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Endocrinology Vol. 141, No. 4 1394-1402
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of Inositol Trisphosphate Receptor Isoform Expression in Glucose-Desensitized Rat Pancreatic Islets: Role of Cyclic Adenosine 3',5'-Monophosphate and Calcium1

Bumsup Lee2 and Suzanne G. Laychock

Department of Pharmacology and Toxicology, State University of New York, School of Medicine and Biomedical Sciences, Buffalo, New York 14214

Address all correspondence and requests for reprints to: Dr. Suzanne Laychock, 102 Farber Hall, Department of Pharmacology and Toxicology, State University of New York School of Medicine, Buffalo, New York 14214. E-mail: laychock{at}acsu.buffalo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The regulation of inositol 1,4,5-trisphosphate receptor (IP3R) messenger RNA (mRNA) and protein expression was investigated in glucose-desensitized rat isolated pancreatic islets. Islets were cultured for 4 days with glucose (11 mM; G-treated) to induce desensitization; IP3R-I mRNA levels were similar to basal (5.5 mM glucose) values, whereas IP3R-II mRNA levels were increased and IP3R-III levels were reduced compared with basal levels. Somatostatin increased the expression of IP3R-II mRNA and reduced the expression of IP3R-III mRNA compared with basal values, but did not significantly affect G-treated islet IP3R expression. When forskolin (FSK), 8-bromo-cAMP, and glucagon-like peptide 1-(7–36) amide were added to G-treated islets after 4 days of culture, IP3R-II mRNA levels were reduced, whereas IP3R-III mRNA levels increased, to levels observed in control islets, within 3 h. The levels of IP3R-I mRNA were unaffected by either somatostatin or FSK. The protein kinase A inhibitor, H-89, and actinomycin D prevented the effects of FSK. A Ca2+ ionophore mimicked the effects of FSK on IP3R mRNA expression, whereas blockade of voltage-dependent Ca2+ channels or chelation of intracellular Ca2+ inhibited the actions of FSK. cAMP also increased IP3R-III mRNA in insulinoma cells. In G-treated islets, FSK slowed IP3R-III mRNA degradation. FSK, but not glucose, stimulated protein kinase A activation in G-treated islets. Thus, cAMP mediates changes in IP3R-II and -III mRNA transcription and stability in glucose-desensitized islets. The regulated expression of IP3R-II and -III mRNA is mediated in part by intracellular Ca2+ availability.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INOSITOL 1,4,5-trisphosphate (IP3) receptor (IP3R) is a primary intracellular calcium release channel in many tissues (1, 2). Within the IP3R family five IP3R isoforms (types I–V) have been characterized at the molecular level, and each type has a unique primary structure and tissue distribution (3). Full-length complementary DNA (cDNA) sequences have been reported for rat IP3R isoforms I (4), II (5), and III (6), and partial sequences for isoforms IV (7) and -V (8). Messenger RNA (mRNA) for IP3R types I, II, and III have been identified in rat pancreatic islets and ß-cell lines (6, 9, 10). IP3R-III mRNA is the most abundant isoform, followed by almost equal levels of types I and II in rat islets and rat ß-cell lines (9, 10). Relative IP3R protein expression for the isoforms parallel mRNA levels in rat islets and ß-cell lines (9, 11).

Expression levels of the IP3R isoforms are unique for different tissues (12). Whereas type III is most abundant in rat islets (9, 11), type I is predominant in mouse islets (8). In addition to the IP3-binding domain on the IP3R, phosphorylation, calmodulin, ATP, and calcium are postulated to modulate activation of the receptors (4, 5). The significance of differences in the expression levels of the isoforms with regard to cellular function has been speculated to pertain to the unique IP3 affinities (5, 13) and calcium sensitivities (14, 15) of the isoforms, and the calcium oscillatory capacity of cells (11, 16). Recently, it was demonstrated for a rat ß-cell line that IP3R-III was activated by increasing calcium levels, and that activation of this isoform by IP3 resulted in the global release of intracellular calcium with consequential depletion of internal calcium stores (11). In contrast, the type I receptor showed a biphasic activation response to increasing calcium concentrations, and calcium release was localized and nonpropagating in non-ß-cells expressing predominantly this isoform (11).

