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An Insulinotropic Effect of Vitamin D Analog with Increasing Intracellular Ca²⁺ Concentration in Pancreatic ß-Cells through Nongenomic Signal Transduction¹

Department of Metabolism and Clinical Nutrition (M.K., S.F., E.M., M.N., J.F., Y.T., Y.O., Y.S.), Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan; Third Department of Internal Medicine (H.I.), Kyorin University School of Medicine, Mitaka, Tokyo 181-8611 Japan; and Department of Biochemistry (A.W.N.), University of California, Riverside, California 92521

Address all correspondence and requests for reprints to: Mariko Kajikawa M.D., Department of Metabolism & Clinical Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: kajikawa{at}metab.kuhp.kyoto-u.ac.jp

	Abstract

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The effect of 1,25-dihydroxylumisterol₃ (1,25(OH)₂lumisterol₃) on insulin release from rat pancreatic ß-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1,25(OH)₂lumisterol₃, a specific agonist of mVDR, dose-dependently augmented 16.7 mM glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i), though not increasing Ca²⁺ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1ß,25-dihydroxyvitamin D₃ (1ß,25(OH)₂D₃), or by a blocker of voltage-dependent Ca²⁺ channels, nitrendipine. Moreover, both [Ca²⁺]_i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1,25(OH)₂lumisterol₃, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca²⁺ influx through voltage-dependent Ca²⁺ channels on the plasma membrane, being also linked to metabolic signals derived from glucose in pancreatic ß-cells. However, further investigations will be needed to discuss physiologically the meaning of insulinotropic effects of vitamin D through mVDR.

	Introduction

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PANCREATIC ß-cells have been found to have a receptor for vitamin D and to be a target of vitamin D (1, 2). Although there are many studies on the effect of vitamin D on insulin secretion (3, 4, 5, 6, 7, 8), the role of vitamin D in ß-cell function is not yet clear. Most investigation has been restricted to the chronic effects of vitamin D and its analogs on insulin secretion, those possibly mediated through a signal transduction system involving the nuclear vitamin D receptor (nVDR) (9). On the other hand, rapid biological responses to vitamin D have been reported in various types of tissues and cells. The stimulation within a few minutes of intestinal Ca²⁺ transduction through plasma membrane, transcaltachia, involves the opening of Ca²⁺ channels (10). In addition, a stimulatory effect on the opening of voltage-dependent Ca²⁺ channels (VDCC) in rat osteosarcoma cells (11, 12) and an increase in the intracellular free Ca²⁺ concentration of osteoblast-like cells (13), as well as other cell types (14, 15, 16), also have been reported. These effects are thought to be mediated by nongenomic action of the hormone, and the membrane vitamin D receptor (mVDR) has been demonstrated to be involved in the signal transduction associated with transcaltachia in basal-lateral membrane of chick intestinal epithelium (17).

The effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), a biologically active metabolite of vitamin D, and its analogs have been examined regarding binding to nVDR and mVDR, through which they might induce genomic and nongenomic responses, respectively. These studies revealed that 6-s-cis-locked analogs of 1,25(OH)₂D₃ are preferred for nongenomic action through putative signal transduction by binding to mVDR (12, 13, 16, 17, 18, 19, 20, 21, 22, 23). Among these 6-s-cis analogs, 1,25-dihydroxylumisterol₃ (1,25(OH)₂lumisterol₃) can efficiently induce transcaltachia in intestinal epithelium and also can stimulate Ca²⁺ uptake in the osteosarcoma cell line via mVDR. This analog, however, failed to activate the genomic action, and it possessed only very weak ability to bind to nVDR (17, 21, 22, 23). On the other hand, another analog, 1ß,25-dihydroxyvitamin D₃ (1ß,25(OH)₂D₃) which is an A-ring diastereomer of 1,25(OH)₂D₃, has been found to block rapid responses induced by 1,25(OH)₂D₃ or 1,25(OH)₂lumisterol₃ and is recognized as a specific antagonist of the nongenomic action (15, 24, 25, 26).

There is only a small number of investigations concerning the rapid nongenomic effect of vitamin D on pancreatic ß-cell function. Among these studies, Sergeev and Rhoten (27) have reported that the administration of 1,25(OH)₂D₃ evoked oscillations of intracellular Ca²⁺ in a pancreatic ß-cell line within a few minutes. However, the detailed mechanism of the nongenomic effect of vitamin D through signal transduction via mVDR in pancreatic ß-cells remains unclear. In this study, we have compared the effect of two vitamin D analogs, 1,25(OH)₂lumisterol₃ and 1ß,25(OH)₂D₃, in the rapid action of vitamin D on insulin secretory mechanisms, and we find evidence of nongenomic stimulation by vitamin D on insulin secretion in normal rat pancreatic ß-cells.

