Pulsatile Insulin Release from Pancreatic Islets with Nonoscillatory Elevation of Cytoplasmic Ca21 Johanna Westerlund, Erik Gylfe, and Peter Bergsten Department of Medical Cell Biology, University of Uppsala, S-751 23 Uppsala, Sweden Abstract early that Ca21 omission (12, 13) or addition of blockers of voltage-dependent Ca21 channels (14) abolishes glucose-stim- The relationship between insulin release and cytoplasmic ulated insulin secretion. Moreover, glucose and other stimula- 21 21 21 Ca concentration ([Ca ]i) was studied in isolated pancre- tors of insulin secretion induce elevation of [Ca ]i in b cells 21 atic islets from ob/ob mice. Although [Ca ]i was low and (15), and insulin secretion from permeabilized b cells is stimu- stable in the presence of 3 mM glucose, basal insulin release lated by a rise in the external Ca21 concentration (16). Since exhibited low amplitude pulsatility, with a frequency of the discovery that glucose induces large-amplitude oscillations 21 1 21 0.3260.04 min . Depolarization by raising K from 5.9 to of [Ca ]i in individual b cells (17), it has been demonstrated 30.9 mM or by the addition of 1 mM tolbutamide caused a that these oscillations spread by gap-junctional coupling to pronounced initial insulin pulse followed by declining neighboring cells (18) and become synchronized in the entire pulses, but there was no change in frequency. This decline pancreatic islet (19). With the development of a sensitive in amplitude of the insulin pulses was prevented in similar ELISA (1), it was possible to verify that these oscillations oc- experiments performed in the presence of 11 mM glucose. cur in synchrony with pulsatile release of insulin (19). Conse- 21 Corresponding measurements of [Ca ]i in islets exposed to quently, there is strong evidence supporting an imperative role tolbutamide or the high K1 concentration revealed stable el- for Ca21 as an initiator of insulin secretion. 21 evations without oscillations. Although the [Ca ]i level is Circulating insulin not only oscillates postprandially; there an important determinant for the rate of secretion, the re- are also regular variations of insulin in the fasting state (4, 5, sults indicate that pulsatile insulin release does not always 20). Since b cells and pancreatic islets show low and stable 21 21 depend on [Ca ]i oscillations. It is suggested that cyclic [Ca ]i at nonstimulatory glucose concentrations (17–19), it is generation of ATP may fuel pulsatile release under condi- tempting to attribute the insulin oscillations in the fasting state 21 tions when [Ca ]i remains stable. (J. Clin. Invest. 1997. to a small basal secretion stimulated by the combined effect of 100:2547–2551.) Key words: insulin secretion • tolbutamide • different nutrients and hormones like glucagon. However, we depolarization • islets of Langerhans • adenosine triphos- also found recently that basal secretion of insulin from individ- phate ual pancreatic islets exposed to nutrient-free medium exhibits 21 pulsatile release of insulin despite a low and stable [Ca ]i (21). Introduction We have now examined further the relationship between 21 [Ca ]i and insulin secretion in individual pancreatic islets de- Pulsatile secretion of insulin has been observed from individ- polarized by exposure to the hypoglycemic sulfonylurea tol- ual pancreatic islets (1) as well as from the isolated perfused butamide or to a high concentration of K1. Both of these con- pancreas (2). It is believed that this pulsatility is causing the ditions are known to be associated with a sustained elevation 21 21 pronounced oscillations of insulin in the portal vein (3, 4) and of [Ca ]i. It was found that this stable elevation of [Ca ]i had the more dampened ones in the periphery (3, 5, 6). Such varia- a dramatic effect in transforming the tiny basal insulin pulses tions are essential for the effectiveness of the hormone as a hy- into much more pronounced ones. This observation seems to poglycemic agent (7–9), and the early disappearance of these indicate a more permissive role for Ca21 in insulin secretion. regular oscillations in type 2 diabetes is associated with insulin Since sulfonylureas are commonly used antidiabetic agents, it resistance and hyperglycemia (10, 11). is advantageous that they do not interfere with pulsatile insulin The regulatory role of the cytoplasmic Ca21 concentration release, which is considered important for keeping the hor- 21 1 ([Ca ]i) in insulin secretion is well-established. It was shown mone receptors upregulated (22). Methods Address correspondence to Dr. Peter Bergsten, Department of Materials. Reagents of analytical grade and deionized water were Medical Cell Biology, University of Uppsala, Box 571, S-751 23 Upp- used. Collagenase, Hepes, and BSA (fraction V) were obtained from sala, Sweden. Phone: 46-18-471-4923; FAX: 46-18-471-4059; E-mail: Boehringer Mannheim GmbH (Mannheim, Germany). Tetramethyl- [email protected] benzidine and insulin peroxidase came from Sigma Chemical Co. (St. Received for publication 10 February 1997 and accepted in revised Louis, MO). The rat insulin standard was from Novo Nordisk A/S form 22 September 1997. (Bagsvaerd, Denmark). IgG-certified microtiter plates were pur- chased from Nunc, Inc. (Roskilde, Denmark). Tolbutamide was a gift 21 21 1. Abbreviation used in this paper: [Ca ]i, cytoplasmic Ca concen- from Hoechst AG (Frankfurt, Germany). The mouse insulin antibod- tration. ies were raised in our laboratory in guinea pigs. General design of experiments. Pancreatic islets were collage- J. Clin. Invest. nase-isolated from overnight-starved ob/ob mice taken from a local 21 © The American Society for Clinical Investigation, Inc. colony (23). Islets used for the combined measurements of [Ca ]i 0021-9738/97/11/2547/05 $2.00 and insulin release were cultured overnight in RPMI 1640 medium Volume 100, Number 10, November 1997, 2547–2551 supplemented with 5.5 mM glucose and 10% FCS, whereas freshly http://www.jci.org isolated islets were used when measuring insulin release alone. All Pulsatile Insulin Release at Stable Cytoplasmic Ca21 2547 perifusions were performed in a medium supplemented with 1 mg/ml albumin and containing (mM): NaCl 125, KCl 5.9, MgCl2 1.2, CaCl2 1.28, and Hepes 25, titrated to pH 7.4 with NaOH. When KCl was in- creased to 30.9 mM, NaCl was isoosmotically reduced. Combined measurements of cytoplasmic Ca21 and insulin release. The combined measurements were performed adhering to a previous protocol (24). A single islet was loaded with the acetoxymethyl ester of fura-2 (2 mM) for 40 min in the presence of 3 mM glucose. After rinsing, the islet was allowed to attach to the central part of a cover- slip coated with poly-L-lysine. The coverslip was part of a 15-ml peri- fusion chamber that was placed on the stage of an inverted micro- scope (Diaphot; Nikon Inc., Melville, NY) within a climate box maintained at 378C. The microscope was equipped for epifluores- cence fluorometry with a 400-nm dichroic mirror, a 3100 ultraviolet fluorite objective, and a photomultiplier. The islet was perifused at a 21 rate of 150–200 ml/min, and [Ca ]i was recorded with dual wave- length fluorometry, with excitation at 340 and 380 nm and emission at 510 nm (25). For determination of insulin, the perifusate was col- lected in 20-s fractions that were cooled immediately on ice. Measurements of insulin release alone. The kinetics of insulin re- lease alone were studied as described previously (1). A single islet was placed in a thermostated (378C) 10-ml chamber and perifused at a rate of 150–200 ml/min with medium containing 3 mM glucose. After 60–75 min of introductory perifusion, the perifusate was collected in 20-s fractions and cooled immediately on ice. Insulin was assayed by a competitive ELISA with the insulin antibody immobilized directly onto the solid phase (1). Amounts of insulin down to 100 amol were 1 21 Figure 1. Effects of K depolarization on [Ca ]i (top) and insulin re- obtained from linear standard curves in semilogarithmic plots. The lease (bottom) of an individual pancreatic islet. The islet was cultured rate of insulin release was normalized to dry weight after freeze-dry- overnight in the presence of 5.5 mM glucose and loaded with the Ca21 ing and weighing the islets on a quartz fiber balance. To assure that indicator fura-2 for an incubation of 40 min in medium containing the amounts of insulin in each fraction were within the linear part of 3 mM glucose. The traces start after subsequent perifusion for 20 min the standard curve, islets weighing between 5 and 10 mg were se- in similar medium lacking indicator. Arrows, Elevation of K1 concen- lected. tration from 5.9 to 30.9 mM. Representative experiment of four. Data analysis. The frequencies of pulsatile insulin release were analyzed by Fourier transformation using Igor software (WaveMet- rics, Lake Oswego, OR). The figures show three point moving aver- ages. Other data are presented as means6SEM. Differences in secre- tory rates and frequencies were evaluated with Student’s t test for paired observations. Results 21 The effects of depolarization on [Ca ]i and insulin release were studied simultaneously in individual pancreatic islets that had been cultured overnight in the presence of 5.5 mM glu- cose. During subsequent perifusion with medium containing 3 mM glucose, increase in the K1 concentration from 5.9 to 30.9 mM (Fig. 1) or addition of 1 mM tolbutamide (Fig. 2) caused 21 elevation of [Ca ]i paralleled by marked stimulation of insulin 21 release. However, whereas the elevation of [Ca ]i was sus- tained, the stimulated release of insulin occurred in pro- nounced pulses, with gradually decreasing amplitudes and fre- quencies of 0.2–0.3 min21.