This laboratory reported recently that glucose stimulation in rat islets and insulinoma cells differentially affected IP3R isoform expression levels (9, 10). After a short duration glucose stimulus, the expression of islet IP3R-III mRNA and protein increased, but after chronic glucose stimulation in an in vitro model of hyperglycemia and glucose-induced desensitization (17), islet mRNA and protein levels for IP3R-III were down-regulated, and those of IP3R-II were up-regulated (10). Moreover, the chronic glucose-induced changes observed in vitro for IP3R-II were mimicked in an in vivo 90% partial pancreatectomy model of hyperglycemia (10). Thus, in islets, as in other tissues (12, 18, 19), IP3R isoform expression levels are independently regulated and responsive to physiological stimuli that modulate changes in cellular Ca2+ levels. The signal transduction mechanisms responsible for the regulation of IP3R expression are not known. The present study was undertaken to test the hypothesis that cAMP and/or Ca2+ mediate chronic changes in IP3R mRNA and protein expression in rat pancreatic islets in an in vitro model of hyperglycemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Collagenase (type P) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). 8-Bromo-cAMP, 8-bromo-cGMP, forskolin, 3-isobutyl-1-methylxanthine (IBMX), BSA, glucagon-like peptide-(7–36) amide (GLP7–36), cycloheximide, and actinomycin D were obtained from Sigma (St. Louis, MO). Nimodipine, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), and ionomycin were obtained from Calbiochem (San Diego, CA). CMRL-1066 and DMEM (pyruvate-free) culture medium, random hexamer, protein kinase A (PKA) assay kit, agarose, and Trizol were purchased from Life Technologies, Inc. (Grand Island, NY). FBS was obtained from Atlanta Biologicals (Norcross, GA). Peroxidase-conjugated antirabbit IgG secondary antibody was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Immobilon-P membrane was purchased from Millipore Corp. (Bedford, MA). Rat insulin for RIA standard was a gift from Eli Lilly & Co. (Indianapolis, IN). [125I]Insulin (porcine) was obtained from NEN Life Science Products (Boston, MA).

Isolation and culture of rat islets
Pancreatic islets from adult male Sprague Dawley rats were isolated using collagenase, as described previously (17). All animal procedures were approved by the institutional animal care and use committee. Isolated islets, devoid of any visible adherent acinar tissue, were either used immediately as freshly isolated (fresh) islets or were cultured for various periods of time, as described previously (10), with the concentrations of glucose indicated in the text. Mannitol was included in 30-min and 2-h control cultures to maintain osmotic equivalence to glucose-stimulated conditions, and cytosine arabinoside (10 µM) was included in all islet incubations 18 h or longer to prevent cell proliferation, as described previously (10). In certain experiments, after the prescribed period of time in culture, islets were treated with various agents as indicated in the text.

Murine ßHC9 cells were cultured at 35 C in DMEM for 4 days at 2.8 mM glucose, as described previously (9). Other additions to the cultures are indicated in the text.

Insulin release
Insulin release was determined for isolated islets after 4 days of culture in the presence or absence of somatostatin. After the change of culture medium on day 2, an aliquot of culture medium was removed from the islets, and another aliquot was removed on day 4. Insulin levels were quantitated by RIA using rat insulin as standard. Insulin levels on day 2 were subtracted from insulin levels on day 4 to determine hormone release over the 2-day interval.

RNA isolations and cDNA synthesis
Total RNA was extracted from rat pancreatic islets and insulinoma cells using Trizol. cDNA was reverse transcribed, as described previously (10), from 1 µg total RNA by random hexamer.

PCR amplification and quantitation of IP3R transcript levels
Polymerization reactions were carried out as described previously (10) using a Hybaid Sprint thermocycler, such that amplification was in the exponential phase. The amplimers were separated by electrophoresis in a 1.5% agarose gel in Tris borate buffer. The gel was stained by ethidium bromide and viewed by Gel Doc 1000 (Bio-Rad Laboratories, Inc.) with density analysis of each PCR fragment by Molecular Analyst software (Bio-Rad Laboratories, Inc.). The image densities of PCR products for IP3R isoforms were compared with the density of coamplified ß-actin to determine the ratio of expression. Values are expressed as relative levels of IP3R mRNA/ß-actin mRNA.

Immunoblotting
Rat pancreatic islets were sonicated, and the protein concentration was determined by Bio-Rad Laboratories, Inc. protein assay using BSA as standard. The sonicates were precipitated with acetone and pelleted by centrifugation at 14,000 x g for 5 min at 4 C. The pellet was resuspended in sample buffer, and the protein (20–30 µg) was separated by 5% SDS-PAGE and transferred to an Immobilon-P membrane. The membrane was blocked with 10% nonfat dried milk, and then incubated with antisera against the IP3R isoforms (antisera were gifts from Dr. R. Wojcikiewicz), as described previously (10, 18, 20). Antisera were generated against synthetic peptides corresponding to the C-termini of rat IP3R-I, -II, or -III and affinity purified to yield antisera specific to their cognate proteins. After washing, the membrane was incubated with peroxidase-conjugated antirabbit IgG secondary antibody. The bound antibody was localized by chemiluminescence, and the density of each band was determined by densitometric scanning using Molecular Analyst software.