	Materials and Methods

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Measurement of insulin release from isolated rat pancreatic islets (28)
Male Wistar rats, weighing 180–230 g, were fed standard lab chow ad libitum, with free access to water, in an air-conditioned room with a 12-h light, 12-h dark cycle, until used in the experiments. Islets of Langerhans were isolated from the rats by collagenase digestion (29). Insulin release from intact islets was monitored using either static incubation or a perifusion system. For static incubation experiments, the islets were preincubated at 37 C for 30 min in Krebs-Ringer bicarbonated buffer (KRBB) supplemented with 3.3 mM glucose, 10 mM HEPES (pH 7.4), and 0.1% BSA. Groups of 5 islets were then batch-incubated for 30 min in 0.7 ml KRBB with test materials. At the end of the incubation period, islets were pelleted by centrifugation (10,000 x g, 3 min), and aliquots of the buffer were sampled. For perifusion experiments, groups of 15 islets were placed in parallel chambers (400 µl each) of a perifusion apparatus and perifused with the same medium at a rate of 0.7 ml/min at 37 C (28). Perifusate samples were collected at the times indicated in the figures. The amount of immunoreactive insulin was determined by RIA, using rat insulin as standard (30). Experiments using the same protocol were repeated 3 times to ascertain reproducibility.

Measurement of insulin release from electrically permeabilized islets (31)
After preincubation, as described above, the islets were washed twice in cold potassium asparate buffer (KA buffer) containing 140 mM KA, 7 mM MgSO₄, 5 mM ATP, 3.3 mM glucose, 2.5 mM EGTA, 30 mM HEPES, and 0.5% BSA (pH 7.0), with CaCl₂ to a Ca²⁺ concentration of 30 nM. The islets were then permeabilized by high voltage discharge (four exposures, each of 450 µsec duration, to an electrical field of 4.0 kV/cm). Groups of five electrically permeabilized islets were then batch-incubated in 0.4 ml KA buffer with various concentrations of Ca²⁺ and test materials, for 30 min at 37 C. At the end of the incubation period, islets were pelleted, and aliquots of the buffer were sampled for immunoreactive insulin, as described above. Experiments using the same protocol were repeated three times to ascertain reproducibility.

Measurement of intracellular Ca²⁺ concentration (28)
Isolated islets were washed in PBS and incubated with 0.25% trypsin and 1 mM EDTA solution (Life Technologies, Inc., Grand Island, NY) for 3 min at 37 C. They were washed in cold PBS again, placed on small glass coverlips (15 x 4 mm), and incubated in KRBB supplemented with 3.3 mM glucose and 0.1% BSA, in humidified air containing 5% CO₂ at 37 C, until used for experiments. Then 1 µM fura-2/acetoxymethyl ester (fura-2/AM; Molecular Probes, Inc., Eugene, OR) was loaded on freshly dispersed islet cells for 30 min at 37 C. A heat-controlled chamber on the stage of an inverted microscope kept at 37 ± 1 C was superfused with KRBB supplemented with 3.3 mM glucose and 10 mM HEPES adjusted to pH 7.4. Ratiometry of emission light (510 nm) elicited by dual-wave excitation lights (340 and 380 nm) was performed on an ARGUS-50 images analyzing system (Hamamatsu Photonics, Hamamatsu, Japan). The 340-nm (f340) and 380-nm (f380) fluorescence signals were detected every 10 sec, and the ratios (f340/f380) were calculated. In vivo calibration was performed, and f340/f380 was converted into calibrated values of intracellular calcium concentration ([Ca²⁺]_i).

Materials
Diazoxide, nitrendipine, mannoheptulose, and KA were purchased from Sigma Chemical Co. (St. Louis, MO), and ATP was purchased from Kohjin (Tokyo, Japan); all other agents were obtained from Nacalai Tesque (Tokyo, Japan). Vitamin D metabolites were first prepared in ethanol and diluted to 1:1000 with buffer, and the same concentration of ethanol was added to control.

Statistical analysis
Results are expressed as means ± SE. Statistical significance was evaluated by unpaired Student’s t test or paired t test. P < 0.05 was considered significant.

	Results

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Effect of 1,25(OH)₂lumisterol₃ on glucose-induced insulin release from pancreatic islets
1,25(OH)₂lumisterol₃ (JN) augmented 16.7 mM glucose-induced insulin release from rat islets in a dose-dependent manner (Fig. 1). 1,25(OH)₂lumisterol₃ (JN) (10^-8 M) augmented high concentrations (more than 11.1 mM) of glucose-induced insulin release; however, at lower concentrations (below 8.3 mM), augmentation was not observed (Table 1a). On the other hand, in the presence of 200 µM diazoxide, which inhibits ATP-sensitive K⁺ (K_ATP) channel closure and produces hyperpolization, 1,25(OH)₂lumisterol₃ (JN) failed to augment insulin secretion, even with high concentrations of glucose (Table 1b).

