Do Oscillations of Insulin Secretion Occur in the Absence of Cytoplasmic Ca2+ Oscillations in  -Cells?

Do Oscillations of Insulin Secretion Occur in the Absence of Cytoplasmic Ca2ⴙ Oscillations in ␤-Cells? Lise L. Kjems, Magalie A. Ravier, Jean-Christophe Jonas, and Jean-Claude Henquin That oscillations of the cytoplasmic free Ca2ⴙ concentration ([Ca2ⴙ]i) in ␤-cells induce oscillations of insulin secretion is not disputed, but whether metabolismdriven oscillations of secretion can occur in the absence of [Ca2ⴙ]i oscillations is still debated. Because this possibility is based partly on the results of experiments using islets from aged, hyperglycemic, hyperinsulinemic ob/ob mice, we compared [Ca2ⴙ]i and insulin secretion patterns of single islets from 4- and 10-month-old, normal NMRI mice to those of islets from 7- and 10-monthold ob/ob mice (Swedish colony) and their lean littermates. The responses were subjected to cluster analysis to identify significant peaks. Control experiments without islets and with a constant insulin concentration were run to detect false peaks. Both ob/ob and NMRI islets displayed large synchronous oscillations of [Ca2ⴙ]i and insulin secretion in response to repetitive depolarizations with 30 mmol/l Kⴙ in the presence of 0.1 mmol/l diazoxide and 12 mmol/l glucose. Continuous depolarization with high Kⴙ steadily elevated [Ca2ⴙ]i in all types of islets, with no significant oscillation, and caused a biphasic insulin response. In islets from young (4-month-old) NMRI mice and 7-month-old lean mice, the insulin profile did not show significant peaks when [Ca2ⴙ]i was stable. In contrast, two or more peaks were detected over 20 min in the response of most ob/ob islets. Similar insulin peaks appeared in the insulin response of 10-month-old lean and NMRI mice. However, the size of the insulin peaks detected in the presence of stable [Ca2ⴙ]i was small, so that no more than 10 –13% of total insulin secretion occurred in a pulsatile manner. In conclusion, insulin secretion does not oscillate when [Ca2ⴙ]i is stably elevated in ␤-cells from young normal mice. Some oscillations are observed in aged mice and are seen more often in ob/ob islets. These fluctuations of the insulin secretion rate at stably elevated [Ca2ⴙ]i, however, are small compared with the large oscillations induced by [Ca2ⴙ]i oscillations in ␤-cells. Diabetes 51 (Suppl. 1):S177–S182, 2002 From the Unité d’Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, Brussels, Belgium. Address correspondence and reprint requests to henquin@endo.ucl.ac.be. Accepted for publication 12 June 2001. L.L.K. and M.A.R. contributed equally to the study. [Ca2⫹]i, cytoplasmic free Ca2⫹ concentration. The symposium and the publication of this article have been made possible by an unrestricted educational grant from Servier, Paris. DIABETES, VOL. 51, SUPPLEMENT 1, FEBRUARY 2002 G lucose induces insulin secretion by activating two pathways, both of which require metabolism of the sugar by ␤-cells (1). The triggering pathway involves membrane depolarization, Ca2⫹ influx, and rise in the cytoplasmic free Ca2⫹ concentration ([Ca2⫹]i). The amplifying pathway increases the efficacy with which Ca2⫹ promotes exocytosis of insulin granules. Insulin secretion is characterized by a pulsatility that is reflected by oscillations of plasma insulin concentration (2– 6). During continuous glucose stimulation, the triggering signal, [Ca2⫹]i, oscillates synchronously in all ␤-cells of each islet, and each of these oscillations induces a pulse of insulin secretion (7). Several in vitro studies have established that oscillations of