PKA activation
Freshly isolated islets were preincubated in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 5.5 mM glucose for 1 h at 37 C under an atmosphere of 95% O2-5% CO2 in a shaking water bath. The islets were then resuspended in Krebs-Ringer bicarbonate buffer with various stimuli and incubated for 10 min. Islets cultured for 4 days in CMRL-1066 containing 5.5 or 11 mM glucose were either untreated or treated with forskolin for 10 min. Fresh or cultured islets were then washed twice in cold PBS and resuspended in extraction buffer before sonication. PKA activity was determined using a commercial kit and is expressed as the percentage of total PKA activity in the presence of cAMP.

cAMP determination
ßHC9 cells were cultured in DMEM as described in the text. cAMP was quantitated in culture medium by RIA, as described previously (21).

Statistical analysis
Values are the mean ± SE. Significant differences between treatment groups were determined by one-way ANOVA, with post-hoc analysis using Student-Newman-Keuls multiple comparison test. P <= 0.05 was accepted as significant. The statistical significance of values expressed as a percentage of the control value was determined using mRNA expression ratio values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of IP3R mRNA and protein expression
It was reported previously that in vivo hyperglycemia or culture of isolated pancreatic islets at an elevated glucose level (11 mM) for 7 days caused no change in IP3R-I mRNA levels, but resulted in the up-regulation of islet IP3R-II mRNA and the down-regulation of IP3R-III mRNA levels (9, 10). Similarly, islets cultured with a continuous glucose stimulus (11 mM) for only 4 days (glucose-treated islets) also showed an increase in IP3R-II mRNA but showed a decrease in the relative levels of IP3R-III mRNA compared with control islets cultured at 5.5 mM glucose (Table 1Go). Protein levels of the IP3Rs paralleled the changes in mRNA levels in response to glucose (Fig. 1Go). To determine whether changes in IP3R mRNA expression levels were regulated by insulin secretion responses during the 4-day culture, islets were cultured in the presence of somatostatin, an inhibitor of ß-cell insulin secretion. Somatostatin failed to affect IP3R-I mRNA expression levels after a 4-day culture with 5.5 or 11 mM glucose (Table 1Go). However, somatostatin significantly increased IP3R-II mRNA levels and reduced IP3R-III mRNA levels compared with basal values; in the presence of 11 mM glucose, mRNA levels were unchanged with somatostatin (Table 1Go). Insulin secretion from islets was determined during the last 2 days of the 4-day culture period, and somatostatin (1 µM) inhibited basal insulin release (5.5 mM glucose) by approximately 80% (Table 2Go). Insulin secretion from islets cultured at 11 mM glucose was increased 42-fold above basal values, but somatostatin inhibited glucose-stimulated insulin release by 59 ± 5% (Table 2Go).


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  		Table 1. Effects of somatostatin on islet IP3R mRNA expression

 


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  		Figure 1. Western blot analysis of the effects of glucose and forskolin on IP3R isoform expression levels in 4-day cultured rat islets. Islets were cultured in with 5.5 mM glucose (5.5G; control) or 11 mM glucose (11G) for 4 days. On day 4, forskolin (FSK; 10 µM) was added to 11G islets for 2 h for IP3R-I and -III determinations or for 3 h for IP3R-II protein determination. Values are the mean ± SE for three to five independent determinations. *, P < 0.05 vs. 5.5G (control), as determined by one-way ANOVA and post-hoc analysis.

 

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  		Table 2. Effects of somatostatin on insulin release