View larger version (20K): [in this window] [in a new window]   Figure 1. Concentration-dependent effect of 1,25(OH)2lumisterol3 (JN) on 16.7 mM glucose-induced insulin secretion from rat pancreatic islets. Values are expressed as means ± SE of five determinations in the same experiment. *, P < 0.05 vs. control (16.7 mM glucose alone).

View this table: [in this window] [in a new window]   Table 1. Effect of 10-8 M 1,25(OH)2 lumisterol3 (JN) on insulin release in the presence of various concentrations of glucose and various concentrations of K+ with or without 200 µM diazoxide

View larger version (28K): [in this window] [in a new window]   Figure 2. Effects of 1,25(OH)2lumisterol3 (JN) on insulin release in perifusion experiments. Values are expressed as means ± SE. G, glucose; A, Time course effect of 10-8 M JN on biphasic 16.7 mM glucose-induced insulin release. Two groups of islets were perifused for 30 min with 3.3 mM glucose (G3.3) and for 30 min with 16.7 mM glucose (G16.7) with (closed circle, n = 6) or without (open circle, n = 6) 10-8 M JN. JN was introduced 15 min before the period of 16.7 mM glucose stimulation. The withdrawal effect of JN for 30 min is also shown. *, P < 0.05 vs. control. B, Effect of 1ß,(OH)2D3 (HL) on JN-induced augmentation of insulin release. HL (10-8 M) was added to the solution 30 min before the addition of 10-8 M JN [open circle, control (n = 5); closed circle, JN (n = 5); open triangle, JN plus HL (n = 5)]. *, P < 0.05 vs. 16.7 mM control; , P < 0.05 vs. 16.7 mM glucose and 10-8 M HL.

Effect of 1,25(OH)₂lumisterol₃ on insulin release in the presence of glucose, diazoxide, and a depolarizing concentration of K⁺

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of 10-8 M 1,25(OH)2lumisterol3 (JN) on insulin secretion in the presence of a depolarizing concentration (30 mM) of K+ and 200 µM diazoxide. A, Effects of JN on insulin secretion in the presence of 30 mM K+ and 200 µM diazoxide. Glucose was present at 3.3 mM or at 16.7 mM. The effect of 10-8 M 1ß,(OH)2D3 (HL), 30 µM nitrendipine, or 20 mM mannoheptulose on the enhancement of insulin release induced by 10-8 M JN in the presence of 200 µM diazoxide, 30 mM K+, and 16.7 mM glucose are also indicated. All experimental conditions are indicated below the graph. §, P < 0.05 vs. control (16.7 mM, 30 mM K+, and 200 µM diazoxide). Values are expressed as means ± SE of five determinations in the same experiment. B, Time course effect of 10-8 M JN on insulin release in the presence of 16.7 mM glucose, 200 µM diazoxide, and a depolarizing concentration of (30 mM) K+. JN was introduced 5 min after the period with 30 mM K+ [open circle, control (n = 5); closed circle JN (n = 5)]. *, P < 0.05 vs. control. Values are expressed as means ± SE of seven determinations in the same experiment. G, Glucose.

Effect of 1,25(OH)₂lumisterol₃ on insulin release in electrically permeabilized islets

View this table: [in this window] [in a new window]   Table 2. Effect of 10-8 M 1,25(OH)2 lumisterol3 (JN) on the Ca2+ dose response of insulin release from electrically permeabilized islets

