An extremely low frequency magnetic field attenuates insulin secretion from the insulinoma cell line, rin-m

An Extremely Low Frequency Magnetic Field Tomonori Sakurai,1 Akira Satake,1 Shoichiro Sumi,1 Kazutomo Inoue,1 and Junji Miyakoshi2* 1Department of Organ Reconstruction, Institute for Frontier Medical Sciences, 2Department of Radiological Technology, School of Health Sciences, Faculty of Medicine, Hirosaki University, Hirosaki, Japan In this study, we investigated the effects of exposure to an extremely low frequency magnetic field(ELFMF) on hormone secretion from an islet derived insulinoma cell line, RIN-m. We stimulatedRIN-m cells to secrete insulin under exposure to an ELFMF, using our established system for theexposure of cultured cells to an ELFMF at 5 mT and 60 Hz, or under sham exposure conditions for 1 hand observed the effects. In the presence of a depolarizing concentration of potassium (45 mM KCl),exposure to ELFMF significantly attenuated insulin release from RIN-m cells, compared to shamexposed cells. Treatment with nifedipine reduced the difference in insulin secretion betweencells exposed to an ELFMF and sham exposed cells. The expression of mRNA encoding synaptosomalassociated protein of 25 kDa (SNAP-25) and synaptotagmin 1, which play a role in exocytosis inhormone secretion and influx of calcium ions, decreased with exposure to an ELFMF in the presenceof 45 mM KCl. These results suggest that exposure to ELFMF attenuates insulin secretion fromRIN-m cells by affecting calcium influx through calcium channels. Bioelectromagnetics 25:160–166,2004.
Key words: 5 mT and 60 Hz; insulinoma cell line; KCl stimulation; calcium channels; synaptosomal associated protein; synaptotagmin 1 ion efflux, and insulin secretion during glucose stimu-lation were reduced when isolated rabbit islets were The possible health effects of exposure to ex- exposed to low frequency pulsed magnetic fields.
tremely low frequency magnetic fields (ELFMFs) have Hayek et al. [1984] reported that exposure to low become a considerable public concern. Several epide- intensity homogeneous magnetic fields inhibited insu- miological studies have shown an association between lin release from isolated newborn rat islets stimulated exposure to ELFMF and elevated risk in children and by high glucose concentration (16.7 mM) and amino- occupationally exposed adults [Savitz and Loomis, phylline (10 mM). Recently, Laitl-Kobierska et al.
1995]. Whether exposure to magnetic fields causes [2002] reported that long term exposure of rats to significant cellular stress remains a contentious issue in ELFMF led to increased synthesis and secretion of Pancreatic islets play a fundamental role in re- gulating the blood glucose levels of the body through Grant sponsor: Research for the Future Program, Japan Society for the secretion of hormones such as insulin, glucagon, the Promotion of Science; Grant sponsor: Ministry of Education somatostatin, and pancreatic polypeptide. The release (for Scientific Research S); Grant number: 13854020.
of insufficient amounts of these hormones is the basis of *Correspondence to: Prof. Junji Miyakoshi, Department of various forms of diabetes. Therefore, it is important to Radiological Technology, School of Health Sciences, Faculty of assess the effects of exposure to ELFMF on pancreatic Medicine, Hirosaki University, 66-1 Hon-cho, Hirosaki, 036-8564, Japan. E-mail: miyakosh@cc.hirosaki-u.ac.jp Studies evaluating the influence of exposure to ELFMF on pancreatic islet function are scarce, and it is Received for review 2 May 2003; Final revision received 4 August2003 often difficult to compare the existing studies because ofthe different research methods used. Previously, Joelly et al. [1983] reported that calcium ion content, calcium Published online in Wiley InterScience (www.interscience.wiley.com).
insulin. Consequently, an association between mag- netic field exposure and pancreatic islet function has not was removed, and the cells were washed twice with 2- [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid One important approach to overcome the pro- (HEPES)-buffered Krebs–Ringer solution (119 mM blems of using islets is the use of insulinoma cell lines.
NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM RIN-m cells are derived from X-ray radiation induced rat insulinoma [Chick et al., 1977; Gazdar et al., 1980] 10 mM HEPES at pH 7.4) containing 0.2% bovine and have been used to investigate the mechanism of serum albumin (BSA), and 3.3 mM D-glucose. The cells insulin secretion [Yada et al., 1989].
were then preincubated in the same buffer at 37 8C In this study, we have investigated the effects of for 30 min. The buffer was then removed, and 0.8 ml exposure to ELFMF on insulin release by the insuli- of basal incubation buffer (HEPES-buffered Krebs– noma cell line, RIN-m, using our previously manufac- Ringer solution containing 0.2% BSA and 3.3 mM tured equipment to expose cultured cells to an ELFMF D-glucose) was added and the cells were incubated at 37 8C for 1 h. After the basal incubation period, wellswere divided into two groups; one is stimulated underexposure to an ELFMF (Fig. 1, Group A), and the other is stimulated under sham exposure condition (Fig. 1, The stimulation was performed as follows: the ELFMF exposure, a sinusoidal magnetic field at a cells were incubated at 37 8C for 1 h in 0.8 ml of frequency of 60 Hz, 5 mT, was performed using a pre- stimulation buffer, which consisted of HEPES buffered viously described magnetic field exposure apparatus Krebs–Ringer solution containing 0.2% BSA, D-glucose, [Miyakoshi et al., 1996; Ding et al., 2000]. The distri- and various reagents, as indicated in the figure legends.
bution of the magnetic density was measured using a At the end of the basal incubation period and stimu- Gauss meter (Model 3251, Yokogawa Electrical Co., lation period, an aliquot of the incubation buffer in each Ltd., Tokyo). Briefly, the ELFMF exposure system well was collected and stored at À20 8C until the insulin consists of a magnetic field generator that uses measurement was performed. The insulin concentration Helmholtz coils built into a CO2 incubator, a transfor- of the samples was measured by enzyme linked im- mer and a thermocontroller. The direction of the field is munosorbent assay (ELISA) using a rat insulin ELISA vertical. The atmosphere in the incubator is maintained kit (Sibayagi Co., Gunma, Japan). Insulin secretion with humidified 95% air plus 5% CO2. The temperature from RIN-m cells stimulated various regents were in the exposure space, which is monitored by thermo- analyzed using the ratio of insulin secretion during the couple sensor probes, is maintained at 37 Æ 0.2 8C. The stimulation period/insulin secretion during basal incu- measured 60 Hz ELFMF exposure during the sham exposure was <0.5 mT. Static magnetic fields other thangeomagnetism were undetectable (<0.1 mT).
After insulin secretion tests were performed using 6-well culture plates, the cells were scraped and the RIN-m cells (obtained from the Dainippon Phar- total RNA was prepared using an ISOGEN isolation kit maceutical Co., Osaka, Japan) were cultured in RPMI- (Nippon Gene Co., Toyama, Japan). Briefly, the cells 1640 medium supplemented with 10% fetal bovine were homogenized in 1 ml of ISOGEN reagent, and serum at 37 8C in humidified 95% air plus 5% CO2. For 0.1 ml of chloroform was added to this mixture. The each experiment, a new vial of frozen cells was thawed, mixture was centrifuged at 15,000g for 15 min at 4 8C, seeded at a density of 1 Â 105 cells/cm2 on 12- or 6-well and the resulting aqueous phase was transferred to cell culture plates and cultured. Cells were used from 0.5 ml of isopropyl alcohol. The resulting precipitate was collected by centrifugation at 15,000g for 10 min at4 8C, and the RNA pellet obtained was washed with 75% ethanol, and then dissolved in diethyl pyrocarbo- Cells were plated on 12-well culture plates at nate-treated water. The amount of RNA was measured a density of 3.5 Â 105 cells/well. Insulin secretion tests using an ultraviolet-visible spectrometer at 260 nm, and were performed 4 days after plating, when the cells were the purity of the RNA obtained was determined from the 80–90% confluent. The culture medium was changed absorbance ratio at 260/280 nm. The ratio at >1.8 was to fresh medium 16 h before the insulin secretion tests, used following reverse transcriptase-polymerase chain and the tests were performed as shown in Figure 1.
