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Endocrinology Vol. 140, No. 5 2173-2182
Copyright © 1999 by The Endocrine Society

ARTICLES

Glucose Regulates Expression of Inositol 1,4,5-Trisphosphate Receptor Isoforms in Isolated Rat Pancreatic Islets1

Bumsup Lee2, Jean-Christophe Jonas3, Gordon C. Weir and Suzanne G. Laychock

Department of Pharmacology and Toxicology (B.L., S.G.L.), School of Medicine and Biomedical Sciences, the State University of New York at Buffalo, Buffalo, New York 14214; and the Research Division (J.-C.J., G.C.W.), Joslin Diabetes Center, and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Isolated rat pancreatic islets were studied to determine the dynamic regulatory effects of glucose stimulation on the expression of messenger RNA (mRNA) and protein levels for inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) isoforms I, II, and III. The relative isoform abundance was: IP3R-III > IP3R-II ~ IP3R-I. Culture of islets with glucose (G; 20 mM) or {alpha}-ketoisocaproic acid for 30 min increased only IP3R-III mRNA expression above control (5.5 mM glucose). 2-Deoxyglucose was without effect. Islet culture for 2 h with G (20 mM) or {alpha}-ketoisocaproic acid reduced IP3R-III mRNA expression levels below control, and cycloheximide blocked the response. Culturing islets for 1 day or 7 days with G (11 mM) reduced the expression of IP3R-III mRNA but increased the expression of IP3R-II mRNA in a time-dependent manner. Cytosine arabinoside lowered cultured islet IP3R-II and -III mRNA levels, but glucose effects remained evident. IP3R-II mRNA levels were also significantly higher in islets from hyperglycemic 90% partial pancreatectomized rats, compared with sham animals. Islet IP3R mRNA expression also showed osmotic sensitivity. Islet IP3R-III protein levels increased after 2 h islet culture at 20 mM G, were unchanged after 1 day culture at 11 mM G, and were lower than control after 7 days culture at 11 mM G. In contrast, IP3R-II levels increased after 1 day and 7 days culture at 11 mM G, whereas IP3R-I protein levels remained unchanged. Thus, G stimulation rapidly increases transcription and expression of IP3R-III mRNA and protein levels in rat islets. However, chronic G stimulation up-regulates IP3R-II mRNA in cultured islets and in islets from partial pancreatectomized rats. Metabolic regulation of IP3R-II and III expression may mediate ß-cell IP3-responsive Ca2+ mobilization and insulin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOSE STIMULATION of pancreatic islet ß-cells initiates a cascade of events resulting in insulin secretion. Glucose-stimulated insulin secretion is dependent on an increase in intracellular Ca2+ concentration ([Ca2+]i) (1, 2). Glucose stimulation not only opens voltage-dependent calcium channels (VDCC), but it also increases phosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP3) production and mobilizes Ca2+ from intracellular IP3-sensitive Ca2+ stores in pancreatic ß-cells (2, 3, 4, 5). IP3, as a second messenger for hormones and neurotransmitters, exerts its action through specific receptors that are ligand-activated, Ca2+-selective channels (6). IP3 receptors (IP3R) are located mainly in endoplasmic reticulum but are also reported to be located in insulin secretory granules and plasma and nuclear membranes (7, 8, 9, 10). Five isoforms of IP3R (-I through -V) have been identified. IP3R complementary DNA (cDNA) clones have been fully sequenced for rat IP3R-I, -II, and -III (11, 12, 13) and partially sequenced for isoforms IV and V (14, 15). IP3R-III is predominantly expressed in rat pancreatic islets and a clonal ß-cell line, RINm5F (13, 16). More than one isoform of IP3R is often found in a single cell type, and different patterns of expression have been found in various tissues, suggesting that different mechanisms regulate IP3R isoforms and that each isoform has a distinct function (13, 15).

Glucose is the primary stimulus for pancreatic islet insulin-secreting ß-cells, and in addition to acute secretory effects, glucose also has long-term modulatory effects on islet ß-cells. Prolonged exposure of pancreatic islets to elevated glucose concentrations both in vivo and in vitro has been shown to impair glucose-stimulated insulin release (17, 18, 19). In an in vitro model of glucose desensitization of insulin secretion, which uses isolated islets cultured for up to 7 days at 11 mM glucose, ß-cell sensitivity to glucose was reduced (desensitized islets) (19, 20). The reduced glucose sensitivity in vitro mimics the response of islets in in vivo hyperglycemic models and is similar to the loss of ß-cell glucose sensitivity in non-insulin-dependent diabetes mellitus (18).

The impaired glucose-stimulated insulin release has been related to the modification of [Ca2+]i (21). With prolonged exposure of islets to elevated glucose levels, the reduced insulin secretory response has been partly ascribed to the reduced level of L-type VDCC messenger RNA (mRNA) and Ca2+ influx through VDCC (21, 22). However, little is known about the regulation of the IP3-sensitive Ca2+ pool in pancreatic islets, except that various stimuli increase IP3 levels and mobilize Ca2+ (23, 24, 25). It has been reported previously that IP3R-I, -II, and -III mRNAs and protein are expressed in rat pancreatic islets and that IP3R-III is the most abundant isoform (7, 16, 26, 27). However, evidence for regulatory properties associated with islet IP3R mRNA or protein is incomplete. A possible regulatory influence of glucose on IP3R protein levels has been suggested for islets from Zucker diabetic fatty (ZDF) rats and RINm5F cells (7). The present study was performed to determine the regulatory effects of glucose stimulation on the expression of mRNA and protein levels for IP3R-I, -II, and -III in rat pancreatic islets.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of rat islets
Pancreatic islets from adult male Sprague Dawley rats were isolated using collagenase (type P; Boehringer Mannheim, Indianapolis, IN), as described previously (19). 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 they were cultured for various periods of time in 35-mm culture dishes (80–150 islets/dish) with 2 ml CMRL-1066 medium (Gibco BRL, Grand Island, NY) containing glucose at the concentrations indicated in the text, 9% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 5% CO2-95% air, at 35 C, as described previously (19). Pancreatic acinar tissue was collected from the collagenase-dispersed pancreas and was cultured as described for islets. Mannitol was included in 30-min and 2-h control cultures to maintain osmotic equivalence to glucose-stimulated conditions. Culture medium was changed every second day.

Partial pancreatectomized (PPx) rats
Four- to 5-week-old Sprague Dawley rats were submitted to a 90–95% pancreatectomy or sham surgery (28, 29). Fed-rat whole-blood glucose levels were measured weekly for 4 weeks with a One Touch II glucometer (Lifescan, Milpitas, CA), at which time the islets were isolated by collagenase digestion of the pancreatic remnants, as previously described (30).

RNA isolations and cDNA synthesis
Total RNA was extracted from rat pancreatic islets and acinar tissue using Trizol (Gibco BRL). cDNA was reverse transcribed from 2 µg total RNA by random hexamer (Gibco BRL) in 20 µl of a solution containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM deoxynucleotide triphosphate (dNTP), and 200 U Superscript II RNase H- RT (Gibco BRL). Reactions were incubated for 1 h at 42 C and then heated to 70 C for 15 min.