Effect of 1,25(OH)₂lumisterol₃ on [Ca²⁺]_i response in pancreatic ß-cells

View larger version (26K): [in this window] [in a new window]   Figure 4. Fluorescence measurement of [Ca2+]i and response to 10-8 M 1,25(OH)2lumisterol3 (JN) in dispersed islet cells. G, glucose. A, [Ca2+]i increase induced by JN in the presence of 30 mM K+, 16.7 mM glucose, and 200 µM diazoxide. The glucose concentration was raised from 3.3 mM to 16.7 mM after 2 min. Cells that responded to 16.7 mM glucose were regarded as ß-cells. The trace during the first 10 min shows the average [Ca2+]i of 19 such cells. A depolarizing concentration (30 mM) of K+ and 200 µM diazoxide, with (open triangle) and without (open and closed circle) 10-8 M 1ß,(OH)2D3 (HL), were then introduced from 10 min. JN (10-8 M) was added from 15 min (closed circle and open triangle). Traces from 10–35 min indicate the average [Ca2+]i of 6 cells to which JN was administered, 6 cells to which JN and HL were administered, and 7 control cells. Vertical bars represent ± SE at 10, 11, 15, 20, 25, 30, and 35 min. *, P < 0.05 vs. 16.7 mM glucose, 30 mM K+, and 200 µM diazoxide; , P < 0.05 vs. 10-8 M JN, 10-8 M HL, 16.7 mM glucose, 30 mM K+, and 200 µM diazoxide. B, Effect of cessation of JN. After 10-min introduction of JN with the same protocol as A, JN was removed from the medium (n = 11). C, No effect of JN on the 30 mM K+-induced [Ca2+]i rise. In the presence of 3.3 mM glucose, 30 mM K+ was introduced from 2 min, then 10-8 M JN was added from 7 min. Traces indicate the average [Ca2+]i of 12 cells to which JN was administered (closed circle) and 14 control cells (open circle). Vertical bars represent ± SE at 0, 2, 5, 7, 15, 20, and 25 min. No significant differences, compared with control, were observed.

View larger version (22K): [in this window] [in a new window]   Figure 5. Effect of Ca2+ channel blockers on 1,25(OH)2lumisterol3 (JN)-induced [Ca2+]i increase. G, glucose. The glucose concentration was raised from 3.3 mM to 16.7 mM after 3 min. A depolarizing concentration (30 mM) of K+ and 200 µM diazoxide were introduced from 8 min, and 10-8 M JN at 13 min was then introduced (double circle) or not (open square). The trace (double circle) from 13–23 min shows the average [Ca2+]i of such cells, which were exposed to JN (21 cells in A, 20 cells in B). A, Nitrendipine suppressed JN-induced [Ca2+]i increase. Nitrendipine (30 µM) was added from 23 min (open circle and open square). The trace from 23–30 min indicates the average [Ca2+]i of 12 cells to which nitrendipine was administrated in the presence of JN (open circle) and 9 cells exposed to JN alone (closed circle). The time course of the average of 11 cells to which nitrendipine alone (without JN) was introduced as a control was also indicated (open square). Vertical bars represent ± SE at 13, 18, 23, 24, 28, and 30 min. B, Diphenylamine-2-carboxylate (DPC) had only a partial inhibitory effect on JN-induced [Ca2+]i increase. DPC (300 µM) was added from 23 min (open triangle). The trace from 23–33 min indicates the average [Ca2+]i of 8 cells to which DPC was administered (open triangle) and 12 control cells (JN alone) (closed circle). Vertical bars represent ± SE at 23, 24, and 30 min. *, P < 0.05 vs. JN alone.

	Discussion

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In the present study, we found that the vitamin D analog, 1,25(OH)₂lumisterol₃, enhanced high concentration of glucose-induced insulin release together with acceleration of Ca²⁺ influx in pancreatic ß-cells. This augmentation was observed within 15 min and was abolished rapidly after removal of the agent. In addition, the effect was completely abolished by the antagonist of nongenomic action (1ß,25(OH)₂D₃), strongly suggesting a nongenomic vitamin D action on insulin release that might be mediated by signal transduction via mVDR.

In pancreatic ß-cells, signals generated through intracellular glucose metabolism close the K_ATP channels on the plasma membrane. The resulting decrease in K⁺ conductance leads to depolarization of the membrane, with subsequent opening of VDCC. Ca²⁺ influx through these channels then increases, leading to a rise in [Ca²⁺]_i, which eventually triggers exocytosis of insulin granules (33). The secretory enhancement by 1,25(OH)₂lumisterol₃ was observed also in the presence of diazoxide (an opener of K_ATP channels), a depolarizing concentration of K⁺, and 16.7 mM glucose (which is known to produce so-called K_ATP channel-independent insulin release through metabolic signaling derived from glucose) (34, 35). On the other hand, both insulin release and [Ca²⁺]_i dynamics were not affected by 1,25(OH)₂lumisterol₃ in the presence of a basal concentration (3.3 mM) of glucose, indicating that 1,25(OH)₂lumisterol₃ itself is not a depolarizing agent, like sulfonylurea, which induces K_ATP channel closure to produce membrane depolarization.