 
The addition of forskolin (10 µM) to 4-day glucose-treated islets reduced IP3R-II mRNA levels (ratio, 0.09 ± 0.01) to basal control (5.5 mM glucose) values (ratio, 0.08 ± 0.01; P > 0.05) within 3 h, whereas IP3R-III mRNA (ratio, 0.22 ± 0.01) increased to levels observed in control islets (ratio, 0.23 ± 0.01; P > 0.05) within 2 h. IP3R-II and -III protein levels paralleled the forskolin effects on mRNA levels and returned to control values, except for glucose-treated islets, in which forskolin stimulation increased IP3R-III levels to slightly higher than 5.5 mM glucose-cultured islet values (Fig. 1Go). The effects of forskolin on the relative levels of IP3R-II and IP3R-III mRNA were time dependent. Forskolin significantly reduced IP3R-II mRNA levels after 2 h, and the inhibitory effect was even more pronounced after 3 h, when it reached 50% of the levels originally observed in the glucose-treated islets (Fig. 2Go). In contrast, forskolin significantly increased IP3R-III mRNA levels within 1 h and evoked a maximal increase in IP3R-III mRNA levels within 2 h (Fig. 2Go). Therefore, hereafter 2- and 3-h cultures were used in assessing the effects of forskolin on IP3R-III and IP3R-II mRNA levels, respectively, unless stated otherwise. The levels of IP3R-I mRNA were unaffected by either a glucose stimulus (Table 1Go) or forskolin (Fig. 2Go). In contrast to islets maintained under hyperglycemic conditions, islets cultured for 4 days at a basal glucose concentration (5.5 mM) showed no significant change in IP3R-II mRNA expression (97 ± 12% of the control; P > 0.05), but there was an increase in IP3R-III mRNA levels to 173 ± 8% of the control level (P < 0.01) after 3 h of incubation with forskolin (10 µM).



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  		Figure 2. Time course of IP3R isoform mRNA changes in response to forskolin. Islets were cultured for 4 days in 11 mM glucose. On day 4, forskolin (10 µM) was added to the islets at time zero, and IP3R-I, -II, and -III mRNA relative expression levels were determined during 3 h of culture. Values are the percent change from time zero in mRNA levels and are the mean ± SE for three independent determinations. *, P < 0.05 vs. time zero values for each isoform, as determined by one-way ANOVA and post-hoc analysis.

 
To confirm that the effects of forskolin were mediated by cAMP, glucose-treated islets were cultured with the cell-permeant cAMP analog, 8-bromo-cAMP, or the guanosine nucleotide analog, 8-bromo-cGMP, for up to 3 h. 8-Bromo-cAMP affected the relative levels of IP3R-II mRNA in glucose-treated islets, mimicking the effects of forskolin and reducing the expression of this isoform; 8-bromo-cGMP was without effect (Fig. 3AGo). GLP7–36 also reduced IP3R-II mRNA levels, similar to forskolin and 8-bromo-cAMP in glucose-treated islets (Fig. 3AGo). The effects of GLP7–36 showed a longer latency than forskolin, and a significant change in IP3R-II mRNA was not observed until 4 h after islet treatment with this stimulus. The addition of H-89 completely prevented the IP3R-II mRNA response to forskolin in glucose-treated islets, without affecting IP3R-II expression in control islets (Fig. 3AGo).



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  		Figure 3. Effects of cyclic nucleotide-generating agents on IP3R isoform mRNA expression. Islets were cultured for 4 days at 11 mM glucose (control). Then, forskolin (FSK; 10 µM), GLP7–36 (0.1 µM), 8-bromo-cAMP (8Br-cAMP; 5 mM), 8Br-cGMP (5 mM), or H-89 (1 µM) was added to the islets for an additional 3 h or 4 h (for GLP7–36) for IP3R-II mRNA determination (A) and 2 h for IP3R-III mRNA determination (B). Values are the mean ± SE for three to eight independent determinations. *, P < 0.05 vs. control, as determined by one-way ANOVA and post-hoc analysis.

 
8-Bromo-cAMP increased IP3R-III mRNA levels in glucose-treated islets to values comparable to the changes observed with forskolin, whereas 8-bromo-cGMP had no effect (Fig. 3BGo). In addition, GLP7–36, which is a physiological incretin for insulin secretion and a stimulus for adenylyl cyclase and cAMP accumulation in islets (21), increased the relative levels of IP3R-III mRNA in glucose-treated islets to values observed in the presence of forskolin and 8-bromo-cAMP (Fig. 3BGo). Moreover, when forskolin was combined with H-89, the effects of forskolin on IP3R-III mRNA levels were completely inhibited (Fig. 3BGo). H-89 alone had no effect on IP3R mRNA expression.

When the transcription inhibitor, actinomycin D, was added to glucose-treated islets in the presence of forskolin, the increase in IP3R-II mRNA levels and the decrease in IP3R-III mRNA levels were prevented (Fig. 4Go). In contrast, inhibition of translation and protein synthesis with cycloheximide did not affect the effects of forskolin on either IP3R-II or -III mRNA levels (Fig. 4Go).



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  		Figure 4. Effect of translational and transcriptional inhibition on IP3R isoform mRNA expression. Islets were cultured for 4 days at 11 mM glucose (control). Then, islets were pretreated with actinomycin D (ActD; 8 µM) or cycloheximide (CHX; 10 µM) for 10 min, as indicated, before the addition of forskolin (FSK; 10 µM) and culture for an additional 3 h for IP3R-II determination (A) or 2 h for IP3R-III mRNA determination (B). Values are the mean ± SE for three independent determinations. *, P < 0.05; {ddagger}, P < 0.01 (vs. control, as determined by one-way ANOVA and post-hoc analysis).

