulin secretion from freshly isolated, control-cultured, and TPA-cultured islets. Three groups of islets were studied: 1st group was studied immediately after collagenase isolation (black-triangle), 2nd group was studied after 22-24 h of culture in CMRL-1066 (open circle ), and 3rd group was studied after 22-24 h of culture in CMRL-1066 + 500 nM TPA (bullet ). All islets were perifused for 30 min in a KRB buffer supplemented with 3 mM glucose. After this, they were stimulated for 40 min with 15 mM glucose (G15).

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 · islet-1 · 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.

Compared with the sustained insulin secretory responses from freshly isolated islets, however, cultured islets exhibited impaired insulin secretory responses to 15 mM glucose stimulation irrespective of whether or not TPA was included in the culture medium (Fig. 4). Sustained rates of secretion from control-cultured islets measured 35-40 min after the onset of stimulation (235 ± 49 pg · islet-1 · min-1) were reduced by ~80% compared with freshly isolated islets (1,050 ± 104 pg · islet-1 · min-1). The sustained secretory responses to 15 mM glucose from islets cultured for 1 day in 500 nM TPA, although greater than those seen from control-cultured islets, were still reduced by ~70% compared with the responses from freshly isolated islets. In both qualitative and quantitative terms, the 30-fold increase above prestimulatory secretion rates evoked from freshly isolated islets in response to 15 mM glucose is comparable to the responses noted in the perfused rat pancreas preparation (10, 15, 16, 31).

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 beta -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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Fig. 5.   Potassium (KCl)-induced insulin secretion from freshly isolated, control-cultured, and TPA-cultured islets. Three groups of islets were studied: 1st group was studied immediately after collagenase isolation (black-triangle), 2nd group was studied after 22-24 h of culture in CMRL-1066 (open circle ), and 3rd group was studied after 22-24 h of culture in CMRL-1066 + 500 nM TPA (bullet ). All islets were perifused for 30 min in a KRB buffer supplemented with 3 mM glucose. After this, they were stimulated for 40 min with 30 mM potassium in continued presence of 3 mM glucose. Data are means ± SE for at least 3 experiments.

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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Fig. 6.   Effects of nitrendipine on potassium (KCl)-induced insulin secretion from TPA-cultured islets. Two groups of islets were studied after 22-24 h of culture in CMRL-1066 + 500 nM TPA. Islets were perifused for 30 min with 3 mM glucose with (dashed line) or without (solid line) 5 µM nitrendipine and for an additional 40 min with 30 mM potassium in continued presence of 3 mM glucose with or without nitrendipine. Secretory data for islets stimulated with potassium alone are same as in Fig. 5. Data are means ± SE for at least 3 experiments.

A similar result was obtained with 15 mM glucose-stimulated islets. Nitrendipine (5 µM) abolished glucose-induced secretion from freshly isolated and control 1-day cultured islets (results not shown). This concentration of the calcium channel antagonist also abolished the response to glucose from TPA-cultured islets (results not shown).

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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Table 1.   Glucose utilization rates in freshly isolated or cultured islets

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 PKCalpha 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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Fig. 7.   Phosphoserine and phosphothreonine patterns of proteins from groups of islets cultured for 22-24 h in presence or absence of 500 nM TPA. After culture period, they were retrieved and sonicated, and 20-23 micrograms of islet protein were prepared for Western blots as described in METHODS and probed with anti-phosphoserine (left, lanes 1 and 2) and anti-phosphothreonine (right, lanes 4 and 5) antibodies. Note enhanced phosphorylation state of bands (indicated by arrows) from islets cultured in presence of TPA. Bands selected show at least 100% increase in density compared with islets cultured in absence of phorbol ester. Densitometric measurements were made using a Visage 2000. A control sample of brain is shown in phosphothreonine blot (lane 3). Blots are representative of 3 experiments.

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-delta 1 and PLC-beta 1. Compared with freshly studied islets, both isozymes were reduced ~50%.


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Fig. 8.   Phospholipase C (PLC)-beta 1 and PLC-delta 1 content in freshly isolated and cultured islets. A and B: groups of islets were analyzed for PLC-beta 1 content immediately after isolation or after 22-24 h of culture in CMRL-1066 medium. Twenty micrograms of protein from fresh or cultured islet sonicates were analyzed as described in METHODS. A: blot with sample of brain run in parallel. B: analysis of 5 paired experiments. C and D: groups of islets were analyzed for PLC-delta 1 content immediately after isolation or after 22-24 h of culture in CMRL-1066 medium. Twenty micrograms of protein from fresh or cultured islet sonicates were analyzed as described in METHODS. C: blot with sample of brain homogenate run in parallel. D: analysis of 5 paired experiments.

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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Fig. 9.   Restorative effect of the PKC activator TPA on glucose-induced release from 1-day control-cultured islets. Two groups of islets (open circle ) were studied after 22-24 h of culture in CMRL-1066 alone. Both groups of islets were perifused for 30 min in a KRB buffer supplemented with 3 mM glucose. After this, they were stimulated for 40 min with 15 mM glucose alone (open circle , solid line) or 15 mM glucose + 500 nM TPA (open circle , dashed line). Data for 15 mM glucose alone are same data presented in Fig. 4. For comparative purposes, secretory responses of freshly isolated islets to combination of 15 mM glucose and 500 nM TPA are also shown (black-triangle). Data are means ± SE for at least 3 experiments.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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 PKCalpha 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 PKCalpha 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 PKCalpha 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 PKCalpha . Our conclusion as to the involvement of PKCalpha 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 beta -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 · h-1 · 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 beta -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, PKCalpha 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 PKCalpha -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 beta -cell (20), this suggests that beta -cell calcium sensitivity has been enhanced by culturing in TPA and that this alteration is sustained despite dramatic reductions in immunoreactive PKCalpha content. This finding is also in accord with the established acute effect of TPA to increase the sensitivity of the beta -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 PKCalpha -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 PKCalpha -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 beta -cell to calcium.

    ACKNOWLEDGEMENTS

These studies were supported by National Institutes of Health Grants DK-41230 and DK-50662.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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