1,25(OH)₂lumisterol₃, therefore, acts distal to membrane depolarization in glucose-induced insulin secretion, and its enhancement of insulin secretion could result from rising [Ca²⁺]_i or augmentation of Ca²⁺ efficacy in the exocytotic system. However, in the presence of a depolarizing concentration of K⁺ with diazoxide, 1,25(OH)₂lumisterol₃ did not enhance insulin release without a high concentration of glucose, and even with a high concentration of glucose, 1,25(OH)₂lumisterol₃ failed to enhance insulin release from diazoxide-hyperpolarized islets. 1,25(OH)₂lumisterol₃-enhanced insulin release, therefore, requires both activated glucose metabolism and plasma membrane depolarization, as suggested also by the observation that mannoheptulose prevents 1,25(OH)₂lumisterol₃ from enhancing insulin secretion, even in the depolarizing condition.

To determine whether this K_ATP channel-independent effect of 1,25(OH)₂lumisterol₃ on insulin release is attributable to the rising [Ca²⁺]_i in depolarized cells or to enhancement of Ca²⁺ efficacy in the exocytotic system of insulin granules, the effect of 1,25(OH)₂lumisterol₃ was examined on [Ca²⁺]_i in dispersed ß-cells and also on insulin release from electrically permeabilized islets in which [Ca²⁺]_i was clamped. [Ca²⁺]_i-clamped experiments showed that Ca²⁺ efficacy in the exocytotic apparatus distal to the Ca²⁺ rise is not affected by 1,25(OH)₂lumisterol₃, in accord with the finding that a calcium sensitizer (such as pimobendan) augmented insulin release in the presence of diazoxide and 30 mM K⁺, even with a basal level of (3.3 mM) glucose (28), whereas 1,25(OH)₂lumisterol₃ did not augment it under the same condition. The analog augmented the [Ca²⁺]_i rise in the presence of 16.7 mM glucose, diazoxide, and 30 mM K⁺. The time courses of [Ca²⁺]_i dynamics and insulin release in this condition were very similar (Fig. 3B and Fig. 4A), and the effects of 1,25(OH)₂lumisterol₃ on the [Ca²⁺]_i rise were also abolished by 1ß,25(OH)₂D₃. On the other hand, 1,25(OH)₂lumisterol₃ affected neither [Ca²⁺]_i nor insulin release under the condition of basal (3.3 mM) glucose, diazoxide, and 30 mM K⁺. 1,25(OH)₂lumisterol₃, therefore, probably enhances insulin release through mVDR, under a depolarizing condition of the plasma membrane in the presence of activated glucose metabolism.

The effect of 1,25(OH)₂lumisterol₃ on [Ca²⁺]_i dynamics and insulin secretion were delayed, although they were all expressed via mVDR. It seems that this phenomenon does not result from distributional delay of the agonist, because in the perifusion experiment shown in Fig. 2A, despite pretreatment of 1,25(OH)₂lumisterol₃ for 15 min before glucose stimulation, it also took approximately 10 min after stimulation with glucose for the effects to appear. Postreceptor signal transduction of 1,25(OH)₂lumisterol₃, therefore, may require a metabolically activated state for more than 10 min for the insulinotropic effect.

Sergeev and Rhoten (27) have reported that the voltage-independent Ca²⁺ channels (VICC) participate in vitamin D-induced [Ca²⁺]_i oscillation, as do the VDCC in RIN cells. It also has been reported that, in pancreatic ß-cells, the VICC play a part in the Ca²⁺ influx that is attenuated by the nonselective cation channel blocker, DPC (32). Using DPC to determine the participation of the VICC in augmentation of [Ca²⁺]_i influx by 1,25(OH)₂lumisterol₃, we found that DPC failed to abolish it completely, whereas nitrendi-pine, a blocker of VDCC, eliminated the effects of 1,25(OH)₂lumisterol₃ on both insulin release and [Ca²⁺]_i. Moreover, plasma membrane depolarization is required for the augmentation of Ca²⁺ influx and insulin release by 1,25(OH)₂lumisterol₃. These results suggest that the mechanism of the insulinotropic effect of 1,25(OH)₂lumisterol₃ is dependent on augmentation of Ca²⁺ influx, mainly through the VDCC.

Insulinotropic agents are categorized into two groups: depolarizing agents, including sulfonylureas and a high concentration of K⁺, which can trigger insulin secretion by membrane depolarization alone; and potentiators, including protein kinase A (PKA) activators and protein kinase C (PKC) activators, which cannot trigger insulin release by themselves but augment that triggered by depolarizing agents (36). Because 1,25(OH)₂lumisterol₃ augments insulin release triggered by glucose or a high concentration of K⁺, and is not a depolarizing agent itself, 1,25(OH)₂lumisterol₃ is a potentiator of insulin secretion, but its mechanism is unique. Other known potentiators, including PKA and PKC activators, increase Ca²⁺ efficacy in the exocytotic system (37, 38, 39, 40), but 1,25(OH)₂lumisterol₃ has no such Ca²⁺-sensitizing effect. The potentiating effect of 1,25(OH)₂-lumisterol₃, therefore, is derived from increasing Ca²⁺ influx under a depolalizing condition, as also has been reported of PKA activators (41, 42) but is controversial in the case of the PKC activators (40, 43, 44, 45).