When islets from PPx animals were studied, total RNA was extracted from islets of individual animals, following the manufacturer’s suggested protocols, using Ultraspec (Biotex Laboratories, Inc., Houston, TX). After quantitation by spectrophotometry, 500 ng RNA was diluted to a final concentration of 0.1 µg/µl, heated at 85 C for 3 min, and then reverse-transcribed into cDNA in 25 µl solution containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 1 mM dNTPs, 50 ng random hexamers, and 200 U Superscript II RNase H- reverse transcriptase (Gibco BRL). Reactions were incubated for 10 min at 25 C, 1 h at 42 C, and 10 min at 95 C. The final cDNA reaction products were then diluted with 50 µl H2O, to a concentration corresponding to 20 ng of starting RNA (20 ng RNA equivalents) per 3 µl, and stored at -80 C.

PCR amplification and quantitation of IP3R transcript levels
The amount of cDNA and the number of PCR cycles were determined based on the preliminary study that PCR amplification was in the exponential phase of the amplification. Polymerization reactions were carried out in a 2400 Thermocycler (Perkin-Elmer Corp., Foster City, CA) in 25 µl reaction mixture containing 0.2 mM dNTPs, 10 pmol of appropriate oligonucleotide primers, PCR buffer (Gibco BRL), 2 mM MgCl2, and 1 U Taq DNA polymerase (Gibco BRL); pancreatic acinar tissue IP3R-I and -II required 1.5 mM MgCl2. The resulting complimentary DNA (cDNA) was then amplified, as indicated with the following (+) and (-) strand oligonucleotide primers, respectively: IP3R-I (423 bp), IP3R-II (390 bp), and IP3R-III (560 bp), and ß-actin (441 bp), as reported previously (27), or for acinar tissue studies IP3R-I (418 bp) 5'-GAGAGAAAGCGCACGCCGAGAG-3' and 5'-GGACATAGCTTAAAGAGGC-AGTC-3'; glyceraldehyde 3-phosphate dehydrogenase (220 bp) 5'-ACCAGGTTGTCTCCTGTGAC-3', and 5'-CTCTCTTGCTCTCAGTATCC-3'. For amplification of IP3R-II and -III cDNAs and ß-actin cDNA, 2.5, 5.0, and 10.0 µl of 1:5 dilution of cDNA were used as templates. For amplification of IP3R-I cDNA, 2.5, 5.0, and 10.0 µl of 1:2.5 dilution of cDNA were used as template. For amplification of glyceraldehyde 3-phosphate dehydrogenase cDNA, 2.5, 5.0, and 10.0 µl of 1:200 dilution of cDNA were used as template. Amounts of cDNA were chosen that were within a linear range for amplification. The amplification conditions were 35 cycles with denaturation for 1 min at 94 C, annealing for 2 min at 55 C, and extension for 3 min at 72 C with the final extension for 7 min. The amplimers were separated by electrophoresis in a 2.0% agarose gel in Tris borate buffer. The gel was stained by ethidium bromide and viewed by Gel Doc 1000 (Bio-Rad Laboratories, Inc., Hercules, CA). The density of each PCR fragment was analyzed by Molecular Analyst software (Bio-Rad Laboratories, Inc.). The image densities of PCR products for IP3R isoform and glyceraldehyde 3-phosphate dehydrogenase were compared with the density of coamplified ß-actin to determine the ratio of expression.

Semiquantitative radioactive multiplex PCR
Polymerization reactions were performed in a 9600 thermal cycler (Perkin-Elmer Corp.) in 50 µl reaction mixture containing 3 µl cDNA (20 ng RNA equivalents), 80 µM cold dNTPs, 2.5 µCi [{alpha}-32P]deoxycycidine triphosphate ([{alpha}-32P]dCTP), 10 pmol of appropriate oligonucleotide primers, GeneAmp PCR buffer, and 5 U AmpliTaq Gold DNA polymerase (Perkin-Elmer Corp.). The IP3R-II was amplified, along with thymine adenine repeat region (TATA)-binding protein (TBP) as control. The following (+) and (-) strand oligonucleotide primers were used to amplify TBP cDNA (190 bp): 5'-ACCCTTCACCAATGACTCCTATG-3' and 5'-CGCTAAACGACGTCAGTAGTA-3'. The conditions used were 10 min at 94 C, to release AmpliTaq Gold activity, followed by 32 cycles of 1 min denaturation at 94 C, 1 min annealing at 55 C, and 1 min extension at 72 C each, and a final 10-min extension step. The IP3R-III was amplified along with {alpha}-tubulin as control. The following (+) and (-) strand oligonucleotide primers were used to amplify {alpha}-tubulin cDNA (451 bp): 5'-CTCGCATCCACTTCCCTC-3' and 5'-CATGCACCCACTCCCGTA-3'. The conditions were as for IP3R-II, but only 27 cycles were performed.

Control experiments, using normal rat islet cDNA, showed that the amount of each amplimer obtained in a multiplex PCR was independent of the presence of the other primers (cross-correlation analysis), excluding the possibility of strong interference between primers. The number of cycles and the final reaction conditions were then adjusted to be in the exponential phase of the amplification of each product. Finally, the amount of each PCR product in a multiplex reaction increases linearly with the amount of starting cDNA (from 2.5 to 80 ng RNA equivalents), ensuring that changes in the ratio of PCR product to control gene product truly reflects a change in mRNA abundance of that gene, relative to the control gene.

After removal of free [{alpha}-32P]dCTP by gel filtration on Probequant G50 microcolumns (Pharmacia Biotech, Alameda, CA), the amplimers were separated on a 4.5 or 6% polyacrylamide gel in Tris borate EDTA buffer. The gel was dried, and the amount of [{alpha}-32P]dCTP incorporated in each amplimer was measured with a phosphoimager and quantified with ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA). The amounts of IP3R isoform PCR products were then expressed, relative to their control gene. All relative values were compared and expressed as a percent of shams tested in the same PCR.

Immunoblotting
Whole-cell homogenates were prepared from rat pancreatic islets by sonication (15 sec at 30 output, Tekmar sonic disrupter) in ice-cold distilled water containing 5 µg/ml leupeptin and 1 µg/ml pepstatin. The protein concentration was determined by protein assay (Bio-Rad Laboratories, Inc.) using BSA (Sigma Chemical Co., St. Louis, MO) as standard. The cell homogenates 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 containing 4% SDS, 30% glycerol, 0.05 M Tris-HCl (pH 6.8), 2% 2-mercaptoethanol, and 0.01% bromphenol blue. Protein (20–30 µg) was separated by 5% SDS-PAGE and transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA). The membrane was blocked with 10% nonfat dried milk in Tween phosphate- buffered saline (TPBS) containing: 138 mM NaCl, 4 mM KCl, 5 mM Na2HPO4·7H2O, 1.5 mM KH2PO4, and 0.1% Tween. The membrane was incubated with antisera against the IP3R isoforms (antisera were a gift from Dr. R. Wojcikiewicz), as described previously (16, 26). Antisera were generated against synthetic peptides corresponding to the C-termini of rat IP3R-I, -II, or -III and were affinity purified to yield antisera specific to their cognate proteins. Antibodies to IP3R-I, -II, and -III were diluted 1:80, 1:100, and 1:50, respectively, for Western blot, as described in previous studies (16, 26). After washing, the membrane was incubated with peroxidase-conjugated antirabbit IgG secondary antibody (Bio-Rad Laboratories, Inc.). The bound antibody was localized by chemiluminescence, and the density of each band was determined by densitometric scanning using Molecular Analyst software.

