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Nutrients Induce Different Ca²⁺ Signals in Cytosol and Nucleus in Pancreatic ß-Cells

¹ Department of Bioengineering, University of Washington, Seattle, Washington
² Institute of Bioengineering, Miguel Hernandez University, San Juan de Alicante, Spain
³ Department of Surgery, National University of Singapore, Singapore

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Specific activation of Ca²⁺-dependent functions is achieved by the particular dynamics and local restriction of Ca²⁺ signals. It has been shown that changes in amplitude, duration, or frequency of Ca²⁺ signals modulate gene transcription. Thus, Ca²⁺ variations should be finely controlled within the nucleus. Although a variety of mechanisms in the nuclear membrane have been demonstrated to regulate nuclear Ca²⁺, the existence of an autonomous Ca²⁺ homeostasis within the nucleus is still questioned. In the pancreatic ß-cell, besides their effect on insulin secretion, Ca²⁺ messages generated by nutrients also exert their action on gene expression. However, the dynamics of these Ca²⁺ signals in relation to nuclear function have been explored little in islet cells. In the current study, Ca²⁺ changes both in the nucleoplasm and in the cytosol of INS-1 and pancreatic ß-cells were monitored using spot confocal microscopy. We show that nutrients trigger Ca²⁺ signals of higher amplitude in the nucleus than in the cytosol. These amplitude-modulated Ca²⁺ signals transmitted to the nucleus might play an important role in the control of gene expression in the pancreatic ß-cell.

Although the regulation of nuclear Ca²⁺ is an issue that has attracted the attention of many investigators, data have not been conclusive (6). Some studies have demonstrated the presence of functional channels and mechanisms in the nuclear membrane of isolated nuclei such as inositol 1,4,5-trisphosphate and ryanodine receptors and Ca²⁺-ATPases, which regulate the release and uptake of Ca²⁺ in the nucleoplasm and nuclear envelope (7,8). These observations have led several groups to postulate that the nucleus is a dynamic Ca²⁺ pool, which could account for the cytosolic-nuclear Ca²⁺ gradients observed in some systems (6). However, independent Ca²⁺ homeostasis within the nucleus different from that in the cytosol has been disputed by the presence of the nuclear pores, which would offer no barrier to Ca²⁺ diffusion, allowing rapid Ca²⁺ equilibration between both compartments (6,9).

In pancreatic ß-cells, Ca²⁺ also signals several functions by means of the above-mentioned spatial and temporal features. Nutrients produce intracellular Ca²⁺ signals that activate insulin secretion but also induce the transcription of several Ca²⁺-dependent genes such as insulin or immediate early genes such as c-fos, nur-77, or c-myc (10–13). Although Ca²⁺ dynamics and its spatial distribution in the ß-cell have been further studied in relation to the exocytotic process (14,15), little has been examined about the pathways that link extracellular messages, Ca²⁺ signals, and nuclear function. In this regard, it would be particularly interesting to analyze nuclear and cytosolic Ca²⁺ dynamics, especially in light of recent findings that have demonstrated the presence of ATP-dependent K⁺ (K_ATP) channels on the nuclear envelope of pancreatic ß-cells, whose blockage triggers nuclear Ca²⁺ transients that affect nuclear function (8). In the current study, we used spot confocal microscopy to evaluate the dynamics of Ca²⁺ signals elicited by glucose, oleate, and K⁺ in the nucleus and in the cytoplasm of INS-1 cells and pancreatic ß-cells. We show that nutrients produce Ca²⁺ elevations of higher amplitude in the nucleus, probably by interaction with nuclear Ca²⁺ release mechanisms.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell isolation and culture.
Islets from adult (8–10 weeks old) Swiss albino male mice (OF1) killed by cervical dislocation in accord with our institutional guidelines were isolated and then dispersed into single cells following a procedure described elsewhere (14). Isolated cells were cultured in RPMI-1640 medium (Invitrogen, Barcelona, Spain) containing 5.6 mmol/l glucose for 12–16 h before experiments. Inmunocytochemistry analysis of cultured cells revealed that around 80% of the preparation was insulin-producing cells (16). In addition, we selected ß-cells according to their large size and low nuclear/cytoplasmic ratio compared with other islet cell types (16). Thus, we expected the majority of cells that we studied in the experiments to be pancreatic ß-cells.