It is not clear why augmentation of the [Ca²⁺]_i rise induced by 1,25(OH)₂lumisterol₃ is observed only in the presence of a stimulatory concentration of glucose. It may be because signal transduction of 1,25(OH)₂lumisterol₃ is linked to the mechanism of modulation of the VDCC by the intracellular glucose metabolism, which was reported by Smith et al. (46).

However, in our preliminary experiments, 1,25-dihydroxyvitamin D₃, a biologically active form of vitamin D, could not augment either glucose-induced, or a depolarizing concentration of K⁺-induced, insulin release. Therefore, further investigations will be needed to discuss physiologically the meaning of insulinotropic effects of vitamin D via mVDR.

In the present study, we find that vitamin D analog has a nongenomic rapid insulinotropic effect via a putative mVDR. This effect is dependent on augmentation of Ca²⁺ influx through plasma membrane, which may also be linked to metabolic signals derived from glucose.

	Acknowledgments

 
The authors thank Mr. H. Imamura for his technical assistance.

	Footnotes

 
¹ This study was supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan; Grants-in Aid for Creative Basic Research (10NP0201) from the Ministry of Education, Science, Sports and Culture of Japan; grants from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF 97 I 00201); and a grant for Diabetic Research from Tsumura & Co., Japan.

² A research fellow of the Japan Society for Promotion of Science.