Statistical analysis
Values are means ± SE. Significant differences between treatment groups were determined by paired Student’s t test or one-way ANOVA with post hoc analysis using Student-Newman-Keuls multiple-comparison test. Values of P <= 0.05 were accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Amplification of rat pancreatic islet mRNA
Freshly isolated rat pancreatic islets expressed mRNA for IP3R-I, -II, and -III (Table 1Go). The sequences of the RT-PCR amplification primers for each IP3R subtype analysis were distinct and corresponded with the reported cDNA sequences of the respective rat receptors (12, 13, 31). IP3R-III was the most abundant subtype expressed in fresh islets, whereas IP3R-I and -II were expressed at similar levels (Table 1Go). In comparison, pancreatic acinar tissue predominantly expressed IP3R-I mRNA (0.83 ± 0.08 IP3R-I/ß-actin mRNA; 84 ± 2% of total) and IP3R-II mRNA (0.15 ± 0.02 IP3R-II/ß-actin mRNA; 15 ± 1% of total), whereas IP3R-III mRNA was least abundant (0.016 ± 0.002 IP3R-III/ß-actin mRNA; 1.7 ± 0.4% of total).


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  		Table 1. IP3R mRNA subtype expression during long-term glucose stimulation

 
However, the relative abundance of the IP3R mRNA isoforms, compared with ß-actin mRNA levels, changed during the 7-day culture of islets at a basal level (5.5 mM) of glucose. In 7-day cultured control islets, although the expression of IP3R-I mRNA did not change (compared with fresh islets), the expression of IP3R-II and -III mRNAs were 4-fold and 2-fold higher than fresh islets, respectively (Table 1Go). The expression of glyceraldehyde 3-phosphate dehydrogenase mRNA also increased, relative to ß-actin mRNA, during 7-day islet culture in comparison with fresh islets (Table 1Go).

Although islet endocrine cells ordinarily proliferate very slowly (32), hyperglycemia has been reported to increase ß-cell mass (33). To determine whether the rapid proliferation of cells, including fibroblasts, contributed to the increase in IP3R mRNA during long-term islet culture, the mitotic inhibitor, cytosine arabinoside (Ara-C), was added (34). In 7-day cultured islets treated with Ara-C, the relative levels of IP3R-I, -II, and -III mRNAs were comparable with those of fresh islets; and IP3R-II and -III mRNAs were significantly (P < 0.05) lower than levels in untreated islets (Table 1Go).

Short-term regulation of IP3R mRNA expression in islets
Acute regulation of expression of the three isoforms of IP3R mRNA was determined in fresh islets and in islets cultured at 5.5 mM glucose (control) or 20 mM glucose (glucose-treated). Osmotic equivalence between the treatment groups was maintained by the addition of mannitol to control islets. After 30 min of culture with a maximally secretagogic concentration of glucose (20 mM), the expression of islet IP3R-III mRNA was 186 ± 20% of control levels (Fig. 1AGo and 2AGo), whereas the expression of IP3R-I was 95 ± 10% of control (0.10 ± 0.01 IP3R-I/ß-actin mRNA; P > 0.05) and expression of IP3R-II was 103 ± 9% of control (0.13 ± 0.01 IP3R-II/ß-actin mRNA; P > 0.05) (Fig. 1AGo). Glucose (20 mM) stimulation for 30 min did not significantly affect pancreatic acinar tissue expression of IP3R-I (107 ± 5% of control), IP3R-II (108 ± 6% of control), or IP3R-III (94 ± 0.1% of control).



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  		Figure 1. IP3R isoforms in pancreatic islets. Isolated islets were cultured for (A) 30 min or (B) 2 h, in the presence of 5.5 mM glucose (C) or 20 mM glucose (G). Total RNA was extracted from the islets, and mRNA levels were determined by RT-PCR for IP3R isoforms I, II, and III, and for ß-actin, as indicated.

 


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  		Figure 2. IP3R-III mRNA expression in islets during short-term stimulation. Isolated islets were incubated for (A) 30 min or (B) 2 h, in the presence or absence of 5.5 mM glucose (C), 20 mM glucose (G), actinomycin D (10 µM) (ActD), cycloheximide (8 µM) (CHX), 2-deoxyglucose (14.5 mM) (DOG), or KIC (10 mM), as indicated. Values are the mean ± SE of IP3R-III mRNA, relative to ß-actin mRNA for the number of independent determinations shown at the base of each bar. Significant differences were determined by one-way ANOVA and Student-Newman-Keuls multiple-comparison test. *, P < 0.05 vs. C for each time point.

 
2-Deoxyglucose (2-DOG), which is transported and phosphorylated but not metabolized in ß-cells, was compared with glucose for effects on IP3R mRNA expression. Culture of islets for 30 min, in the presence of 2-DOG (20 mM), did not evoke any significant changes in IP3R-III mRNA expression (Fig. 2AGo). A similar lack of effect of 2-DOG (20 mM) after 30 min was also noted for IP3R-I/ß-actin mRNA expression (102 ± 8% of control; P > 0.05) and IP3R-II/ß-actin mRNA expression (107 ± 4% of control; P > 0.05). Control islets were treated with an osmotically equivalent concentration of mannitol to sugar.

Islets were also treated with actinomycin D, an inhibitor of transcription, or with cycloheximide to inhibit translation. IP3R-III mRNA expression in islets treated with actinomycin D did not respond to a 30-min glucose stimulus (Fig. 2AGo). In contrast, IP3R-III mRNA expression was stimulated by glucose in the presence of cycloheximide, and the increase in expression was similar to that evoked by glucose in the absence of inhibitor (Fig. 2AGo).

In contrast to the changes in expression observed at 30 min, islets cultured for 2 h at 20 mM glucose had a 42 ± 8% reduction in IP3R-III mRNA expression, compared with control values (Fig. 1BGo and 2BGo), whereas IP3R-II mRNA expression was 96 ± 6% of control (0.15 ± 0.01 IP3R-II/ß-actin mRNA; P > 0.05) (Fig. 1BGo). When islets were cultured in the presence of cycloheximide and 20 mM glucose for 2 h, IP3R-III mRNA expression (104 ± 7% of control) was not different from control values (Fig. 2BGo). Cycloheximide did not significantly affect IP3R-III mRNA expression under basal conditions (97 ± 5% of control) (Fig. 2BGo). ß-Actin mRNA expression was similar in control and glucose-treated islets after 30 min or 2 h of culture (Fig. 1Go, A and B).

