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Endocrinology Vol. 138, No. 8 3161-3165
Copyright © 1997 by The Endocrine Society

ARTICLES

Oscillatory Signaling and Insulin Release in Human Pancreatic ß-Cells Exposed to Strontium1

Bo Hellman, Erik Gylfe, Peter Bergsten, Eva Grapengiesser, Alf Berts, Yi-Jia Liu, Anders Tengholm and Johanna Westerlund

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden

Address all correspondence and requests for reprints to: Prof. Bo Hellman, Department of Medical Cell Biology, Biomedicum, Box 571, S-751 23 Uppsala, Sweden.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oscillatory signaling and insulin release were studied in isolated pancreatic islets and ß-cells obtained from human cadaveric organ donors. Taking advantage of Sr2+ as an analog for Ca2+, it was possible to demonstrate glucose-induced rhythmic activity in individual ß-cells identified by immunostaining. Glucose-induced slow oscillations of Sr2+ (frequency, 0.1–1.0/min) were sometimes seen at a sugar concentration as low as 3 mM. Addition of 20 nM glucagon resulted in a broadening of the oscillations or in their transformation into sustained elevation. Moreover, the presence of glucagon resulted in the appearance of short transients of Sr2+, which disappeared after exposure to the intracellular Ca2+-adenosine triphosphatase inhibitor thapsigargin. Digital image analyses indicated that slow oscillations can be synchronized among cells in small aggregates and intact islets. The rhythmic activity in the glucose-stimulated ß-cell had its counterpart in pulsatile insulin release when single islets were perifused with a Sr2+-containing medium. It is concluded that the human ß-cell has oscillatory signaling for insulin release similar to that observed in experimental animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOSE stimulation of insulin release can be attributed to an increase in cytoplasmic Ca2+ in the pancreatic ß-cell due to opening of voltage-dependent Ca2+ channels in the plasma membrane (1, 2). This rise of cytoplasmic Ca2+ is usually manifested as slow oscillations from a basal level (3). There are reasons to believe that the rhythmic Ca2+ signals, after being synchronized within and between the islets, generate pulsatile insulin release responsible for the cyclic variations of the hormone in the blood (4, 5).

Although most of the basic studies of insulin release have been performed in experimental animals, oscillatory Ca2+ signaling is probably also important for human ß-cells. Cyclic variations in circulating insulin are well established (6, 7, 8, 9), and it has been reported that isolated human islets respond to glucose with cytoplasmic Ca2+ oscillations (10, 11, 12) and pulsatile release of insulin (10, 13). To date, cytoplasmic Ca2+ has been measured in human ß-cells identified by their responses to glucose and sulfonylureas (10, 14). However, recent studies indicate that this identification procedure does not discriminate between ß-cells and somatostatin-producing {delta}-cells (15).

In the present report evidence will be provided for glucose-induced rhythmic activity in human ß-cells identified by immunostaining for insulin. Taking advantage of previous observations that replacement of Ca2+ with its analog Sr2+ facilitates the demonstration of a rhythmic behavior (16), it is shown that the human ß-cell has oscillatory signaling for insulin release similar to that observed in experimental animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Reagents of analytical grade and deionized water were used. Collagenase, HEPES, and BSA (fraction V) were obtained from Boehringer Mannheim (Mannheim, Germany). Fura-2/acetoxymethyl ester was purchased from Molecular Probes (Eugene, OR), and material for insulin immunostaining was provided by Dako Corp. (Carpenteria, CA). IgG-certified microtiter plates were purchased from Nunc (Roskilde, Denmark). Tetramethylbenzidine, insulin peroxidase, and thapsigargin were obtained from Sigma Chemical Co (St. Louis, MO). Rat insulin standard and crystalline porcine glucagon were gifts from Novo Nordisk (Bagsvaerd, Denmark), and tolbutamide was donated by Hoechst (Frankfurt/Main, Germany).