Received January 12, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Clark SA, Stumpf WE, Sar M, DeLuca HF, Tanaka Y 1980 Target cells for 1,25-dihydroxyvitamin D₃ in rat pancreas. Cell Tissue Res 209:515–520[Medline]
2. Ishida H, Norman AW 1988 Demonstration of high affinity receptor for 1,25-dihydroxyvitamin D₃ in rat pancreas. Mol Cell Endocrinol 60:109–117[CrossRef][Medline]
3. Chertow BS, Sivitz WI, Baranetsky NG, Clark SA, Waite A, DeLuca HF 1983 Cellular mechanism of insulin release: the effect of vitamin D deficiency and repletion on rat insulin secretion. Endocrinology 113:1511–1518[Abstract]
4. Kadowaki S, Norman AW 1984 Dietary vitamin D essential for normal insulin secretion from the rat perfused pancreas. J Clin Invest 73:759–766
5. Labriji-Mestaghanmi H, Billaudel B, Garnier PE, Malaisse WJ, Sutter BJC 1988 Vitamin D and pancreatic islet function. I. Time course for changes in insulin secretion and content during vitamin D deprivation and repletion. J Endocriol Invest 11:577–584
6. Tanaka Y, Seino Y, Ishida M, Yamaoka K, Satomura K, Yabuuchi H, Seino Y, Imura H 1986 Effect of 1,25-dihydroxyvitamin D₃ on insulin secretion: direct or mediated? Endocrinology 118:1971–1976[Abstract]
7. Beaulieu C, Kestekian R, Havrankova J, Gascon-Barre’ M 1993 Calcium is essential in normalizing intolerance to glucose that accompanies vitamin D depletion in vivo. Diabetes 42:35–43[Abstract]
8. Lee S, Clark SA, Gill RK, Christakos S 1994 1,25-dihydroxyvitamin D₃ and pancreatic ß-cell function: vitamin D receptors, gene expression, and insulin secretion. Endocrinology 134:1602–1610[Abstract]
9. Lowe KE, Maiyar AC, Kushner PJ, Norman AW 1984 Vitamin D-mediated gene expression. Crit Rev Eukaryot Gene Expr 2:65–109
10. Nemere I, Zhou L-X, Norman AW 1993 Nontranscriptional effects of steroid hormone. Receptor 3:277–291[Medline]
11. Caffrey JM, Farach-Carson MC 1989 Vitamin D₃ metabolites modulate dihydropyridine-sensitive Ca²⁺ currents in clonal rat osteosarcoma cells. J Biol Chem 264:20265–20274[Abstract/Free Full Text]
12. Farach-Carson MC, Sergeev IN, Norman AW 1991 Nongenomic action of 1,25 dihydroxyvitamin D₃ in rat osteosarcoma cells: structure-function studies using ligand analogs. Endocrinology 129:1876–1884[Abstract]
13. Civitelli R, Kim YS, Gunsten SL, Fujimori A, Huskey M, Avioli LV, Hruska KA 1990 Nongenomic activation of the calcium message system by vitamin D metabolites in osteoblast-like cells. Endocrinology 127:2253–2262[Abstract]
14. Tornquist K, Tashjian AH 1989 Dual action of 1,25 dihydroxy cholecalciferol on intracellular Ca²⁺ in GH4C1 cells: evidence for effect of voltage dependent Ca²⁺ channel and Na⁺/Ca²⁺ exchange. Endocrinology 124:2765–2776[Abstract]
15. Baran DT, Sorensen AM, Honeyman TW, Ray R, Holick MF 1990 1,25-dihydroxyvitamin D₃-induced increments in hepatocyte cytosolic calcium and lysophoshatidylinositol: inhibition by pertussis toxin and 1ß,25-dihydroxyvitamin D₃. J Bone Miner Res 5:517–524[Medline]
16. Tien XY, Brasitus TA, Qasawa BM, Norman AW, Sitrin MD 1993 Effect of 1,25(OH)₂D₃ and its analog on membrane phosphoinositide turn over and [Ca²⁺]_i in Caco-2 cells. Am J Physiol 265:G143–G148
17. Nemere I, Dormanen MC, Hammond MW, Okamura WH, Norman AW 1994 Identification of specific binding protein for 1,25-dihydroxyvitamin D₃ in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia. J Biol Chem 269:23750–23756[Abstract/Free Full Text]
18. Dusso AS, Negrea L, Gunawardhana S, Lopez-Hilker S, Finch J, Mori T, Nishi Y, Slatopolosky E, Brown AJ 1991 On the mechanism for the selective action of vitamin D analogs. Endocrinology 128:1687–1692[Abstract]
19. Norman AW, Okamura WH, Farach-Carson MC, Allewaert K, Branisteanu D, Nemere I, Muralidharan KR, Bouillon R 1993 Structure-function studies of 1,25-dihydroxyvitamin D₃ and the vitamin D endocrine system. J Biol Chem 268:13811–13819[Abstract/Free Full Text]
20. Dormanen MC, Bishop JE, Hammond MW, Okamura WH, Nemere I, Norman AW 1994 Nonnuclear effects of the steroid hormon 1,25-dihydroxyvitamin D₃ analogs are able to functionally differentiate between nuclear and membrane receptors. Biochem Biophys Res Commun 201:394–401[CrossRef][Medline]
21. Okamura WH, Midland MM, Hammond MW, Abd.Rahman N, Dormanen MC, Nemere I, Norman AW 1995 Chemistry and conformation of vitamin D molecules. J Steroid Biochem Mol Biol 53:603–613[CrossRef][Medline]
22. Norman AW, Bishop JE, Collins ED, Seo E-G, Satchell DP, Dormanen MC, Zanello SB, Farach-Carson MC, Bouillon R, Okamura WH 1996 Differing shapes of 1,25-dihydroxyvitamin D₃ function as ligands for the D-binding protein, nuclear receptor and membrane receptor: a status report. J Steroid Biochem Mol Biol 56:13–22[CrossRef][Medline]
23. Norman AW, Okamura WH, Hammond MW, Bishop JE, Dormanen MC, Bouillon R, van Baelen H, Ridall AL, Daane E, Khoury R, Farach-Carson MC 1997 Comparison of 6-s-cis- and 6-s-trans-locked analog of 1,25-dihydroxyvitamin D₃ indicates that 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis- nor 6-s-trans-locked analogs are preferred for genomic biological responses. Mol Endocrionol 11:1518–1531[Abstract/Free Full Text]