Cells from the pancreatic ß-cell line INS-1 were grown in monolayer cultures as follows (12). When cells reached 80% confluence, they were washed twice with Krebs-Ringer bicarbonate buffer at pH 7.4, containing 5.6 mmol/l glucose and 0.07% BSA. Cells were subsequently incubated 2 more days in RPMI-1640 medium containing 5 mmol/l glucose and 10% FCS, but the second-day cells were trypsinized and transferred to poly-L-lysine-treated coverslips.

Fatty acids were prepared as previously described (12). Defatted BSA was used as a control condition (12).

Spot confocal microscopy in single islet cells.
Cells were loaded with 2 µmol/l FLUO-3 acetoxymethyl ester (Molecular Probes, Leiden, the Netherlands) for 30 min at room temperature. Under these conditions, a homogenous distribution of the dye in the cytosol and in the nucleus was confirmed in the majority of cells (83%) by imaging cultures as described earlier (14). Cells were perfused at a rate of 0.5 ml/min in a Krebs-Ringer buffer (in mmol/l): 119 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, 5 HEPES, and 3 glucose gassed with a mixture of 95% O₂, 5% CO₂ (pH = 7.4). Ca²⁺ concentration ([Ca²⁺]) changes were measured using spot confocal microscopy, which excels in measuring localized Ca²⁺-induced fluorescence elevations because of its remarkable signal-to-noise ratio over conventional systems (8,14,17). The spot illumination-detection configuration has been previously described in detail (14,17). Briefly, a laser-illuminated pinhole (10 µm) was focused onto a spot through the objective either on the nucleus or the bulk cytosol of an isolated islet cell (Fig. 1A). The full-width half-maximal dimension of the illumination spot is 0.6 µm, whereas the depth of field is 1.1 µm. Thus, the predicted detection volume is about 0.6 x 0.6 x 1.1 µm³, which is 1.2 and 0.075% of the nuclear and cell volume (assuming a radius of 2 and 5 µm, respectively). Changes in fluorescence in this small volume were detected with a photodiode (HR008; UDT, Hawthorne, CA), which was connected to an Axopatch-200A amplifier (50 G feedback; Axon Instruments, Foster City, CA). The laser illumination time was 30 ms. We minimized the number of measurements on the same spot to prevent probe photo-bleaching. Under our experimental conditions and sampling rate, bleaching was insignificant. Autofluorescence was determined to be <2%. Nuclear Ca²⁺ was measured by positioning the spot in the center of the nucleus, identified easily by transmitted light microscopy, whereas cytosolic Ca²⁺ was recorded by placing the spot in a cytosolic area adjacent to the nuclear periphery (separation 1 µm). Distances were measured from acquired images using a charge coupled device (CCD) camera (14).

View larger version (32K): [in this window] [in a new window]   FIG. 1. Nuclear and cytosolic [Ca2+] changes in cultured cells induced by different stimuli. A: Hybrid phase-contrast images of a pancreatic ß-cell (left) and an INS-1 cell (right) showing the location of the fluorescence spot focused in the nucleus (bar = 10 µm). B and C: Isolated cells were stimulated with 40 mmol/l K+ (), 16.7 or 22 mmol/l glucose (G) (•) for ß-cells or INS-1 cells, respectively, and 0.5 mmol/l oleate in 3 mmol/l glucose (Ol) () for the time indicated by the line. Ca2+ changes in the nucleoplasm or in the cytoplasm of different pancreatic ß-cells (B) and INS-1 cells (C) were monitored by spot confocal microscopy. These records are representative of more than seven cells for each condition from at least three different preparations. D and E: The amplitude of these [Ca2+] changes, measured as the mean [Ca2+] increase during the response, is shown for ß-cells (D) and INS-1 cells (E). Data are means ± SE (*P < 0.05, unpaired t test). HG, high glucose (16.7 mmol/l for ß-cells and 22 mmol/l for INS-1 cells); LG, low glucose (3 mmol/l). Means were obtained from 7–19 cells for each condition.