{alpha}-Ketoisocaproic acid (KIC) was also investigated for effects on IP3R mRNA expression in islets. KIC stimulation of islets for 30 min evoked a significant increase (143 ± 6% of control) in IP3R-III mRNA expression levels (Fig. 2AGo); whereas after 2 h of KIC stimulation, IP3R-III mRNA expression was reduced to 69 ± 12% of control (Fig. 2BGo). After 30 min or 2 h stimulation with KIC (10 mM), IP3R-I/ß-actin mRNA ratios were 102 ± 8% of control (P > 0.05) and 112 ± 5% of control (P > 0.05), respectively; and IP3R-II/ß-actin mRNA ratios were 105 ± 4% and 107 ± 3% of control (P > 0.05), respectively.

Long-term regulation of IP3R mRNA expression in cultured islets
Islets were cultured for 1–7 days to characterize the long-term effects of a physiological, but hyperglycemic, concentration of glucose (11 mM) on IP3R mRNA expression. After 1 day of culture at 11 mM glucose, islet IP3R-II mRNA expression significantly increased, by 37 ± 9% of control, whereas IP3R-III mRNA expression was reduced by 30 ± 2% of control (Fig. 3AGo and Table 1Go). When islets were cultured for 7 days in the absence of Ara-C, glucose (11 mM) stimulation increased the expression of IP3R-II mRNA to 90 ± 20% above control, whereas IP3R-III mRNA expression was reduced by 42 ± 3%, and IP3R-I mRNA expression did not respond to changes in glucose concentration (Fig. 3BGo and Table 1Go). After 7 days of culture in the presence of Ara-C, glucose (11 mM) stimulation of islet IP3R-II mRNA expression was increased to 183 ± 39% above control, in contrast to IP3R-III mRNA being reduced by 40 ± 4% (Fig. 3CGo and Table 1Go). Although the presence of Ara-C reduced the ratio of IP3R/ß-actin mRNA in control and glucose-stimulated islets by 55 ± 4%, compared with the ratio in islets in the absence of Ara-C, glucose-induced changes in the expression of IP3R-II and -III mRNAs remained highly evident (Fig. 3CGo and Table 1Go). No attempt was made in these studies to determine whether the level of ß-actin mRNA was affected by islet culture; therefore, valid comparisons are only made between paired islet samples cultured for the same time period.



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  		Figure 3. Islet IP3R isoform mRNA expression during chronic glucose stimulation. Isolated islets were cultured for (A) 1 day (d), or (B and C) 7 d, in the presence of 5.5 mM glucose (C) or 11 mM glucose (G) in the absence (A and B) or presence (C) of Ara-C (10 µM). Islet IP3R-I, -II, or -III mRNAs, ß-actin mRNA, or glyceraldehyde 3-phosphate dehydrogenase (GPDH) mRNA expression levels were determined by RT-PCR and visualized by ethidium bromide staining on agarose gel.

 
The linear cDNA amplification of ß-actin showed no significant differences (n = 3, P > 0.05) between 7-day control and glucose (11 mM)-treated islets (Y = 505 + 404x, r = 0.99, P < 0.01, n = 3), and expression levels of ß-actin mRNA were similar between control and glucose-treated islets. There also was no significant difference in the relative expression of glyceraldehyde 3-phosphate dehydrogenase mRNA (n = 3, P > 0.05) between 7-day control and glucose (11 mM)-cultured islets (Fig. 3BGo and Table 1Go).

IP3R mRNA expression in PPx rat islets
Isolated islets from 90% PPx rats also showed evidence of regulation of IP3Rs. Hyperglycemic blood glucose levels in the PPx rats were characterized as being of intermediate (6–8.1 mmol/liter) to high (8.4–19.8 mmol/liter) (Fig. 4Go). IP3R-II mRNA levels were significantly elevated in islets from rats displaying intermediate and high hyperglycemia, compared with sham operated controls with euglycemia; and the mRNA levels paralleled the increases in blood glucose in the PPx rats (Fig. 4Go). In contrast, IP3R-III mRNA was not significantly different in islets from rats displaying intermediate or high hyperglycemia, compared with sham animals (Fig. 4Go).



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  		Figure 4. Blood glucose levels and IP3R isoform mRNA in islets from 90% PPx rats. Four weeks after 90% pancreatectomy or sham surgery (A), whole-blood glucose levels were determined in the fed state with a glucometer, and pancreatic islets were isolated for total RNA extraction. The mRNA transcript ratios for (B) IP3R-II/TBP and (C) IP3R-III/{alpha}-tubulin were compared in sham and PPx islets using a semiquantitative radioactive multiplex RT-PCR. Values are means ± SE for five to eight independent determinations. Significant differences between groups were determined by one-way ANOVA followed by Student-Newman-Keuls multiple-comparison test. *, P < 0.01 vs. sham animals.

 
IP3R mRNA expression in response to changes in osmotic conditions
Because changes in glucose concentration necessitate changes in osmotic potential of the islet, the effects of osmotic conditions on IP3R mRNA expression were characterized. Compared with control levels of IP3R mRNA at 5.5 mM glucose in the absence of mannitol, the addition of 5 mM mannitol did not evoke a significant change in the expression of IP3R-I, -II, or -III after 7-day culture (Fig. 5Go). When mannitol was increased to 10 mM, small (but significant) increases in IP3R-I mRNA (144 ± 5% of control) and IP3R-II mRNA (116 ± 5% of control) were observed (Fig. 5Go). After the addition of 20 mM mannitol, IP3R-I, -II, and -III mRNA expression levels were significantly increased, to 149 ± 11%, 200 ± 17%, and 144 ± 8% of control values, respectively (Fig. 5Go).



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  		Figure 5. Effects of osmotic conditions on IP3R isoform mRNA expression. Isolated islets were cultured for 7 days in the presence of 5.5 mM glucose and increasing concentrations of D-mannitol, as indicated. Total RNA was extracted from the islets, and IP3R isoform and ß-actin mRNAs were quantitated by RT-PCR. Values are the mean ± SE of IP3R-I (•), -II ({blacksquare}), and -III ({blacktriangleup}) mRNAs, relative to ß-actin mRNA, for three or four independent determinations. Significant differences were determined by one-way ANOVA and Student-Newman-Keuls multiple-comparison test. *, P < 0.05 vs. 5.5 mM glucose alone, within each treatment group.