Preparation of intact islets and single cells
Collagenase-isolated human islets of Langerhans were obtained from the Central Unit of ß-Cell Transplant, Vrije Universiteit Brussels (Brussels, Belgium) (10). The islets were taken from seven cadaveric organ donors, 34–49 yr old. After transport to Uppsala, the islets were cultured at 37 C in an atmosphere of 5% CO2 in humidified air in RPMI 1640 medium supplemented with 10% FCS, 100 IU penicillin, 100 µg/ml streptomycin, and 10 µg/ml gentamicin and containing 5.6 mM glucose. Three to 7 days after isolation, some of the islets were dispersed into single cells by suction into a narrow capillary. The single cells were then allowed to attach to circular 25-mm cover glasses during culture for another 1–3 days. Subsequent experiments with intact islets or single cells were performed at 37 C with a basal medium containing 125 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 5 mM SrCl2, 0.5 mg/ml albumin, and 25 mM HEPES. NaOH was used for adjusting the pH to 7.40.

Measurements of cytoplasmic Sr2+
The indicator fura-2 was introduced into the cells during 30–40 min incubation at 3 mM glucose with fura-2/acetoxymethyl ester at a concentration of 1 µM (cells attached to coverglasses) or 2 µM (free floating islets). Before measuring cytoplasmic Sr2+, the fura-2-loaded islets were placed in the center of a coverglass coated with poly-L-lysine. The coverglasses with attached cells or islets were then used as bottoms of an open chamber placed on the stage of an inverted microscope and superfused at a rate of 0.75 mL/min at 37 C. Thapsigargin, which sticks to plastic, was added directly to the superfusion chamber. The perifusion flow was then interrupted for 2–3 min to ascertain an effect of the drug.

As the Sr2+ complex of fura-2 has spectral properties similar to those of Ca2+ (17), it was possible to measure its cytoplasmic concentration using dual wavelength fluorometry according to the principles of Grynkiewicz et al. (18). Fluorescence emitted at 510 nm was recorded with a photomultipler or an intensified video camera (19). The latter approach made it possible to determine whether oscillatory Sr2+ signaling is synchronized among cells situated in clusters or in different parts of intact islets. Autofluorescense was negligible and not compensated for. In accordance with our previous measurements of Sr2+ in pancreatic ß-cells (16), the cytoplasmic concentration of this ion was presented as the 340/380 nm fluorescence excitation ratio.

Identification of ß-cells
Measurements of Sr2+ were followed by immunostaining for insulin of oscillating cells remaining in position in the perifusion chamber. The cells were fixed during 5-min exposure to 95% ethanol, rinsed with water, and then stained using a peroxidase-antiperoxidase technique (20).

Measurements of insulin release
Perifusate from individual islets was collected in 20-sec fractions and immediately cooled on ice. Insulin was measured as previously described (21), using competitive enzyme-linked immunosorbent assay with the insulin antibody immobilized directly onto the solid phase. The rate of insulin release was normalized to dry weight after freeze-drying and weighing the islets on a quartz fiber balance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Slow oscillations of Sr2+ (frequency, 0.1–1.0/min) were seen in individual ß-cells perifused with a glucose-containing medium. The frequencies and amplitudes of these oscillations remained essentially unaffected after raising the glucose concentration from 3 to 20 mM (Fig. 1AGo). When not present at 3 mM glucose, the rhythmic activity often appeared in response to 20 mM of the sugar (Fig. 1BGo). Addition of glucagon resulted in prolongation of the oscillations (Fig. 1AGo) or their transformation into a sustained elevation (Fig. 1BGo). Moreover, the presence of glucagon was often accompanied by the appearance of short (<10 sec) transients superimposed on the slow oscillations (Figs. 2Go and 3Go). These transients disappeared when the intracellular Ca2+-adenosine triphosphatase activity was blocked with thapsigargin. Addition of tolbutamide resulted in a prompt rise in the cytoplasmic Sr2+ concentration in a medium lacking glucose (Fig. 3Go).