Nuclei preparation.
Nuclei were isolated according to procedures published earlier (8). Briefly, isolated islet cells were suspended in an intracellular solution (in mmol/l: 125 KCl, 2 K₂PO₄, 40 HEPES, 0.1 MgCl₂, at pH 7.2, and 100 nmol/l Ca²⁺) and disrupted by brief sonication. Nuclei were separated by centrifugation, resuspended in the intracellular solution, and then allowed to attach onto glass chambers. Isolated nuclei were loaded with the Ca²⁺ probe Calcium Green-1 dextran (30 µg/ml; 30 min at 4°C; Molecular Probes). For further details, see Quesada et al. (8).

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Nutrients trigger Ca²⁺ signals of higher amplitude in the nucleoplasm.
Spot confocal microscopy has been successfully applied in pancreatic ß-cells, skeletal muscle cells, and neurons to monitor Ca²⁺ dynamics in local intracellular domains (8,14,17). We used this technique to measure [Ca²⁺] changes either in the cytosol or in the nucleus of INS-1 cells and pancreatic ß-cells exposed for 5 min to 40 mmol/l K⁺, 16.7 or 22 mmol/l glucose, and 0.5 mmol/l oleate in 3 mmol/l glucose (Fig. 1A–C). All stimuli led to a [Ca²⁺] rise in both intracellular compartments. When we examined the Ca²⁺ responses, we found no differences in duration between those generated in the nucleus and in the cytosol in any of the two types of insulin-secreting cells (P > 0.05; unpaired t test). However, significant differences in amplitude were obtained when comparing nuclear and cytosolic [Ca²⁺] mean values in both kinds of cells (Fig. 1D and E). Whereas K⁺ induced similar elevations of [Ca²⁺] in both intracellular compartments, glucose and oleate produced Ca²⁺ signals of higher amplitude in the nucleus (Fig. 1D and E).

It has been shown that Ca²⁺ probes exhibit enhanced absorbance and fluorescence emission in the nucleoplasm (18). Accordingly, we performed two independent in situ calibrations in the nucleus and in the cytosol to get comparable values between both cell locations (9) (see RESEARCH DESIGN AND METHODS). The differences in amplitude observed between the two compartments cannot arise from calibration artifacts, otherwise such differences should be reflected concomitantly with all the stimuli and not just with glucose and oleate. Similar [Ca²⁺] values in the nucleus and in the cytosol were obtained at rest with 3 mmol/l glucose as well as with stimulatory conditions with 40 mmol/l K⁺. These data also support the reliability of the calibration, because Ca²⁺ is expected to be equilibrated between both compartments in such conditions in ß-cells (19) (see DISCUSSION). Furthermore, the same observations have been confirmed in two different kinds of insulin-producing cells with similar phenotypes and physiological responses.

Potential involvement of nuclear Ca²⁺ release mechanisms.
Plasma membrane depolarization, either by a rise in extracellular K⁺ or by stimulatory glucose as a result of an ATP/ADP increase and K_ATP channel closure, leads to massive Ca²⁺ influx in the cytosol by activation of voltage-dependent Ca²⁺ channels (10). Likewise, oleate can produce a cytosolic increase of [Ca²⁺], probably by direct action on plasma membrane K_ATP channels or voltage-dependent Ca²⁺ channels (20,21). However, this classic pathway is not sufficient to explain the higher values of amplitude of the Ca²⁺ signals induced by both nutrients in the nucleus. In Fig. 2, we show that tolbutamide, which closes K_ATP channels, can trigger Ca²⁺ transients in isolated ß-cell nuclei. This result confirms previous findings that propose the existence of a new signaling pathway, linking extracellular messages with nuclear function by means of nuclear K_ATP channels. Their blockage results in depolarization of the nuclear membrane and the release of Ca²⁺ from the nuclear envelope to the nucleoplasm, probably through ryanodine channels or a voltage-sensitive mechanism (8).