insulin secretion are temporally correlated with [Ca2⫹]i oscillations (7–10). Because insulin secretion also closely followed [Ca2⫹]i oscillations imposed by repetitive depolarizations of ␤-cells with high K⫹ and was stable during sustained elevation of [Ca2⫹]i (11,12), we concluded that Ca2⫹ is the direct regulator of insulin pulsatility. Dissociations between the two phenomena, however, have been reported and have prompted the suggestion that Ca2⫹ has only a permissive role, whereas a cyclic metabolic signal drives oscillations of secretion even in the presence of stable [Ca2⫹]i (13,14). Therefore, we directly tested this possibility by imposing metabolic oscillations in single islets. Although oscillations of metabolism could induce oscillations of insulin secretion in the absence of [Ca2⫹]i oscillations (through the amplifying pathway), their efficacy was clearly less than that of [Ca2⫹]i oscillations (12). The hypothesis that oscillations of insulin secretion can occur in the absence of [Ca2⫹]i oscillations is based partly on experiments using islets from aged (8 –12 months) ob/ob mice (14) that are leptin-deficient, obese, hyperglycemic, and hyperinsulinemic (15–17). In contrast, our previous experiments have been performed with young (3– 4 months), normoglycemic mice. In the present study, we have thus measured simultaneously insulin secretion and [Ca2⫹]i in single islets from different types of mice. Islet [Ca2⫹]i was stably elevated by a sustained depolarization with high K⫹. The insulin secretion profiles were then subjected to mathematical analysis to assess the presence of oscillations and determine the contribution of these oscillations to the whole responses. RESEARCH DESIGN AND METHODS Preparation. The experiments were performed with pancreatic islets isolated from three types of mice: noninbred female NMRI mice from a local S177 Ca2ⴙ OSCILLATIONS IN ␤-CELLS AND INSULIN SECRETION TABLE 1 Characteristics of the mice 7 months 10 months Lean (⫹/⫹ or ob/⫹) 7 months 10 months 6 7 (7–9) 73 ⫾ 3.4* 9.9 ⫾ 0.8* 342 ⫾ 73* 12 10 (10–11) 69 ⫾ 2.1* 10.7 ⫾ 1.0* 197 ⫾ 48* 8 7 (6–8) 26 ⫾ 1.0 6.8 ⫾ 0.4 1.1 ⫾ 0.2 ob/ob n Age (months) Body weight (g) Blood glucose (mmol/l) Plasma insulin (ng/ml) 6 10 (10–11) 26 ⫾ 1.4 6.7 ⫾ 0.3 1.9 ⫾ 0.4 NMRI Young Old 8 4 33 ⫾ 0.5 6.3 ⫾ 0.2 0.8 ⫾ 0.2 5 10 (9–10) 32 ⫾ 2.9 5.8 ⫾ 0.6 1.2 ⫾ 0.2 Data are modes (range) or means ⫾ SE. The mice were killed by decapitation in the fed state. *P ⬍ 0.01 or less vs. all other groups (Newman-Keuls test). colony and noninbred female obese ob/ob mice or their lean littermates (ob/⫹ or ⫹/⫹) from the Umea colony (provided by J. Sehlin, University of Umea, Sweden). After their transfer from Sweden to Brussels, the mice were allowed to adapt to their novel environment for at least 2 weeks. Because Swedish ob/ob mice are traditionally used as islet donors at an age between 8 and 12 months (8,18,19), lean and NMRI mice were grown until a similar advanced age for comparison. The animals were killed by decapitation in the fed state. Blood glucose was measured with a Glucometer (Bayer AG, Zurich, Switzerland), and plasma was saved for insulin assay. After digestion of the pancreas with collagenase, the islolated islets were hand-picked (11) and cultured for 16 –24 h in RPMI medium containing 5.5 or 10 mmol/l glucose. Solutions. The medium used for islet isolation and for the experiments after islet culture was a bicarbonate-buffered solution that contained (in mmol/l): NaCl 120, KCl 4.8, CaCl2 2.5, MgCl2 1.2, and NaHCO3 24. It was gassed with O2/CO2 (94%/6%) to maintain a pH of 7.4 and was supplemented with 1 mg/ml BSA. When