 
Effects of cAMP-elevating agents on ßHC9 cell IP3R-III mRNA levels
The effects of cAMP-elevating agents on IP3R-III expression were also investigated in a murine insulinoma cell line, ßHC9, which was previously reported to exhibit glucose-sensitive IP3R-III mRNA expression (9). This cell line responded to forskolin and a phosphodiesterase inhibitor, IBMX, with significant increases in cAMP (Fig. 5AGo). In addition, forskolin and IBMX increased IP3R-III mRNA levels in ßHC9 cells (Fig. 5BGo). Moreover, 8-bromo-cAMP raised IP3R-III mRNA to levels observed in the presence of forskolin, whereas 8-bromo-cGMP did not have any effect on type III mRNA expression (Fig. 5BGo).



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  		Figure 5. Effects of cyclic nucleotides on IP3R-III mRNA expression in ßHC9 cells. ßHC9 cells were cultured at 2.8 mM glucose (control) for 4 days. Then, forskolin (FSK; 10 µM), IBMX (0.1 mM), 8-bromo-cAMP (8Br-cAMP; 5 mM), or 8Br-cGMP (5 mM) was added to the cells, and culture was continued for 2 h. A, cAMP levels were determined by RIA. B, IP3R-III mRNA expression levels in cells were determined. Values are the mean ± SE for three independent determinations. *, P < 0.05 vs. control, as determined by one-way ANOVA and post-hoc analysis.

 
Effects of calcium agonist and antagonists on IP3R mRNA expression
To determine whether calcium mediated the islet IP3R expression response to forskolin, glucose-treated islets were cultured with a calcium ionophore, ionomycin, and calcium-mobilizing antagonists. Ionomycin partially mimicked the effect of forskolin on IP3R-II mRNA expression and mimicked the effects of forskolin on IP3R-III mRNA expression (Fig. 6Go, A and B). In glucose-treated islets incubated with nimodipine to block voltage-dependent Ca2+ channels or with BAPTA to chelate intracellular calcium, there was partial antagonism of the effect of forskolin on IP3R-II mRNA expression in glucose-treated islets (Fig. 6AGo), in contrast to the total inhibition of the forskolin-stimulated increase in IP3R-III mRNA (Fig. 6BGo). Nimodipine alone did not affect IP3R-II or -III mRNA expression levels, and BAPTA did not affect IP3R-III mRNA expression levels, although BAPTA did reduce IP3R-II mRNA levels even in the absence of forskolin in glucose-treated islets (Fig. 6Go, A and B).



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  		Figure 6. Effects of a calcium channel blocker, ionophore, and an intracellular Ca2+ chelator on IP3R mRNA responses to forskolin stimulation. Islets were cultured for 4 days at 11 mM glucose (control). Then, the islets were pretreated with nimodipine (NIM; 1 µM) or BAPTA-AM (BAPTA; 10 µM) for 10 min before the addition at time zero of forskolin (FSK; 10 µM) or ionomycin (1 µM). The culture was continued for 3 h for determination of IP3R-II mRNA (A) and 2 h for determination of IP3R-III mRNA (B). Values are the mean ± SE for three to eight independent determinations. *, P < 0.05 vs. control; {ddagger}, P < 0.01 vs. FSK (as determined by one-way ANOVA and post-hoc analysis).

 
Effects of forskolin on IP3R mRNA stability
To determine whether cAMP affected mRNA stability, islets were exposed to forskolin for 3 h after a 4-day culture at 5.5 mM glucose. Then, actinomycin D was added to the islets, and mRNA levels were determined over a 6-h period. IP3R-II mRNA levels in forskolin-treated islets (slope, -8.8± 0.4) declined at a faster rate (P < 0.02) than in control islets (slope, -4.7 ± 0.9; Fig. 7AGo). In contrast, the rate of decline in IP3R-III mRNA levels in forskolin-treated islets (slope, -3.3 ± 0.4) was not significantly different from that in 5.5 mM glucose-cultured control islets (slope, -6.6 ± 1.8; Fig. 7CGo). The rate of mRNA decay in glucose-treated islets was not significantly different from the control value for IP3R-II or -III, and subsequent treatment with forskolin did not significantly affect IP3R-II mRNA stability (Fig. 7BGo), but did reduce the degradation rate of IP3R-III mRNA (slope, -1.6 ± 0.2) compared with the control value (slope, -4.7 ± 0.5; Fig. 7DGo).