24. Norman AW, Nemere I, Muralidharan KR, Okamura WH 1992 1ß,25-(OH)₂-vitamin D₃ is an antagonist of 1,25-(OH)₂-vitamin D₃ stimulated transcalthachia (the rapid hormonal stimulation of intestinal calcium transport). Biochem Biophys Res Commun 189:1450–1456[CrossRef][Medline]
25. Norman AW, Bouillon R, Farach-Carson MC, Bishop JE, Zhou LX, Nemere I, Zhao J, Muralidharan KR, Okamura WH 1993 Demonstration that 1ß,25-dihydroxyvitamin D₃ is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1,25-dihydroxyvitamin D₃. J Biol Chem 268:20022–20030[Abstract/Free Full Text]
26. Bhatia M, Kirjland JB, Meckling-Gill KA 1995 Monocytes differentiation of acute promyelocytic leukemia cells in response to 1,25-dihydroxyvitamin D₃ is independent of nuclear receptor binding. J Biol Chem 270:15962–15965[Abstract/Free Full Text]
27. Sergeev IN, Rhoten WB 1995 1,25-dihydroxyvitamin D₃ evokes oscillation of intracellular calcium in pancreatic ß-cell line. Endocrinology 136:2852–2861[Abstract]
28. Fujimoto S, Ishida H, Kato S, Okamoto Y, Tsuji K, Mizuno N, Ueda S, Mukai E, Seino Y 1998 The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca²⁺ sensitivity in ß-cells. Endocrinology 139:1133–1140[Abstract/Free Full Text]
29. Sutton R, Peters M, Mcshane P, Gray DWR, Morris PJ 1986 Isolation of rat pancreatic islets by ductal injection of collagenase. Transplantation 42:689–691[Medline]
30. Tsuji K, Taminato T, Usami M, Ishida H, Kitano N, Fukumoto H, Koh G, Kurose T, Yamada Y, Yano H, Seino Y, Imura H 1988 Characteristic features of insulin secretion in the streptozotocin-induced NIDDM rat model. Metabolism 37:1040–1044[CrossRef][Medline]
31. Okamoto Y, Ishida H, Tsuura Y, Yasuda, K, Kato S, Matsubara H, Nishimura M, Mizuno N, Ikeda H, Seino Y 1995 Hyperresponse in calcium-induced insulin release from electrically permeabilized pancreatic islets of diabetic GK rats and its defective augmentation by glucose. Diabetologia 38:772–778[CrossRef][Medline]
32. Silva AM, Rosario LM, Santos RM 1994 Background Ca²⁺ influx mediated by dihydropyridine- and voltage-insensitive channel in pancreatic ß-cells. J Biol Chem 269:17095–17103[Abstract/Free Full Text]
33. Henquin JC 1994 Cell biology of insulin secretion. In: Kahn CR, Weir GC (eds) Joslin’s Diabetes Mellitus. Lea & Febiger, Philadelphia, pp 56–80
34. Gembal M, Gilon P, Henquin JC 1992 Evidence that glucose can control insulin release independently from its action on ATP-sensitive K⁺ channel in mouse B cell. J Clin Invest 89:1288–1295
35. Gembal M, Detimary P, Gilon P, Gao Z-Y, Henquin JC 1993 Mechanism by which glucose can control insulin release independently from its action on adenosine triphosphate-sensitive K⁺ channel in mouse B cell. J Clin Invest 91:871–880
36. Ashcroft FM, Aschroft SJH 1992 Mechanism of insulin secretion. In: Ashcroft FM, Aschroft SJH (eds) Insulin: Molecular Biology to Pathology. IRL Press, Oxford, pp 97–150
37. Tamagawa T, Niki H, Niki A 1985 Insulin release independent of a rise in cytosolic free Ca²⁺ by forskolin and phorbol ester. FEBS Lett 183:430–432[CrossRef][Medline]
38. Jones PM, Stutchfield J, Howell SL 1985 Effect of Ca²⁺ and a phorbol ester on insulin secretion from islets of Langerhans permeabilised by high-voltage discharge. FEBS Lett 191:102–106[CrossRef][Medline]
39. Ämmälä C, Ashcroft FM, Rorsman P 1993 Calcium-independent potentiation of insulin release by cyclic AMP in single ß-cells. Nature 363:356–358[CrossRef][Medline]
40. Ämmälä C, Eliasson L, Bokvist K, Berggren PO, Honkanen RE, Sjöholm Å, Rorsman P 1994 Activation of protein kinase and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic ß-cells. Proc Natl Acad Sci USA 91:4343–4347[Abstract/Free Full Text]
41. Rajan AS, Hill RS, Boyd III AE 1989 Effect of rise in cAMP levels on Ca²⁺ influx through voltage-dependent Ca²⁺ channel in HIT cells. Diabetes 38:874–80[Abstract]
42. Yaekura K, Kakei M, Yada T 1996 cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca²⁺ in rat pancreatic ß-cells. Diabetes 45:295–301[Abstract]
43. Henquin JC, Bozem M, Schmeer W, Nenquin M 1987 Distinct mechanism for two amplification system of insulin release. Biochem J 246:393–399[Medline]
44. Arkhammar P, Nilsson T, Welsh M, Welsh N, Berggren PO 1989 Effects of protein kinase C activation on regulation of the stimulus-secretion coupling in pancreatic ß-cells. Biochem J 264:207–215[Medline]
45. Yada T, Russo LL, Sharp GWG 1989 Phorbor ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca²⁺ concentration. J Biol Chem 264:2455–2462[Abstract/Free Full Text]
46. Smith PA, Rorsman P, Ashcroft FM 1989 Modulation of dihydropyridine-sensitive Ca²⁺ channels by glucose metabolism in mouse pancreatic ß-cells. Nature 342:550–553[CrossRef][Medline]

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