Fig. 1. The outline of insulin secretion tests. Preincubation and basal incubation were performedwithoutexposure to anextremelylow frequencymagnetic field (ELFMF).After wellswere dividedintotwo groups, one group is stimulated under exposure to an ELFMF (Group A), and the other group isstimulatedunder sham exposure (Group B). At the end ofthe basalincubation period and stimulationperiod, an aliquot of the incubation buffer in each well was collected, and the insulin concentration ofthe samples was measured by enzyme linked immunosorbent assay (ELISA).
in a thermal cycler at 95 8C for 45 s, at 59 8C for 45 s, cDNA synthesis was performed using a (dT) and at 72 8C for 90 s. b-Actin expression was used to oligo primer and ThermoScript RT (Invitrogen, Carls- normalize the input template cDNA in a semi-quanti- bad, CA), according to the manufacturer’s instructions.
tative PCR reaction. Serial half dilutions of cDNA were PCR was performed using Taq polymerase (TaKaRa amplified to ensure analysis of products in the linear Co, Shiga, Japan). PCR mixtures contained cDNA, 1Â PCR buffer, 2.5 mM MgCl2, 400 mM dNTP, 2.5 U/50 ml of Taq polymerase, and 1 mM sense and antisenseprimers. The primers were as follows, insulin 1: forward 50-ATGGCCCTGTGGATGCGCTT-30, reverse 50-TA- agarose gel with 0.1 mg/ml ethidium bromide, and the GTTGCAGTAGTTCTCCAGCT-30, insulin 2: forward quantification of the PCR products was performed by densitometry of the band intensity using a Kodak Digi- tal Science IS 440 CF System and 1D Image Analysis associated protein of 25 kDa (SNAP-25): forward 50- Software ver. 3.5 (Eastman Kodak Co., Rochester, NY).
GGTTCCTTAACTAAGCACCACTGACTT-30, reverse50-TTTCCCGGGCATCGTTTGTTACC-30, tagmin 1: forward 50-ATGGCTGTGTATGACTTTGA- Statistical comparisons were performed by analy- sis of variance and, when appropriate, using the Stu- GTCGTT-30, b-actin: forward 50-ATGGTGGGTATG- dent’s t-test. Experimental results were presented as the mean Æ SE, and studies were repeated at least three CTCTCAGTC-30. The reaction mixture was incubated Insulin Secretion From RIN-m CellsUnder Sham Conditions Under sham conditions, incubation with stimula- tion buffer containing a high concentration of potas-sium (45 mM KCl) caused insulin release to increaseapproximately 14 fold, compared to the basal incuba-tion conditions. In contrast, both a normal (3.3 mM)and a high (16.7 mM) concentration of D-glucose didnot increase insulin release, compared to the basal in-cubation conditions. Phorbol-12-myristate-13-acetate(PMA) also increased insulin release from RIN-m cellsby approximately 15 fold (Table 1). These results arein agreement with previous reports describing RIN-mcells and their subcloned cell line, RINm5F [Praz et al.,1983; Bhathena et al., 1984; Yada et al., 1989].
Insulin secretion from RIN-m cells in the stimula- tion buffer containing a normal concentration of D-glucose did not increase under exposure to an ELFMF, Fig. 2. Relative insulin release from RIN-m cells under exposure compared to the sham exposed cells. On the other hand, to an ELFMF or under sham conditions, measured after1h incuba-tion with 3.3 mM in the presence of 45 mM KCl, exposure to ELFMF D-glucose, or 3.3 mM D-glucose plus 45 mM KCl, or 3.3 mM D-glucose plus 1 mM phorbol-12-myristate-13-acetate significantly attenuated insulin release from RIN-m (PMA). Data represent the mean Æ SE (3.3 mM D-glucose, n ¼ 3; cells by approximately 30%, compared to sham exposed 45 mM KCl and PMA, n ¼ 5). **P < .01.