 
IP3R subtype protein expression in islets
Fresh islets expressed IP3R-I, -II, and -III, as determined by Western blot analysis (Fig. 6AGo). IP3R-III appeared on immunoblot as a doublet; however, the upper band is specifically associated with the IP3R and was used for quantitative purposes. Moreover, the lower band was consistently less evident, compared with the upper band, after long-term islet culture (Fig. 6Go, A and D). After 2 h of islet culture, glucose (20 mM) increased the expression level of IP3R-III to 199 ± 24% of control (5.5 mM glucose plus 14.5 mM D-mannitol) (n = 3; P < 0.01), whereas IP3R-I and -II protein levels were 105 ± 6% and 93 ± 4% of control, respectively (n = 3; P > 0.05) (Fig. 6BGo). After 1 day of islet culture, glucose (11 mM)-stimulation increased the expression of IP3R-II to 159 ± 8% of control (n = 3; P < 0.02), whereas IP3R-I and IP3R-III expression was unchanged at 100 ± 6% and 102 ± 8% of control, respectively (n = 3; P > 0.05) (Fig. 6CGo). After 7 days of islet culture at 11 mM glucose, the expression level of IP3R-I was not changed, compared with control (5.5 mM glucose), whereas the level of IP3R-II was significantly increased, to 236 ± 48% of control (n = 4; P < 0.02), in contrast with a 51 ± 4% decrease (n = 4; P < 0.01) in IP3R-III protein expression (Fig. 6DGo).



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  		Figure 6. Western blot of IP3R isoforms in isolated islets. Islets were either freshly isolated (A) or cultured for 2 h (B), 1 day (d) (C), or 7 days (D) at the glucose concentrations indicated. Cell homogenates were analyzed by SDS-PAGE and immunoblot for IP3R isoforms I, II, or III, as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IP3R subtypes I, II, and III are encoded by distinct genes (12, 13, 31), and different cell types often contain more than one isoform expressed in different proportions (26). The functional significance of IP3R diversity seems to be related to an IP3-binding domain (11) and a modulatory domain including phosphorylation sites and binding sites for modulators such as ATP, Ca2+, and calmodulin (11, 12). The three receptor subtypes have been reported to have different affinities for IP3 (II > I > III) (12, 35). In addition, Ca2+ can inhibit IP3 binding to IP3R-I but stimulate (at intermediate Ca2+ levels) or inhibit (at high Ca2+ levels) IP3 binding to IP3R-III (36, 37); regulation of IP3R-II has not been reported. Calmodulin can also inhibit IP3 binding to IP3R-I but not type III (38). The Ca2+-regulated property of the IP3Rs may contribute to coordinated cycles of receptor activation and oscillations in intracellular Ca2+ levels (39). Multiple IP3-sensitive Ca2+ pools, regulated by specific receptor subtypes, have also been proposed to mediate the differential responsiveness of cells to IP3 (35). In addition, IP3Rs have been associated with the apoptotic process (40). Thus, the differential expression of IP3Rs likely contributes to the signal transduction mechanisms of cells that exquisitely regulate Ca2+mobilization and related cellular events.

The expression of IP3R-I, -II, and -III mRNAs in mouse islets and insulinoma cell lines has been previously described, and IP3R-III is the major isoform in rat insulinoma cells (13, 15, 26, 27). The expression of the IP3R isoforms in tissues, including pancreatic islets and RINm5F insulinoma cells, is responsive to stimuli such as glucose/feeding/diabetes, phorbol ester, vitamin D3, retinoic acid, and cholecystokinin, among others (7, 13, 16, 41). However, little is known of the regulation of transcription of these receptors. The present study is the first to demonstrate that glucose stimulation of isolated islets has a profound and complex effect on the expression of IP3R isoform mRNAs and protein levels. Glucose, at a maximally insulin secretagogic concentration, evoked a rapid, marked, and selective increase in islet IP3R-III mRNA. The early response to glucose was not uniform among the isoforms and suggested a selective regulatory mechanism(s). The biphasic nature of the glucose response was noted after 2 h of glucose stimulation when IP3R-III mRNA fell to less than control levels, whereas IP3R-I and -II mRNAs remained unaffected. KIC, an insulin secretagogue and mitochondrial fuel which bypasses glycolysis, affected early IP3R mRNA expression similarly to glucose, suggesting that changes in the energy state of the islet mediate changes in IP3R expression. In contrast, 2-DOG, which is not metabolized and would be expected to inhibit glucose phosphorylation and metabolism, failed to affect IP3R mRNA expression. Therefore, an event(s) distal to glucose phosphorylation seems to play a role in IP3R-III induction. The maintenance of insulin mRNA levels in islets has also been found to correlate with metabolic flux (42). In contrast to islets, the rank order of abundance of pancreatic acinar tissue IP3R mRNA was: type I > type II > type III, as described previously (15). Moreover, pancreatic acinar tissue IP3R isoform expression was not affected by glucose stimulation. Therefore, it is unlikely that remnants of acinar tissue in the islet isolates contributed to islet IP3R mRNA expression responses.

Insight regarding the mechanisms mediating the glucose response was gleaned from experiments demonstrating that inhibition of transcription prevented the 30-min glucose-induced increase in IP3R-III mRNA, whereas the inhibition of translation had no effect on this phase of stimulation. In contrast, postranslational events seem to mediate the reduced level of IP3R-III mRNA observed after 2 h, but not 30 min, of glucose stimulation. Proteins synthesized during glucose stimulation may affect IP3R-III gene transcriptional events directly, or they may participate in destabilization of the mRNA. The induction of labile proteins has been shown to accelerate the degradation of other species of mRNA (43, 44). Alternatively, certain glucose-desensitizing events in islets, after continuous glucose stimulation (18, 19, 45, 46), may block the glucose effects modulating the transcriptional changes in IP3R-III. Future studies will attempt to answer these important questions. The regulation of IP3R-III transcripts in cultured islets may have functional significance, because this is the most abundant receptor isoform in pancreatic islets and ß-cell lines (13, 26), and IP3 binding to this receptor is sensitive to changes in Ca2+ levels (36, 37), making it an excellent candidate for modulating ß-cell Ca2+ oscillations.

It was also observed that IP3R-II and -III and glyceraldehyde 3-phosphate dehydrogenase mRNA levels were elevated during long-term culture of islets at basal glucose levels. The growth of fibroblasts in 7-day cultured islets may account for the increase in basal IP3R mRNA levels, because Ara-C, a pyrimidine antimetabolite and inhibitor of DNA synthesis and cell proliferation, eliminates the growth of fibroblasts (34) and restored IP3R mRNA levels in 7-day islets, to levels observed in fresh islets. Additional support for the fibroblast proliferation theory accounting for the elevation of basal mRNAs in islets is the report that IP3R-II and -III, but not -I, are expressed in COS-1 cells, a fibroblast-like cell-line (26). And, indeed, only islet IP3R-II and -III mRNAs responded to culture. Alternatively, islet cell turnover may somehow contribute to this phenomenon.

During long-term islet culture, glucose stimulation was associated with a significant decrease in IP3R-III mRNA, whereas IP3R-II mRNA levels increased. These changes in the transcription level of IP3R-II and -III mRNAs seemed to be directly related to glucose stimulation, rather than cell proliferation, because similar changes occurred irrespective of the presence of Ara-C. The specificity of the glucose response was reflected by the lack of response in glyceraldehyde 3-phosphate dehydrogenase mRNA expression. The mechanism(s) accounting for the late up-regulation of IP3R-II mRNA and the down-regulation of IP3R-III mRNA in response to glucose is unknown. However, it is possible that changes in transcription rate or mRNA stability play a role.