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  		Figure 7. Forskolin effects on mRNA stability. Islets were cultured for 4 days at 5.5 mM glucose (5.5G) or 11 mM glucose (11G). Then, the islets were cultured for either 3 h for IP3R-II mRNA determination (A and B) or 2 h for IP3R-III mRNA determination (C and D) in the absence (control; •) or presence ({blacksquare}) of forskolin (10 µM). Actinomycin D (8 µM) was added to the islets at time zero, and the culture was continued for up to 6 h. Values are expressed as percent changes from time zero and are the mean ± SE for three or four independent determinations.

 
PKA activity in isolated islets
To determine whether glucose and forskolin activation of adenylyl cyclase, cAMP synthesis, and PKA activation was altered during chronic glucose stimulation, islet PKA activity was determined. In freshly isolated islets, glucose stimulation significantly increased the percentage of activated PKA by about 36%, and forskolin (1 µM) stimulated activation by about 346% above basal values (Table 3Go). After 4 days of culture, glucose-treated islet PKA activation was not significantly different from the control (5.5 mM glucose) value, although forskolin-stimulated PKA activation was significantly higher than the control value (Table 3Go). Total PKA activities in fresh islets or islets cultured 4 days at 5.5 and 11 mM glucose were 508 ± 83, 396 ± 53, and 456 ± 49 pmol phosphate incorporated/min·µg protein, respectively, and there were no significant differences among the values.


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  		Table 3. Protein kinase A activation in rat islets

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The second messengers, IP3 and cAMP, are increased in isolated rat islets in response to glucose as well as other stimuli (22). IP3 binds to receptors classed as ligand-activated Ca2+-selective channels found in cell membranes (1, 23, 24, 25, 26). Functionally, IP3 has been reported to release Ca2+ from intracellular storage sites in ß-cells (27, 28), including a plasma membrane vesicle fraction distinct from microsomes (29). cAMP, which alone is not an efficient stimulus for insulin release, strongly potentiates insulin secretion in response to glucose (22, 30). Interestingly, when islets are chronically stimulated with glucose for 4–7 days, the insulin secretory response (17, 31), IP3 synthesis (32), and cAMP generation (21) responses are glucose desensitized. Moreover, this laboratory has reported previously that in 7-day glucose-desensitized islets, IP3R-II mRNA and protein levels were up-regulated, whereas IP3R-III mRNA and protein levels were reduced (10). In in vivo studies, islets from hyperglycemic rats showed an increase in IP3R-II mRNA correlated with blood glucose levels (10). As long term hyperglycemic responses in islets in vitro are most relevant to islet responses in vivo in noninsulin-dependent diabetes mellitus and to the hyperglycemic 90% partial pancreatectomized rats previously studied (10), the present study explored the relationship among IP3R mRNA regulation, cAMP and other putative second messengers in islets during chronic glucose stimulation in vitro.

As reported previously for 7-day cultured islets (10), when islets were cultured with 11 mM glucose for only 4 days, there was an increase in IP3R-II mRNA expression and a decrease in IP3R-III mRNA expression, which were paralleled by changes in IP3R protein levels. Somatostatin was evaluated as an agent reported to inhibit cAMP formation and insulin secretion in islets (33) to determine whether it would evoke changes in IP3R expression. Somatostatin significantly inhibited insulin release under basal and glucose-treated conditions. These changes correlated with an increase in basal IP3R-II mRNA and a decrease in IP3R-III mRNA levels. Although the somatostatin-evoked decrease in basal insulin secretion suggests that reduced secretory activity may account for changes in IP3R expression levels, the correlated changes in IP3R mRNA levels may be related to the cAMP-lowering effects of somatostatin in islets as described by others (33). As cAMP generation and insulin secretion responses of glucose-desensitized islets were previously reported to be significantly impaired (17, 21, 31), in 4-day glucose-treated islets the up-regulation of IP3R-II and down-regulation of IP3R-III mRNA expression may be related to the reduced secretory response as well as to altered cAMP generation or other glucose-desensitizing effects, including IP3R gene transcription factors. The ability of somatostatin and glucose desensitization (21) to reduce islet cAMP levels may be the basis for their effects on IP3R mRNA levels. The failure of somatostatin to augment the glucose-treated changes in IP3R mRNA levels is not readily explained. Previously, somatostatin at the concentration used in the present study failed to affect the loss of insulin gene expression associated with chronic glucose stimulation (34). Perhaps modulatory changes induced during glucose desensitization are sufficient to maximally affect transcriptional activity of the IP3R genes. In contrast, IP3R-I mRNA levels were unaffected by somatostatin- or glucose-induced desensitization, suggesting that neither cAMP nor changes in insulin secretion has an effect on transcriptional regulation of this isoform. Thus, changes in insulin secretion and/or endogenous cAMP levels appear to modulate IP3R-II and -III mRNA levels.