View larger version (38K): [in this window] [in a new window]   FIG. 2. Nuclear Ca2+ transients induced by blockage of nuclear KATP channels. A: Hybrid phase-contrast image showing the location of the fluorescence spot focused on an isolated nucleus from a ß-cell, loaded with the fluorescent probe Calcium Green-1 dextran (bar = 4 µm). B: Closure of nuclear KATP channels with tolbutamide resulted in Ca2+ release from the nuclear envelope to the nucleoplasm. Traces represent three different cases.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

Given that gene transcription is sensitive to amplitude-modulated [Ca²⁺] changes (1–3), we propose that nutrients could signal nuclear functions by ensuring an appropriate transitory level of [Ca²⁺] within the nucleoplasm higher than that possibly required for the activation of cytosolic functions. An exciting challenge in the future would be to find whether these amplitude-modulated Ca²⁺ signals in the nucleus are able to regulate gene expression in the pancreatic ß-cell, which still remains elusive, and how these signals are integrated within the islets of Langerhans.

	ACKNOWLEDGMENTS

 
This work was supported in part by grants from the Ministerio de Ciencia y Tecnología (PM99-0142 and GEN 2001-4748-CO5-05), Fundació Marató TV3 (99-1210), European Foundation for the Study of Diabetes, Instituto Carlos III (RETIp GO3/171, RGDM GO3/212, TERCEL GO3/210, and RCMN C03/08), and Generalitat Valenciana (GV99-139-1-04).

The authors thank E. Pérez, N. Illera, and A. Pérez for technical assistance.

	FOOTNOTES

 
This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educational grant from Les Laboratoires Servier.

Address correspondence and reprint requests to Bernat Soria, MD, Institute of Bioengineering, Miguel Hernandez University, San Juan Campus, Carretera Alicante-Valencia Km 87, 03550 Alicante, Spain. E-mail: bernat.soria{at}umh.es

Received for publication February 28, 2003 and accepted in revised form May 12, 2003