Correlation between IP3R mRNA expression in cultured islets and islets isolated from a well-characterized rat model of in vivo hyperglycemia was also sought. Hyperglycemia, associated with the rat PPx model, has been previously reported to affect insulin secretion (28). In the present study, hyperglycemia in the PPx model correlated with increased expression of islet IP3R-II mRNA. These results suggest that islet IP3R-II is regulated by glucose, both during culture and in vivo, although other factors affecting the islet after PPx cannot be ruled out at this time. The lack of significant effect of PPx on islet IP3R-III mRNA expression correlates with the lack of stimulatory effect of glucose on this isoform in cultured islets, and it supports the hypothesis that IP3R-III is acutely regulated by glucose and is likely to play a role in short-term (rather than long-term) modulation of insulin secretion. The reduction in IP3R-III mRNA in glucose-stimulated cultured islets, and lack of glucose response in this isoform in highly hyperglycemic PPx rat islets, suggest that a reduced level of gene transcripts may regulate the receptor levels in islet cells. A reduction in gene transcripts for the glucose transporter and insulin has been previously associated with hyperglycemia in the PPx model, and related to a reduction in transcription factor PDX-1 (29, 47). Perhaps key transcription factors sensitive to glucose play a role in regulation of IP3R gene transcription.

In addition to glucose, osmotic changes altered IP3R mRNA expression during long-term culture. The results showed that up to 15.5 mM (mannitol plus glucose) and higher was associated with modest increases in the relative levels of IP3R isoform mRNA. Thus, it is important to recognize the potential contribution of osmolar and metabolic regulation of IP3R mRNA expression. Osmotic regulation has been described as regulating gene expression in mammalian cells, yeast, and bacteria (48). It is not known whether osmotic potential played any role in the increased expression of IP3R-II mRNA in PPx rat islets.

IP3R protein levels also correlated with mRNA expression, albeit protein expression lagged mRNA level changes, as expected. This is the first study demonstrating an acute effect of glucose stimulation to increase islet IP3R-III levels. Up-regulation of IP3R-III may modulate calcium regulation in islet cells, because IP3R-III has been reported to function as a capacitative calcium entry channel, specifically localizing to the plasma membrane and facilitating calcium entry across this membrane (49). The early increase in islet IP3R-III also correlates with elevated ß-cell calcium levels during glucose stimulation (50). Previous studies indicated that IP3 releases calcium from nonmitochondrial storage sites in ß-cells, including endoplasmic reticulum (24, 51). An IP3-sensitive calcium store in HIT insulinoma cells was also located to a cell-surface derived membrane vesicle fraction distinct from the microsomal fraction (25). The latter studies provide evidence supporting the hypothesis of a calcium entrance compartment for ß-cells, which allows for filling of calcium storage pools through plasma membrane-associated microvilli.

In a biphasic manner, chronic glucose stimulation down-regulated IP3R-III levels in parallel with the decline in mRNA. Previous studies with this islet model have shown that chronic glucose stimulation also down-regulates IP3 production, which may further contribute to compromised glucose-stimulated insulin secretion (19, 52). Bell and co-workers (7) and this laboratory (27) recently reported that glucose stimulation of rat RINm5F and mouse ßHC9 insulinoma cells resulted in an increase in IP3R-III protein levels but that these levels were maintained throughout several days of glucose stimulation. However, tumor cell transcriptional/translational control may be expected to differ from that of primary cells. The reduction in IP3R-III mRNA during long-term glucose stimulation suggests that this is a limiting event in islet IP3R expression, although receptor degradation and translational control cannot be ruled out. In preliminary studies, IP3R-III mRNA levels were similar in 7-day glucose-stimulated cultured islets incubated for an additional 2–24 h in the presence or absence of cycloheximide (personal observations, B. Lee), suggesting that postranslational events are not primarily responsible for the decline in this mRNA during long-term glucose stimulation.

Potentially, each of the IP3Rs can be down-regulated during stimulation of phosphoinositide hydrolysis, IP3 production, and IP3R stimulation (26, 39, 53, 54). However, the down-regulation of IP3R-II is limited, compared with isoforms -I and -III in some cell types, because of resistance to degradation (26). This is the first study demonstrating the unique response of IP3R-II levels, which increased during chronic (but not short-term) glucose stimulation and correlated with mRNA expression. The biological relevance of this observation is not known.

Little is known about the precise mechanism of regulation of IP3Rs. Although elevated blood glucose levels seem to increase the levels of IP3R-III in islets of diabetic rats and rats refed after a period of fasting (7), and in vitro glucose stimulation increased IP3R-III expression in rat RINm5F insulinoma cells (7, 27), the regulation of IP3R-I and -II have not been previously reported. In addition, controversy exists regarding the subcellular localization of the IP3R-III because of the lack of specificity of the polyclonal antibody used in some studies (7, 55). Some receptor isoforms may compensate for the loss of others, perhaps in different subcellular locations, where they have specialized functions such as the nucleus, endoplasmic reticulum, plasma membrane, and secretory granules (7, 9, 10, 14, 56). For instance, overexpression of the IP3R-III in insulin-secreting TC-3 cells resulted in the creation of a unique IP3 responsive Ca2+ pool in secretory granules (57). Heterotrimeric receptor complexes also exist for IP3R-I, -II, and -III (16), suggesting that the ratios of the receptor subtypes modulate agonist-selective changes in activity.

In summary, the present studies are the first to demonstrate that islet IP3R-I, -II, and -III transcripts are differentially regulated in response to glucose stimulation. These results may be a reflection of the different cell types within the islets, including approximately 70–90% insulin-secreting ß-cells, and 10–30% non-ß-cells (58), and their responses to glucose. The relative expression levels of IP3R subtypes among islet {alpha}-, {delta}-, and pancreatic polypeptide-secreting PP-cells remain to be characterized. Moreover, the rapid changes in IP3R-III mRNA expression, in response to glucose, suggest that immediate-early transcription factor genes couple the metabolic and nuclear responses. Immediate-early genes such as c-fos, c-jun, and Egr-1 encode proteins including growth factors, receptors, cytoskeletal proteins, and transcription factors (59). In the insulin-secreting ß-cell line, INS-1, the activation of mitogen-activated protein kinase has been correlated with glucose induction of early response genes junB, nur77, and zif268 (60). Signal transduction pathways that modulate early gene regulation include the protein kinase C pathway, but cAMP also potentiates the response in INS-1 cells (59, 60). Both protein kinase C and cAMP mediate glucose responses in islets (2, 61, 62) and may participate in IP3R gene regulation. However, unlike some early response genes that require glucose phosphorylation but not metabolism for the inductive effect of glucose (63, 64), IP3R-III induction required glucose metabolism or mitochondrial stimulation. The data also suggest that changes in the pattern of the IP3R transcripts play a role in the glucose-induced desensitization of insulin secretion that occurs in this in vitro model (19). Thus, modulation of islet IP3R gene expression is likely to play a role in acute responses to metabolic stimulation, as well as to chronic adaptive responses in glucose-stimulated insulin secretion.