the concentration of KCl was raised to 30 mmol/l, that of NaCl was decreased accordingly. During the experiments, the solutions were supplemented with 0.1 mmol/l diazoxide to suppress the spontaneous [Ca2⫹]i oscillations otherwise induced by glucose and to ensure complete control of membrane potential and [Ca2⫹]i by the concentration of extracellular K⫹. Simultaneous measurements of [Ca2ⴙ]i and insulin secretion. All experiments were performed with single islets as previously reported (7,11,12). In brief, one islet was loaded with fura-PE3 during 2 h incubation at 37°C in a medium containing 2 ␮mol/l fura-PE3 acetoxymethylester and the same glucose concentration (5.5 or 10 mmol/l) as that during the culture. The islet was then transfered into a perifusion chamber (110 ␮l) placed on the stage of an inverted microscope. [Ca2⫹]i was monitored by microspectrofluorimetry (20) at a resolution of one measure every 3.12 s. The flow rate was 1.8 ml/min and the effluent fractions were collected at 30-s intervals. Insulin was measured in duplicate in 400-␮l aliquots of the effluent fractions. The characteristics of the radioimmunoassay have been reported elsewhere (11). The size of the islet used for the experiment was estimated from its largest and smallest diameters measured on the screen of the recording system. The volume was then calculated assuming an ovoid shape. Control experiments without islets. Because false oscillations of insulin secretion could result from assay noise, experiments were performed without islets. Rat insulin was added at concentrations ranging from 22 to 310 pg/ml to the perifusion buffer. In some experiments, the medium was run through the recording systems and insulin was assayed in 40 consecutive fractions collected at 30-s intervals; in other experiments, the assay was repeated 40 times with the same medium. Otherwise, the characteristics of the assay were identical to those for the real experiments. The 15 insulin concentration profiles so obtained were subjected to the same mathematical analysis as the secretion profiles. Data analysis. Significant pulses of insulin secretion were identified by the cluster analysis, an objective computerized peak-detection algorithm (21,22). Cluster analysis determines statistically significant up- and downstrokes in serial time series and provides information about the frequency and amplitude of these oscillations. The t statistics used for evaluating significant up- and downstrokes were taken as 3, and the corresponding estimated cluster size of 1 and 1 in the nadirs and peaks were defined using signal-free insulin profiles. Data presentation. The figures show representative experiments, and the tables show means ⫾ SE. Statistical comparisons between means were performed by unpaired t test or ANOVA followed by a Newman-Keuls test as appropriate. RESULTS AND DISCUSSION Characteristics of the mice. Our previous experiments have consistently been performed with islets isolated from NMRI mice 3– 4 months of age. In contrast, the ob/ob mice S178 used by others to test the pulsatility of insulin secretion were much older (8 –10 months) (8). The mice were therefore subdivided into six groups that were agematched except for the “young NMRI” that corresponded FIG. 1. Effects of repetitive depolarizations on cytoplasmic free Ca2ⴙ concentration ([Ca2ⴙ]i) and insulin secretion measured simultaneously in a single islet from a 7-month-old ob/ob mouse. The medium contained 12 mmol/l glucose (G) and 0.1 mmol/l diazoxide (Dz) throughout, whereas the concentration of Kⴙ (K) was changed between 4.8 and 30 mmol/l every 2 min as