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  		Figure 1. Oscillations of cytoplasmic Sr2+ in individual ß-cells during perifusion with 3 and 20 mM glucose. Addition of 20 nM glucagon resulted in prolongation of the oscillations (A) or their transformation into sustained elevation (B). Data shown are from a representative experiment of five performed.

 


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  		Figure 2. Oscillations of cytoplasmic Sr2+ in individual ß-cells during perifusion with 20 mM glucose. The addition of 20 nM glucagon resulted in the appearance of short transients superimposed on the top of the oscillations. These transients disappeared after a period of short exposure to 100 nM thapsigargin (TH). Data shown are from a representative experiment of three performed.

 


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  		Figure 3. Oscillations of cytoplasmic Sr2+ in an individual ß-cell during perifusion with 20 mM glucose. Glucagon, thapsigargin (TH), and tolbutamide (TOL) were present at concentrations of 20 nM, 100 nM, and 100 µM, respectively, during the periods indicated. B–D show at an expanded time scale sections marked with the corresponding letters in A.

 
The Sr2+ signals were synchronized in clusters of ß-cells. The synchronization included both the oscillations obtained when raising the glucose level to 11 mM or more and the increase seen in response to tolbutamide (Fig. 4Go). Also, intact islets showed rhythmic activity with slow oscillations. The proportion of islets with such a rhythmicity was 61% at 3 mM glucose (19 of 31 islets). At 11 mM glucose, the corresponding proportion was 91% (32 of 35 islets). The oscillations were often well synchronized among different parts of the islets (Fig. 5Go).



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  		Figure 4. Synchronization of cytoplasmic Sr2+ oscillations in a cluster of three ß-cells exposed to different concentrations of glucose. Tolbutamide was added at the end of the experiment at a concentration of 100 µM. The variations in cytoplasmic Sr2+ are shown for each of the cells illustrated in the lower right. The oscillatory Sr2+ signals obtained with rise of the glucose concentration were synchronized in all seven studied clusters.

 


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  		Figure 5. Oscillations of cytoplasmic Sr2+ in an intact islet during perifusion with 11 mM glucose. The traces exemplify synchronization among the regions illustrated in the lower right. Data shown are from a representative experiment of three performed.

 
Insulin release from individual islets was markedly stimulated when the glucose concentration was raised from 3 to 11 mM during perifusion with a Sr2+-containing medium. The glucose effect was manifested as secretory pulses with a frequency of 0.2–0.5/min (Fig. 6Go).



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  		Figure 6. Pulsatile release of insulin from an intact islet after raising the glucose concentration from 3 to 11 mM. Data shown are from a representative experiment of six performed.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the discovery in 1988 that glucose induces cytoplasmic Ca2+ oscillations in individual mouse ß-cells (3), the proportion of oscillating cells observed in similar experiments has gradually increased (5). This increase reflects the awareness that even minor damage to the ß-cells results in transformation of the oscillatory response into a sustained elevation of cytoplasmic Ca2+ (4, 5). By improving the procedures for isolating islets and cells and minimizing the exposure to UV light during the measurements of cytoplasmic Ca2+, it has become evident that an oscillatory response is the normal reaction to physiological stimulation not only of ß-cells from different animals, but also of glucagon-secreting {alpha}-cells and somatostatin-releasing {delta}-cells (20, 22). It is apparent that gentle isolation conditions can be used when studying pancreatic islets from the thin pancreas in animals such as mice and rats. Isolation of adequately responding human ß-cells are complicated not only by their location within a solid and fibrous pancreas, but also because of the difficulty of avoiding ischemia during the period preceding the isolation of the islets. Due to these problems and the limited access to human material, the present study was focused on the demonstration of oscillatory activities rather than on providing information about the proportion of ß-cells with such a response.