Levels of cAMP increase within minutes in freshly isolated islets in response to glucose or GLP stimulation, and forskolin is a potent adenylyl cyclase-activating agent in islets (21). Forskolin, 8-bromo-cAMP, and GLP7–36 had similar effects: lowering IP3R-II mRNA levels and elevating IP3R-III mRNA levels in 4-day glucose-desensitized islets. Each of these stimuli effectively negated the effects of chronic glucose stimulation on IP3R mRNA expression. The response to GLP7–36 took up to 4 h to be expressed, probably because the endogenous levels of cAMP in response to GLP7–36 would be expected to be much lower than those evoked by forskolin, and the resulting transcriptional changes more gradual. Moreover, inhibition of PKA prevented the response to forskolin. These results suggest that cAMP mediates the regulation of IP3R mRNAs in glucose-desensitized islets. It was previously reported that adenylyl cyclase is desensitized during chronic glucose stimulation (21), and the present results show that PKA activation in response to glucose stimulation is compromised in 4-day glucose-treated islets. As total PKA activity and forskolin-stimulated PKA activity are not significantly different in 4-day glucose-treated islets compared with control islets, the results suggest that cAMP production and signal transduction in response to glucose stimulation are impaired in the glucose-treated group. Thus, the down-regulation of cAMP responses during chronic glucose stimulation may lead to derangements in IP3R mRNA regulation, which are overcome when cAMP levels are artificially elevated for a brief time. Previous studies also demonstrated that IP3R-III mRNA expression in ßHC9 cells is regulated (9), and in the present study elevations in cAMP by 8-bromo-cAMP, forskolin, or phosphodiesterase inhibition were associated with increased ßHC9 cell IP3R-III mRNA expression levels. However, the previous study also showed that unlike islets, insulinoma cells do not show the long term IP3R mRNA desensitization response to glucose (9), suggesting an aberrant pathway(s) in the regulation of IP3R regulation in these cell lines or that other islet factors, such as glucagon or somatostatin, play a role in this regulation.

Functionally, the changes in IP3R-II and IP3R-III mRNA levels in glucose-treated cultured islets may be related to the progressive loss in glucose sensitive insulin secretion observed in this model of glucose desensitization (17). It has been reported that among the three isoforms, IP3R-I stimulation results in localized nonpropagating increases in intracellular Ca2+, whereas IP3R-II is the most sensitive to IP3 and is required for long lasting Ca2+ oscillations, and IP3R-III is the least sensitive to IP3 and Ca2+ and is responsible for generating monophasic, all or none, Ca2+ transients (11, 35). In the islet, loss of IP3R-III may compromise the acute changes in intracellular Ca2+ that regulate glucose-stimulated insulin secretion (36). It has been reported that fast intracellular Ca2+ oscillations in islets depend upon mobilization of Ca2+ from intracellular stores, and that culture suppressed those oscillations, but an increase in cAMP generation increased the oscillations (37). Thus, it appears that cell culture and cAMP modulate changes in intracellular Ca2+ levels in islets with dynamics similar to the changes observed in IP3Rs. Perhaps, the maintenance of cAMP generation in chronic glucose-challenged islets would be beneficial in maintaining glucose-sensitive insulin secretory responses related to IP3R levels and intracellular Ca2+ mobilization. Glucose stimulates adenylyl cyclase activity in intact islets, a response probably mediated by changes in local glucagon release (38) as well as changes in Ca2+/calmodulin (39, 40). The present study illustrates that cAMP "tone" may have important consequences during the long term exposure of islets to glucose.

cAMP signals through activation of PKA and the cAMP response element-binding protein (CREB) transcription pathway, and these mechanisms probably play a role in regulating IP3R mRNA transcription. Newly transcribed IP3R mRNA appeared to primarily mediate the effects of forskolin, as actinomycin D inhibited the responses of IP3R-II and -III mRNAs to forskolin, whereas protein synthesis inhibition did not affect the regulatory changes. As 8-bromo-cGMP did not mimic the cAMP responses, the results suggest that cAMP generated from forskolin stimulation of adenylyl cyclase specifically mediates transcriptional changes in mRNA. cAMP has been reported to synergize with glucose in the transcriptional activation of immediate-early response genes, including c-fos, c-jun, JunB, zif-268, and nur-77, in the ß-cell line INS-1 (41). In that study it was suggested cAMP plays the role of a competence factor for the induction of immediate-early response genes, which increase within a 1- or 2-h period. As the recovery of IP3R mRNA levels in glucose-treated islets occurred within 2–3 h after forskolin treatment, it is possible that cAMP-sensitive immediate-early response genes play a role in the response.