cells. When PMA was included in the stimulation buf-fer, insulin release was slightly, but not significantly,enhanced by exposure to ELFMF (Fig. 2). These resultssuggest that exposure to an ELFMF in the presence of concentrations of the calcium channel blocker, nifedi- chemical reagents affects insulin secretion, but that pine. Under exposure to an ELFMF and under sham exposure to ELFMF alone is insufficient to stimulate conditions, insulin release decreased in a dose-depen- insulin secretion. These findings are consistent with our dent manner. In the presence of low concentrations of previous reports showing that exposure to an ELFMF nifedipine (5, 50, or 500 nM), insulin secretion was in combination with chemical reagents enhanced the significantly attenuated under exposure to an ELFMF, effects of exposure to ELFMF on cells [Miyakoshi et al., compared to sham exposed cells. In contrast, there was no difference in insulin release between cells exposed toan ELFMF and those under sham exposure at high Effects of Nifedipine Treatment on Insulin concentrations of nifedipine (5 or 50 mM). The amount of insulin secretion in the presence of 5 nM nifedipine Insulin release from RIN-m cells was assessed in under sham conditions was approximately equal to that the presence of 45 mM KCl after treatment with various in the absence of nifedipine under exposure to ELFMF(Fig. 3). These results suggest that ELFMF attenuatedinsulin secretion from RIN-m cells is related to an effect TABLE 1. Insulin Secretion From RIN-m Cells in Response to The effects of an ELFMF on mRNA expression in RIN-m cells were investigated using semi-quantitative RT-PCR. The fragments amplified for insulin 1, insulin 2, SNAP-25, synaptotagmin 1, and b-actin had the ex- pected sizes (331, 333, 493, 456, and 500 base pairs Fig. 3. Nifedipine inhibition of insulin release from RIN-m cells sti-mulated by 3.3 mM D-glucose and 45 mMKClunderexposure to anELFMF or under sham conditions.To study the effect of nifedipine,cells were incubated with the indicated concentrations of nifedi-pine for 30 min, and then incubated for 1 h with stimulation buffercontaining 3.3 mM D-glucose and 45 mM KCl. Each point repre- Fig. 4. A: Reverse transcription-polymerase chain reaction (RT- sents the mean Æ SE (50 mM nifedipine, n ¼ 3; other concentra- PCR) of insulin 1, insulin 2, synaptosomal associated protein of 25 kDa (SNAP-25), and synaptotagmin 1 after an insulin secretiontest under exposure to an ELFMF or under sham conditions.
Twofold serial dilutions of cDNA were amplified for 25 (insulin 1), (bp), respectively), and PCR products were undetect- 29 (insulin 2 and SNAP-25), 33 (synaptotagmin 1) or 26 (b-actin) able when reverse transcription was performed without cycles. Lanes 1^ 3 represent serial dilutions of cDNA sample.
reverse transcriptase (ThermoScript RT). Semi-quanti- Lane1was the most concentrated sample in each series. B: Semi- tative RT-PCR was performed under conditions where quantitative RT-PCR analysis of the effects of exposure to anELFMF on expression of mRNA encoding insulin 1, insulin 2, the amplification reaction for the PCR products was SNAP-25, and synaptotagmin 1. Insulin secretion tests were per- within the linear range. For example, the increase in formed using stimulation buffer containing 3.3 mM D-glucose or the optical density of the amplified PCR products for 3.3 mM D-glucose and 45 mM KCl. Data represent the mean Æ SE b-actin was linear between at least cycles 24 and 29. A (insulin 1 and insulin 2, n ¼ 3; SNAP-25, n ¼ 5; synaptotagmin 1, reduced intensity of products was observed at each dilution step (Fig. 4A). This result confirmed that theRT-PCR was performed in the exponential portion of In the presence of 45 mM KCl, exposure to an Recently, human exposure to ELFMFs from vari- ELFMF significantly reduced expression of the mRNA ous electrical appliances has increased significantly.
encoding SNAP-25 and synaptotagmin 1 by approxi- Thus, the possible health effects of exposure to ELFMF mately 36 and 23%, respectively, compared to sham have become a considerable public concern. On the exposed cells. Insulin 2 mRNA expression was slightly, other hand, occurrence of diabetes mellitus has increas- but not significantly, reduced by exposure to ELFMF ed progressively in recent years. Insufficient pancreatic (approximately 10%), and insulin 1 mRNA expression islet function is the basis of various forms of diabetes.
did not decrease. Insulin 2, SNAP-25, and synaptotag- These two factors motivated us to investigate the effects min 1 mRNA expression increased when RIN-m cells of exposure to ELFMF on the function of insulin were stimulated by 45 mM KCl, compared to 3.3 mM secreting cells. If exposure to ELFMF is deleterious to insulin secreting cells, it is essential that this is demonstrated and communicated as soon as possible. In brane. SNARE proteins play a role in this tethering/ contrast, if exposure to ELFMF is beneficial to insulin docking process between secretory granules and the secreting cells, it might be possible to utilize exposure plasma membrane. SNAP-25 is a SNARE protein that to ELFMF for medical applications. For example, an is expressed in pancreatic islets and is involved in in- ELFMF might be used in diabetes mellitus to decrease sulin release [Sadoul et al., 1995; Wheeler et al., 1996].
blood glucose levels, and to increase insulin levels SNAP-25 mRNA expression was significantly increas- in blood [Laitl-Kobierska et al., 2002]. Because of these ed when chromaffin cells [Garcia-Palomero et al., 2000; issues, the assessment of the effects of exposure to Montiel et al., 2003] and rat granulosa cells [Grosse ELFMF on insulin secretion is very important.