indicated. The horizontal line across the traces corresponds to the calculated nadir of the oscillations. The gray area under the recorded signal and above zero corresponds to the “total area”; the hatched gray area below the nadirs corresponds to the nonpulsatile fraction; the gray area above the nadirs corresponds to the pulsatile fraction. The percentage of pulsatility is the fraction of pulsatile over total area. DIABETES, VOL. 51, SUPPLEMENT 1, FEBRUARY 2002 L.L. KJEMS AND ASSOCIATES TABLE 2 Cluster analysis of the [Ca2⫹]i and insulin secretion profiles during repetitive stimulations of the islets with pulses of 30 mmol/l K⫹ ob/ob mouse islets (n ⫽ 7) [Ca2⫹]i Insulin [Ca2⫹]i (nmol/l) Insulin secretion rate (pg/min) Number of peaks (n per 22 min) Peak interval (min) Peak amplitude (fold above nadir) Pulsatile [Ca2⫹]i and insulin secretion (%) 193 ⫾ 9 — 5⫾0 4⫾0 2.29 ⫾ 0.1 32.5 ⫾ 1.3 — 125 ⫾ 51 5⫾0 4⫾0 15.6 ⫾ 4.7* 66.8 ⫾ 4.2* Young NMRI mouse islets (n ⫽ 10) [Ca2⫹]i Insulin 185 ⫾ 8 — 5⫾0 4⫾0 2.58 ⫾ 0.16 37.3 ⫾ 2.5 — 108 ⫾ 13 5⫾0 4⫾0 4.26 ⫾ 0.13 49.1 ⫾ 1.4 Data are means ⫾ SE for 7 ob/ob mouse islets (different mice of 8 –11 months) and 10 young NMRI mouse islets. In each experiment, a single islet was stimulated by five 2-min pulses of 30 mmol/l K⫹ separated by 2-min rest periods in 4.8 mmol/l K⫹, according to the protocol illustrated in Fig. 1. Both [Ca2⫹]i and insulin secretion profiles were subjected to cluster analysis. *P ⬍ 0.05 or less vs. NMRI mouse islets (unpaired t-test). to our usual model. As expected, ob/ob mice were much heavier than the others. They were slightly hyperglycemic and markedly hyperinsulinemic (Table 1), with plasma insulin concentrations 200- to 300-fold higher than in controls, as previously reported for these Swedish ob/ob mice (17,23). These characteristics can be attributed to their deficiency in leptin (16) and marked insulin resistance (24). There was no difference between lean and FIG. 2. Effects of a sustained depolarization on cytoplasmic free Ca2ⴙ concentration ([Ca2ⴙ]i) and insulin secretion measured simultaneously in single islets from ob/ob mice studied after culture in 5.5 mmol/l glucose. A and B: Islets from 10-month-old ob/ob mice. C and D: Islets from 7-month-old ob/ob mice. Cluster analysis of the last 20 min of the experiment identified significant peaks, the size of which is indicated by the gray areas: no significant peak (A); two peaks (B); five peaks (C). The insulin profile shown in D is one example of the irregular secretory responses that were exceptionally (7 of 106) recorded in the presence of a stable [Ca2ⴙ]i elevation. Note that the scale for insulin secretion is different from that of other panels. K4.8, Kⴙ concentration 4.8 mmol/l; K30, Kⴙ concentration 30 mmol/l. DIABETES, VOL. 51, SUPPLEMENT 1, FEBRUARY 2002 S179 Ca2ⴙ OSCILLATIONS IN ␤-CELLS AND INSULIN SECRETION NMRI mice except for a slightly higher body weight of the latter. On average, islets isolated from ob/ob mice were much larger than those from lean and NMRI mice (15), but purportedly the biggest ones were not taken for the experiments. The estimated size of the islets used was (mm3 䡠 10⫺3): 8.07 ⫾ 0.4 (n ⫽ 53) for ob/ob mice, 3.85 ⫾ 0.2 (n ⫽ 24) for lean mice, and 4.43 ⫾ 0.2 (n ⫽ 33) for NMRI mice. Effects of forced [Ca2ⴙ]i oscillations. Alternating between 4.8 and 30 mmol/l K⫹ in a medium containing glucose and diazoxide, to depolarize islet cells repetitively, caused oscillations of [Ca2⫹]i (Fig. 1). Each of these oscillations triggered a pulse of insulin secretion in islets from NMRI mice (not illustrated) (11,12) and in islets from ob/ob mice (Fig. 