cAMP is also known to affect mRNA stability (42, 43, 44). In the present study forskolin induced a significant increase in glucose-treated islet IP3R-III mRNA stability, which may contribute to the increase in mRNA expression observed after forskolin stimulation. The results also show that cAMP-induced changes in mRNA stability do not account for forskolin’s ability to reverse the effect of glucose-induced desensitization on IP3R-II mRNA expression. Perhaps, proteins induced during forskolin stimulation are responsible for reduced IP3R-II mRNA stability in 5.5 mM glucose-cultured islets. However, as glucose treatment alone did not significantly affect IP3R-II or -III mRNA stability, it is not likely that changes in stability account for increased mRNA levels for this isoform during glucose desensitization. Changes in transcriptional activity appear to be primarily responsible for forskolin’s effects on IP3R-II expression.

In addition to forskolin, ionomycin reduced IP3R-II mRNA levels and increased islet IP3R-III mRNA levels, suggesting that increased cytosolic Ca2+ levels mediated the forskolin responses. Further support for this hypothesis comes from the inhibition of forskolin effects on IP3R mRNA levels by the Ca2+ antagonists nimodipine and BAPTA. Nimodipine was used in this study because it is an L-type Ca2+ channel blocker, and cAMP is reported to induce phosphorylation of L-type Ca2+ channels and potentiate their voltage-dependent activation in islets (45). BAPTA also antagonized the glucose effects on IP3R-II mRNA levels, which suggests that intracellular Ca2+ levels mediate the glucose response of this isoform. Increased intracellular Ca2+ in pancreatic islet cell lines purportedly leads to phosphorylation by Ca2+/calmodulin-dependent protein kinases of the transcription factor CREB, leading to transcriptional activation of genes linked to CRE (46, 47). The cAMP- and Ca2+-mediated regulation of IP3R gene transcription suggests that CREB activation plays a role in regulation of these genes. However, the mechanism is complex, as forskolin and ionomycin had differential effects on glucose-treated islet IP3R-II and -III mRNA levels. One explanation for these responses may be that CREB is also a transcription inhibitor of some genes (48). Moreover, it is possible that the induction of inducible cAMP early repressor transcription factor inhibits cAMP response element activation and down-regulates IP3R-II gene transcription in islets (49). Ca2+ can also have a direct modulatory role in IP3R gene transcription. Ca2+ has been reported to activate transcription through stimulation of the c-jun N-terminal protein kinase signaling pathway along with activation of Ca2+/calmodulin-dependent protein kinases and CREB-binding protein (50). Ca2+ can also stimulate certain isoforms of adenylyl cyclase, which may lead to increased cAMP levels in islets and changes in protein kinase activities related to transcriptional activity.

In summary, cAMP and Ca2+ play regulatory roles in the expression of IP3Rs. Regulation may be related to Ca2+ mobilization in response to glucose stimulation, cAMP and/or PKA, and Ca2+-calmodulin-dependent protein kinase activation. In addition, early gene regulation, including cAMP-responsive element modulator and other transcription factors, may mediate the IP3R mRNA transcriptional regulation. IP3Rs are known to be phosphorylated by several kinases, including PKA, protein kinase G, Ca2+-calmodulin-dependent protein kinase, protein kinase C, and tyrosine kinases (3, 51), but it is not known how phosphorylation might regulate the expression of this receptor family. The down-regulation of the adenylyl cyclase response and changes in IP3R expression in glucose-desensitized islets suggest that cAMP- and Ca2+-regulated transcription factors play a role in IP3R gene regulation. Thus, changes in IP3R regulation might be related to changes in Ca2+ mobilization and glucose-sensitive insulin release in hyperglycemia associated with type 2 diabetes mellitus. Support for this hypothesis is provided by a study showing that aberrant endoplasmic reticulum Ca2+ sequestration and, hence, mobilization capacity in db/db mice are responsible for the defective insulin secretion in this mouse model (52).


   Acknowledgments
 
The authors thank Jill Platten for her assistance with portions of this project.


   Footnotes
 
1 This work was supported in part by grants from the American Diabetes Association and the NIH (DK-25705). Back

2 Recipient of a fellowship from the Juvenile Diabetes Foundation International. Back

Received August 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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