   Acknowledgments
 
The authors would like to thank Jill Platten and Wendy Hasenkamp for their technical assistance on aspects of this project.


   Footnotes
 
1 This work was supported, in part, by grants (to S.G.L.) from the National Institutes of Health (DK-25705) and the American Diabetes Association (R-A48). Back

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

3 Recipient of a fellowship from the Belgian American Educational Foundation. Back

Received October 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wollheim CB, Sharp GWG 1981 Regulation of insulin release by calcium. Physiol Rev 61:914–973[Free Full Text]
  2. Laychock SG 1990 Glucose metabolism, second messengers and insulin secretion. Life Sci 47:2307–2316[CrossRef][Medline]
  3. Hellman B, Gylfe E, Wesslen N 1986 Inositol 1,4,5-trisphosphate mobilizes glucose-incorporated calcium from pancreatic islets. Biochem Int 13:383–389[Medline]
  4. Laychock SG 1983 Identification and metabolism of polyphosphoinositides in isolated islets of Langerhans. Biochem J 216:101–106[Medline]
  5. Vadakekalam J, Rabaglia ME, Chen Q-H, Metz SA 1996 Role for GTP in glucose-induced phospholipase C activation in pancreatic islets. Am J Physiol 271:E85–E95
  6. Berridge MJ 1993 Inositol trisphosphate and calcium signalling. Nature 361:315–325[CrossRef][Medline]
  7. Blondel O, Moody MM, Depaoli AM, Sharp AH, Ross CA, Swift H, Bell GI 1994 Localization of inositol trisphosphate receptor subtype 3 to insulin and somatostatin secretory granules and regulation of expression in islets and insulinoma cells. Proc Natl Acad Sci USA 91:7777–7781[Abstract/Free Full Text]
  8. Ross CA, Meldolesi J, Milner TA, Satoh T, Supattapone S, Snyder SH 1989 Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons. Nature 339:468–470[CrossRef][Medline]
  9. Khan AA, Steiner JP, Klein MG, Schneider MF, Snyder SH 1992 IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Science 257:815–818[Abstract/Free Full Text]
  10. Malviya AN, Rogue P, Vincendon G 1990 Stereospecific inositol 1,4,5-[32P]trisphosphate binding to isolated rat liver nuclei: evidence for inositol trisphosphate receptor-mediated calcium release from nucleus. Proc Natl Acad Sci USA 87:9270–9274[Abstract/Free Full Text]
  11. Mignery GA, Südhof TC 1990 The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor. EMBO J 9:3893–3898[Medline]
  12. Südhof TC, Newton CL, Archer III BT, Ushkaryov YA, Mignery GA 1991 Structure of a novel InsP3 receptor. EMBO J 10:3199–3206[Medline]
  13. Blondel O, Takeda J, Janssen H, Seino S, Bell GI 1993 Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues. J Biol Chem 268:11356–11363[Abstract/Free Full Text]
  14. Ross CA, Danoff SK, Schell MJ, Snyder SH, Ullrich A 1992 Three additional inositol 1,4,5-trisphosphate receptors: molecular cloning and differential localization in brain and peripheral tissues. Proc Natl Acad Sci USA 89:4265–4269[Abstract/Free Full Text]
  15. De Smedt H, Missiaen L, Parys JP, Bootman MD, Mertens L, Van Den Bosch L, Casteels R 1994 Determination of relative amounts of inositol trisphosphate receptor mRNA isoforms by ratio polymerase chain reaction. J Biol Chem 269:21691–21698[Abstract/Free Full Text]
  16. Wojcikiewicz RJH, He Y 1995 Type I, II and III inositol 1,4,5-trisphosphate receptor co-immunoprecipitation as evidence for the existence of heterotrimeric receptor complexes. Biochem Biophys Res Commun 213:334–341[CrossRef][Medline]
  17. Anello M, Rabuazzo AM, Degano C, Caltabiano V, Patane G, Vigneri R, Purrello F 1996 Fast reversibility of glucose-induced desensitization in rat pancreatic islets: evidence for an involvement of ionic fluxes. Diabetes 45:502–506[Abstract]
  18. Leahy JL, Weir GC 1988 Evolution of abnormal insulin secretory responses during 48-h in vivo hyperglycemia. Diabetes 37:217–222[Abstract]
  19. Xia M, Laychock SG 1993 Insulin secretion, myo-inositol transport and Na+,K+-ATPase activity in glucose-desensitized rat islets. Diabetes 42:1392–1400[Abstract]
  20. Bolaffi JL, Bruno L, Heldt A, Grodsky GM 1988 Characteristics of desensitization of insulin secretion in fully in vitro systems. Endocrinology 122:1801–1808[Abstract]
  21. Boschero AC, Bordin S, Herchuelz A, Lebrun P 1990 Effects of glucose on 45Ca2+ outflow, cytosolic Ca2+ concentration and insulin release from freshly isolated and cultured adult rat islets. Cell Calcium 11:603–609[CrossRef][Medline]
  22. Iwashima Y, Pugh W, Depaoli AM, Takeda JJ, Seino S, Bell GI, Polonsky KS 1993 Expression of calcium channel mRNAs in rat pancreatic islets and down-regulation after glucose infusion. Diabetes 42:948–955[Abstract]
  23. Wolf BA, Florholmen J, Turk J, McDaniel ML 1988 Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion. J Biol Chem 263:3565–3575[Abstract/Free Full Text]
  24. Biden T, Prentki M, Irvine RF, Berridge MJ, Wollheim CB 1984 Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ from permeabilized insulin-secreting cells. Biochem J 223:467–473[Medline]
  25. Lange K, Brandt U 1993 The IP3-sensitive calcium store of HIT cells is located in a surface-derived vesicle fraction. FEBS Lett 320:183–188[CrossRef][Medline]
  26. Wojcikiewicz RJH 1995 Type I, II and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J Biol Chem 270:11678–11683[Abstract/Free Full Text]
  27. Lee B, Bradford PG, Laychock SG 1998 Characterization of inositol 1,4,5-trisphosphate receptor isoform mRNA expression and regulation in rat pancreatic islets, RINm5F cells and ßHC9 cells. J Mol Endocrinol 21:31–39[Abstract]
  28. Bonner-Weir S, Trent DF, Weir GC 1983 Partial pancreatectomy in the rat and subsequent defect in glucose-induced insulin release. J Clin Invest 71:1544–1553
  29. Zangen DH, Bonner-Weir S, Lee CH, Latimer JB, Miller CP, Habener JF, Weir GC 1997 Reduced insulin, GLUT2, and IDX-1 in ß-cells after partial pancreatectomy. Diabetes 46:258–264[Abstract]
  30. Montana E, Bonner-Weir S, Weir GC 1993ß-cell mass and growth after syngeneic islet cell transplantation in normal and streptozotocin-diabetic C57BL/6 mice. J Clin Invest 91:780–787