Previous studies have indicated that Sr2+ is a useful analog for Ca2+ when demonstrating oscillatory activities in mouse ß-cells (16). Compared with the glucose-induced oscillations of cytoplasmic Ca2+, those of Sr2+ are more stable and do not transform as easily into sustained elevation. The reason why a replacement of Ca2+ with Sr2+ facilitates the demonstration of a rhythmic behavior is open for discussion. As the slow oscillations probably reflect periodic variations in the membrane potential (4, 5), it is pertinent to ask whether proportionately more Sr2+ than Ca2+ enters the ß-cells via voltage-dependent channels. Part of the Ca2+ uptake into ß-cells from mice and rats has been reported to be regulated by the state of filling of intracellular calcium stores (23, 24, 25). Studies of mast cells have shown that Sr2+ is one among several blockers of such a capacitative entry of Ca2+ (26).

The human ß-cells reacted to a rise in the glucose concentration in a manner similar to that of mouse ß-cells (16) by responding with slow oscillations of cytoplasmic Sr2+. These oscillations, like those of Ca2+, probably reflect periodic entry of the ion due to cyclic variations in membrane potential. Such depolarizations can be expected to have a metabolic origin, depending on the closure of ATP-sensitive K+ channels (4, 5). Electrophysiological studies of human ß-cells (27) have revealed cyclic variations in the K-ATP current, with frequencies equivalent to those observed for the oscillations of Ca2+ and now for Sr2+.

This report is the first description of rhythmic activity in human ß-cells adequately identified by immunostaining. Selection based on cytoplasmic Ca2+ responses to glucose or sulfonylurea stimulation will not discriminate between ß-cells and somatostatin-producing {delta}-cells (15). Nevertheless, measurements of cytoplasmic Ca2+ in such a heterogeneous cell population (10) have provided data similar to those presented here. The observation that ß-cells react similar to cells selected on the basis of a functional response to glucose and sulfonylureas is not surprising when taking into account that {delta}-cells mimic ß-cells with regard to oscillatory Ca2+ activities (15, 22).

Studies at 3 mM glucose have indicated low and stable cytoplasmatic concentrations of Ca2+ and Sr2+ in mouse ß-cells (5, 16) and of Ca2+ in human islet cells responsive to sulfonylureas (10). The present observations of oscillatory activity in human ß-cells exposed to 3 mM glucose can probably be explained by the use of Sr2+ as an analog for Ca2+ in combination with prolonged culture. Most mouse ß-cells exhibit Sr2+ oscillations in the presence of 7 mM glucose, whereas 11 mM of the sugar is usually required for a corresponding Ca2+ response (16). Moreover, it was previously shown that culture for 1 week is associated with a leftward shift of the dose-response curve for glucose-induced insulin release from mouse islets (28). As the proportion of human ß-cells and islets responding with oscillations increased with the rise in the glucose concentration, there are reasons to believe that, as in mice and rats (4, 5), a major effect of glucose is to transform the resting ß-cell into its functionally active oscillatory state.

Addition of glucagon not only modifies the existing oscillatory activity in the glucose-stimulated ß-cell, but also resulted in the appearance of pronounced transients of Sr2+. As these transients are effectively removed by the Ca2+-adenosine triphosphatase inhibitor thapsigargin, it is likely that they reflect mobilization of Sr2+ from intracellular stores. Although the present data do not allow definite conclusions about the mechanisms involved, it is important to note that glucagon has a similar action on mouse ß-cells. Analyzing the latter effect in more detail, it has been proposed that depolarization-dependent formation of inositol trisphosphate results in intracellular mobilization of Ca2+ when the inositol trisphosphate receptors are sensitized by cAMP (16).

The important conclusion from this report is that the human ß-cell has similar oscillatory signaling for insulin release as observed in experimental animals. Accordingly, the cyclic variations in circulating insulin can be supposed to reflect an intrinsic ability of the ß-cell to produce secretory signals synchronized within and among the islets. In providing information about a rhythmic behavior equivalent to that seen with Ca2+ and Sr2+ in experimental animals (5, 16), the present study supports the idea that it is the recruitment of ß-cells into the oscillatory state rather than alterations in the amplitude and frequency of the signal that determine the insulin secretory response to glucose.