Abbreviations: K_ATP channel, ATP-dependent K⁺ channel

	REFERENCES

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Berridge MJ, Lipp P, Bootman MD: The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol1 :11 –21,2000[Medline]
2. Chawla S: Regulation of gene expression by Ca²⁺ signals in neuronal cells. Eur J Pharmacol447 :131 –140,2002[Medline]
3. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI: Differential activation of transcription factors induced by Ca²⁺ response amplitude and duration. Nature386 :855 –858,1997[Medline]
4. Hardingham GE, Chawla S, Johnson CM, Bading H: Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature385 :260 –265,1997[Medline]
5. Hardigham GE, Arnold FJL, Bading H: Nuclear calcium signalling controls CREB-mediated gene expression triggered by synaptic activity. Nat Neurosci4 :261 –267,2001[Medline]
6. Santella L, Carafoli E: Calcium signalling in the cell nucleus. FASEB J11 :1091 –1109,1997[Abstract]
7. Gerasimenko OV, Gerasimenko JV, Tepikin AV, Petersen OH: ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca²⁺ from the nuclear envelope. Cell80 :439 –444,1995[Medline]
8. Quesada I, Rovira JM, Martin F, Roche E, Nadal A, Soria B: Nuclear K_ATP channels trigger nuclear Ca²⁺ transients that modulate nuclear function. Proc Natl Acad Sci U S A99 :9544 –9549,2002[Abstract/Free Full Text]
9. al-Mohanna FA, Caddy KW, Bolsover SR: The nucleus is insulated from large cytosolic calcium ion changes. Nature367 :745 –750,1994[Medline]
10. Prentki M, Matchinsky FM: Ca²⁺, cAMP and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev67 :1185 –1248,1987[Free Full Text]
11. Ban N, Yamada Y, Someya Y, Ihara Y, Adachi T, Kubota A, Watanabe R, Kuroe A, Inada A, Miyawaki K, Sunaga Y, Shen ZP, Iwakura T, Tsukiyama K, Toyokuni S, Tsuda K, Seino Y: Activating transcription factor-2 is a positive regulator in CaM kinase IV-induced human insulin gene expression. Diabetes49 :1142 –1148,2000[Abstract]
12. Roche E, Buteau J, Aniento I, Reig JA, Soria B, Prentki M: Palmitate and oleate induce the immediate-early response genes c-fos and nur-77 in the pancreatic beta-cell line INS-1. Diabetes48 :2007 –2014,1999[Abstract]
13. Jonas JC, Laybutt DR, Steil GM, Trivedi N, Pertusa JG, Van de Casteele M, Weir GC, Henquin JC: High glucose stimulates early response gene c-Myc expression in rat pancreatic beta cells. J Biol Chem276 :35375 –35381,2001[Abstract/Free Full Text]
14. Quesada I, Martin F, Soria B: Nutrient modulation of polarized and sustained submembranal Ca²⁺ microgradients in mouse pancreatic islet-cells. J Physiol525 :159 –167,2000[Abstract/Free Full Text]
15. Bokvist K, Eliasson L, Ammala C, Renstrom E, Rorsman P: Co-localization of L-type Ca²⁺ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells. EMBO J14 :50 –57,1995[Medline]
16. Quesada I, Nadal A, Soria B: Different effects of tolbutamide and diazoxide in , ß and cells within intact islets of Langerhans. Diabetes48 :2390 –2397,1999[Abstract]
17. Escobar AL, Monck JR, Fernandez JM, Vergara JL: Localization of the site of Ca²⁺ release at the level of a single sarcomere in a skeletal muscle fibers. Nature367 :739 –741,1994[Medline]
18. Perez-Terzic C, Stehno-Bittel L, Clapham DE: Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3. Cell Calcium21 :275 –282,1997[Medline]
19. Brown GR, Kohler M, Berggren PO: Parallel changes in nuclear and cytosolic calcium in mouse pancreatic beta-cells. Biochem J325 :771 –778,1997
20. Warnotte C, Gilon P, Nenquin M, Henquin JC: Mechanisms of the stimulation of insulin release by saturated fatty acids: a study of palmitate effects in mouse beta-cells. Diabetes43 :703 –711,1994[Abstract]
21. Liu GX, Hanley PJ, Ray J, Daut J: Long-chain acyl-coenzyme A esters and fatty acids directly link metabolism to K_ATP channels in the heart. Circ Res88 :918 –924,2001[Abstract/Free Full Text]
22. Martin F, Pintor J, Rovira JM, Ripoll C, Miras-Portugal MT, Soria B: Intracellular diadenosine polyphosphates: a novel second messenger in stimulus-secretion coupling. FASEB J12 :1499 –1506,1998[Abstract/Free Full Text]
23. Ropero AB, Fuentes E, Rovira JM, Ripoll C, Soria B, Nadal A: Non-genomic actions of 17beta-oestradiol in mouse pancreatic beta-cells are mediated by a cGMP-dependent protein kinase. J Physiol521 :397 –407,1999[Abstract/Free Full Text]
24. Gilon P, Jonas JC, Henquin JC: Culture duration and conditions affect the oscillations of cytoplasmic calcium concentration induced by glucose in mouse pancreatic islets. Diabetologia37 :1007 –1014,1994[Medline]
25. Antoine MH, Lebrun P, Herchuelz A: Cell culture conditions influence glucose-induced [Ca²⁺]_i responses in isolated rat pancreatic B cells. Adv Exp Med Biol426 :227 –230,1997[Medline]

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Quesada, I. Articles by Soria, B. Search for Related Content PubMed PubMed Citation Articles by Quesada, I. Articles by Soria, B. Social Bookmarking                What's this?