  31. Mignery GA, Newton CL, Archer III BT, Südhof TC 1990 Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem 265:12679–12685[Abstract/Free Full Text]
  32. Bonner-Weir S, Trent DF, Honey RN, Weir GC 1981 Responses of neonatal rat islets to streptozotocin: limited B-cell regeneration and hyperglycemia. Diabetes 30:64–69[Abstract]
  33. Bonner-Weir S, Deery D, Leahy JL, Weir GC 1989 Compensatory growth of pancreatic B-cells in adult rats after short-term glucose infusion. Diabetes 38:49–53[Abstract]
  34. Lee VM, Shelanski ML, Greene LA 1980 Characterization of antisera raised against cultured rat sympathetic neurons. Neuroscience 5:2239–2245[CrossRef][Medline]
  35. Newton CL, Mignery GA, Südhof TC 1994 Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J Biol Chem 269:28613–28619[Abstract/Free Full Text]
  36. Cardy TJA, Traynor D, Taylor CW 1997 Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+. Biochem J 328:785–793
  37. Yoneshima H, Miyawaki A, Michikawa T, Furuichi T, Mikoshiba K 1997 Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors. Biochem J 322:591–596
  38. Cardy TJ, Taylor CW 1998 A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors. Biochem J 334:447–455
  39. Hajnoczky G, Thomas AP 1994 The inositol trisphosphate calcium channel is inactivated by inositol trisphosphate. Nature 370:474–477[CrossRef][Medline]
  40. Marks AR 1997 Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 272:H597–H605
  41. Kirkwood K, Dziak R, Bradford PG 1996 Inositol trisphosphate receptor gene expression and hormonal regulation in osteoblast-like cell lines and primary osteoblastic cell cultures. J Bone Miner Res 11:1889–1896[Medline]
  42. Welsh M, Brunstedt J, Hellerstrom C 1986 Effects of D-glucose, L-leucine, and 2-ketoisocaproate on insulin mRNA levels in mouse pancreatic islets. Diabetes 35:228–231[Abstract]
  43. Attardi B, Winters SJ 1993 Decay of follicle-stimulating hormone-beta messenger RNA in the presence of transcriptional inhibitors and/or inhibin, activin, or follistatin. Mol Endocrinol 7:668–680[Abstract]
  44. Quintela M, Senaris RM, Dieguez C 1997 Transforming growth factor-ßs inhibits somatostatin messenger ribonucleic acid levels and somatostatin secretion in hypothalamic cells in culture. Endocrinology 138:4401–4409[Abstract/Free Full Text]
  45. Laychock SG 1995 Impaired cyclic AMP response to stimuli in glucose-desensitized rat pancreatic islets. Mol Cell Endocrinol 113:19–28[CrossRef][Medline]
  46. Leahy JL, Cooper HE, Deal DA, Weir GC 1986 Chronic hyperglycemia is associated with impaired glucose influence on insulin secretion. A study in normal rats using chronic in vivo glucose infusions. J Clin Invest 77:908–915
  47. Weir GC, Sharma A, Zangen DH, Bonner-Weir S 1997 Transcription factor abnormalities as a cause of beta cell dysfunction in diabetes: a hypothesis. Acta Diabetol 34:177–184[CrossRef][Medline]
  48. Burg MB, Kwon ED, Kültz D 1996 Osmotic regulation of gene expression. FASEB J 10:1598–1606[Abstract]
  49. Putney Jr JW 1997 Type 3 inositol 1,4,5-trisphosphate receptor and capacitative calcium entry. Cell Calcium 21:257–261[CrossRef][Medline]
  50. Ashcroft FM, Proks P, Smith PA, Ammala C, Bokvist K, Rorsman P 1994 Stimulus-secretion coupling in pancreatic beta cells. J Cell Biochem [Suppl] 55:54–65
  51. Prentki M, Corkey BE, Matschinsky FM 1985 Inositol 1,4,5-trisphosphate and the endoplasmic reticulum Ca2+ cycle of a rat insulinoma cell line. J Biol Chem 260:9185–9190[Abstract/Free Full Text]
  52. Xia M, Hokin LE, Laychock SG 1994 Rat pancreatic islets cultured at hyperglycemic glucose concentrations have altered phosphoinositide turnover, inositol phosphate production, and ATP levels. Endocrine 2:741–747
  53. Qian N-X, Russell M, Buhl AM, Johnson GL 1994 Expression of GTPase-deficient G alpha 16 inhibits Swiss 3T3 cell growth. J Biol Chem 269:17417–17423[Abstract/Free Full Text]
  54. Honda Z, Takano T, Hirose N, Suzuki T, Muto A, Kume S, Mikoshiba K, Itoh K, Shimizu T 1995 Gq pathway desensitizes chemotactic receptor-induced calcium signaling via inositol trisphosphate receptor down-regulation. J Biol Chem 270:4840–4844[Abstract/Free Full Text]
  55. Ravazzola M, Halban PA, Orci L 1996 Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited. Proc Natl Acad Sci USA 93:2745–2748[Abstract/Free Full Text]
  56. Yoo SJ, Albanesi JP 1990 Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles. J Biol Chem 265:13446–13448[Abstract/Free Full Text]
  57. Blondel O, Bell GI, Moody M, Miller RJ, Gibbons SJ 1994 Creation of an inositol 1,4,5-trisphosphate-sensitive Ca2+ store in secretory granules of insulin-producing cells. J Biol Chem 269:27167–27170[Abstract/Free Full Text]
  58. Hellerstrom C 1977 Growth pattern of pancreatic islets of animals. In: Volk B, Wellmann KF (eds) The Diabetic Pancreas. Plenum Press, New York, pp 61–97
  59. Gashler A, Sukhatme VP 1995 Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol 50:191–224[Medline]
  60. Frödin M, Sekine N, Roche E, Filloux C, Prentki M, Wollheim CB, Van Obberghen E 1995 Glucose, other secretagogues, and nerve growth factor stimulate mitogen-activated protein kinase in the insulin-secreting ß-cell line, INS-1. J Biol Chem 270:7882–7889[Abstract/Free Full Text]
  61. Tian YM, Urquidi V, Ashcroft SJ 1996 Protein kinase C in beta-cells: expression of multiple isoforms and involvement in cholinergic stimulation of insulin secretion. Mol Cell Endocrinol 119:185–193[CrossRef][Medline]
  62. Zawalich WS, Zawalich KC 1996 Signal transduction in isolated islets from the ob/ob mouse - enhanced sensitivity of protein kinase C to stimulation. Biochem Biophys Res Commun 223:618–623[CrossRef][Medline]
  63. Brun T, Roche E, Kim KH, Prentki M 1993 Glucose regulates acetyl-CoA carboxylase gene expression in a pancreatic beta-cell line (INS-1). J Biol Chem 268:18905–18911[Abstract/Free Full Text]
  64. Marie S, Diaz-Guerra MJ, Miquerol L, Kahn A, Iynedjian PB 1993 The pyruvate kinase gene as a model for studies of glucose-dependent regulation of gene expression in the endocrine pancreatic beta-cell type. J Biol Chem 268:23881–23890[Abstract/Free Full Text]



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