   Footnotes
 
1 This work was supported by grants from the Swedish Medical Research Council (12X-562, 12X-6240, and 12X-11203), the Swedish Diabetes Association, Novo-Nordisk Foundation, Novo Nordisk Pharma, and the Family Ernfors Foundation. This study made use of human islets prepared by the Central Unit of ß-Cell Transplant, supported by a shared costs action of the European Community. Back

Received January 15, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hellman B, Andersson T, Berggren PO, Flatt P, Gylfe E, Kohnert KD 1979 The role of calcium in insulin secretion. In: Hormones and Cell Regulation. Dumont J, Nunez J (eds) Elsevier-North Holland, Amsterdam, vol 3:69–96
  2. Wollheim CB, Sharp GW 1981 Regulation of insulin release by calcium. Physiol Rev 61:914–973[Free Full Text]
  3. Grapengiesser E, Gylfe E, Hellman B 1988 Glucose-induced oscillations of cytoplasmic Ca2+ in the pancreatic ß-cell. Biochem Biophys Res Commun 151:1299–1304[CrossRef][Medline]
  4. Hellman B, Gylfe E, Grapengiesser E, Lund PE, Berts A 1992 Cytoplasmic Ca2+ oscillations in pancreatic ß-cells. Biochim Biophys Acta 1113:295–305[Medline]
  5. Hellman B, Gylfe E, Bergsten P, Grapengiesser E, Lund PE, Saha S, Berts A, Dryselius S, Tengholm A, Liu YJ, Eberhardson M 1994 Pulsatile Ca2+ signalling and insulin release. In: Flatt PR, Lenzen S (eds) Insulin Secretion and Pancreatic ß-Cell Research. Smith-Gordon, London, pp 221–228
  6. Lefèbvre PJ, Paolisso G, Scheen AJ, Henquin JC1987 Pulsatility of insulin and glucagon release: physiological significance and pharmacological implications. Diabetologia 30:443–452
  7. Hansen BC, Jen KC, Pek SB, Wolfe RA 1982 Rapid oscillations in plasma insulin, glucagon and glucose in obese and normal weight humans. J Clin Endocrinol Metab 54:785–792[Abstract/Free Full Text]
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  9. Sonnenberg GE, Hoffman RG, Johnson CP, Kissebah PH 1992 Low- and high-frequency insulin secretion pulses in normal subjects and pancreas transplant recipients: role of extrinsic innervation. J Clin Invest 90:545–553
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  11. Kindmark H, Köhler M, Arkhammar P, Efendic S, Larsson O, Linder S, Nilsson T, Berggren PO 1994 Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance. Diabetologia 37:1121–1131[Medline]
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  15. Hellman B, Berts A, Liu YJ, Gylfe E 1996 Ca2+ signalling in somatostatin-producing cells from the human pancreas. Diabetologia [Suppl 1] 39:A118
  16. Liu YJ, Grapengiesser E, Gylfe E, Hellman B 1996 Crosstalk between the cAMP and inositol trisphosphate-signalling pathways in pancreatic ß-cells. Arch Biochem Biophys 334:295–302[CrossRef][Medline]
  17. Kwan CY, Putney Jr JW 1990 Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool. J Biol Chem 265:678–684[Abstract/Free Full Text]
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  21. Bergsten P, Hellman B 1993 Glucose-induced amplitude regulation of pulsatile insulin secretion from individual pancreatic islets. Diabetes 42:670–674[Abstract]
  22. Berts A, Ball A, Dryselius S, Gylfe E, Hellman B 1996 Glucose stimulation of somatostatin-producing islet cells involves oscillatory Ca2+ signalling. Endocrinology 137:693–697[Abstract]
  23. Leech CA, Holz GG, Habener JF 1994 Voltage-independent calcium channels mediate slow oscillations of cytosolic calcium that are glucose-dependent in pancreatic ß-cells. Endocrinology 135:365–372[Abstract]
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  27. Dryselius S, Lund PE, Gylfe E, Hellman B 1994 Variations in ATP-sensitive K+ channel activity provide evidence for inherent metabolic oscillations in pancreatic ß-cells. Biochem Biophys Res Commun 205:880–885[CrossRef][Medline]
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