et al., 2000] were stimulated to release neurotransmit- Studies evaluating the influence of exposure to ters, and these events were closely related to the influx ELFMF on pancreatic islet function are scarce, and it is often difficult to compare the studies that have been Synaptotagmin 1 is a calcium ion sensor protein performed because of the different research methods that is located on the membrane of insulin containing used. Hence, an association between magnetic field secretory granules [Lang et al., 1997]. It is thought to be exposure and pancreatic islet function has not been play a role in the fusion between secretory granules and demonstrated unequivocally. Furthermore, the use of the plasma membrane according to the elevation of the islets results in significant methodological difficulties, cellular calcium concentration [Gerber and Sudhof, due to cellular heterogeneity, limited availability, and rapid deterioration of function. In order to circumvent In this work, the expression of mRNA encoding these problems in the current study, we used insulinoma SNAP-25 and synaptotagmin 1 decreased under ex- cells instead of islets. To our knowledge, this is the first posure to an ELFMF in the presence of 45 mM KCl.
investigation in which insulinoma cells have been used From these findings, we conclude that exposure to to examine the effects of exposure to ELFMF on insulin ELFMF attenuates insulin secretion by reducing the secreting cells. The advantages of using insulinoma influx of calcium ions through calcium channels. It has cells were the ease of generation of large quantities of been reported that calcium ion content, calcium ion functional cells, and the stability of the resulting cell efflux, and insulin secretion during glucose stimulation was reduced when isolated rabbit islets were exposed to We investigated the insulinoma cell line, RIN-m.
low frequency pulsed magnetic fields [Joelly et al., It has been reported that PMA, which acts via the PKC 1983]. It has also been reported that exposure to pathway, induces insulin secretion [Yada et al., 1989].
ELFMF affected neurite growth via voltage gated On the basis of the insulin secretory response of RIN-m calcium channels [Morgado-Valle et al., 1998] and af- cells to 45 mM KCl and PMA, we thought that RIN-m fected the differentiation of neuroblastoma cells by cells were suitable for a model to evaluate the effects of antagonizing the shift in cell membrane surface charges exposure to ELFMF on the potassium induced insulin and increasing intracellular calcium levels [Tonini et al., secretion pathway and the PKC cascade. A high con- 2001]. Our present findings are in agreement with these centration of D-glucose induces the closure of ATP de- pendent potassium channels in insulin secreting cells, In the current study, exposure to an ELFMF which in turn induces membrane depolarization, open- slightly increased PMA stimulated insulin secretion, ing of voltage dependent calcium channels, and insulin but no statistically significant difference was observed secretion. It has been reported that treatment with high at 5 mT and 60 Hz. We have previously reported that concentrations of potassium bypasses the ATP depen- exposure to ELFMF at 50 Hz and 400 mT, but not dent potassium channels of insulin secreting cells and at 5 mT, enhanced the expression of a neuron deriv- also induces insulin secretion [Hohmeier et al., 2000].
ed orphan receptor gene induced by treatment with In the current study, insulin secretion induced by forskolin and PMA [Miyakoshi et al., 1998]. We also 45 mM KCl was attenuated by approximately 30% reported that the suppression of heat shock protein 70 under exposure to an ELFMF, compared to sham ex- was observed at a magnetic density of 50 mT, but not at posure. Treatment with nifedipine reduced the differ- 5 or 0.5 mT [Miyakoshi et al., 2000]. These previous ence in insulin secretion between ELFMF exposed and results indicate the dependence of the effects of ex- sham exposed cells. Recent investigations have clarifi- posure to ELFMF on the strength of the magnetic field, ed the molecular mechanisms of exocytosis in neuro- and they are consistent with the results of the current transmitter release or hormone secretion [Jones and Persaud, 1998; Gerber and Sudhof, 2002]. In the exo- The results presented here suggest that insulin cytotic release process, secretory granules are docked at secretion decreases under exposure to ELFMF. Hence, the site of exocytosis and fused with the plasma mem- it might be desirable for diabetic patients who have insufficient insulin secretion from pancreatic islets to Joelly WB, Hinshaw DB, Knierim K. 1983. Magnetic field effects on calcium efflux and insulin secretion in isolated rabbit In summary, we investigated the effects of ex- islets of Langerhans. Bioelectromagnetics 4:103–106.
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