1). Cluster analysis identified the five [Ca2⫹]i and insulin oscillations that were imposed at 4-min intervals in both types of islets (Table 2). The peak amplitude quantifies the increase between the nadir and the maximum of the oscillation. For [Ca2⫹]i, it was similar in both types of islets, but for insulin secretion, it was larger in ob/ob than NMRI islets. The area under the oscillations and above the nadir (the pulse mass) was expressed as a percentage of the total signal (above zero) to estimate the degree of pulsatility of the whole response. For the experiment shown in Fig. 1, the relative pulsatility of [Ca2⫹]i was 36.3%, and that of insulin secretion, 57.5%. A similar difference was consistently observed (Table 2, last line), because the oscillations of [Ca2⫹]i are superimposed on a relatively large, steady, basal [Ca2⫹]i (Fig. 1). It is clear that the pulsatile fraction of the response is also influenced by the frequency of the oscillations; for insulin secretion, it would increase if the resting periods were long enough to permit a return to or close to zero. Effects of a sustained [Ca2ⴙ]i elevation. In total, 106 experiments, each using a single islet, were performed according to the protocol illustrated in Fig. 2. The perifusion medium contained 12 mmol/l glucose and 0.1 mmol/l diazoxide throughout, whereas the concentration of K⫹ was steadily raised from 4.8 to 30 mmol/l between 5 and 40 min. This resulted in an abrupt rise in [Ca2⫹]i in all experiments. In ob/ob islets cultured in 5.5 mmol/l glucose, the [Ca2⫹]i rise displayed an initial brief peak followed by a broader hump before stabilizing in a slowly increasing plateau (Fig. 2). After culture in 10 mmol/l glucose, an initial overshoot of [Ca2⫹]i also occurred in ob/ob mouse islets and in islets of NMRI or lean mice, but two distinct phases were only rarely seen. There was no significant difference between mean [Ca2⫹]i in the different groups: as typical examples, between 0 –5 and 20 – 40 min, [Ca2⫹]i increased from 97 ⫾ 6 to 255 ⫾ 11 nmol/l in young NMRI islets, from 98 ⫾ 4 to 251 ⫾ 13 nmol/l in islets from 7-month-old lean mice, and from 100 ⫾ 6 to 260 ⫾ 10 nmol/l in islets from 7-month-old ob/ob mice. Only data from the last 20 min of each experiment were subjected to cluster analysis. No significant [Ca2⫹]i peak was detected in any of the experiments. We can thus consider that [Ca2⫹]i was stably elevated in all islets under these conditions. The vast majority of islets showing a prompt [Ca2⫹]i rise in response to 30 mmol/l K⫹ also rapidly secreted insulin. S180 FIG. 3. Examples of insulin secretion profiles during the last 20 min of stimulation of single islets with 30 mmol/l Kⴙ, according to the protocol shown in Fig. 2. Significant peaks are denoted by arrows and their sizes by the gray areas. Only 6 of 106 experiments were discarded because of a sluggish or inexistent secretory response. Rarely, insulin secretion was characterized by a very irregular pattern despite a regular [Ca2⫹]i rise (Fig. 2D). This pattern was DIABETES, VOL. 51, SUPPLEMENT 1, FEBRUARY 2002 L.L. KJEMS AND ASSOCIATES TABLE 3 Cluster analysis of the insulin secretion profiles during steady-state stimulation of the islets with 30 mmol/l K⫹ Cultured at 5.5 mmol/l glucose ob/ob 7 months 10 months Islets (n) Insulin secretion rate (pg/min) No. of peaks (n per 20 min) No. of islets showing: 0–1 peak ⬎1 peak Peak interval (min) Peak amplitude (fold above nadir) Pulsatile insulin secretion (%) Cultured at 10 mmol/l glucose ob/ob Lean (ob/⫹ or ⫹/⫹) NMRI 7 months 10 months 7 months 10 months Young Old 9 13 11 13 308 ⫾ 65 227 ⫾ 39 212 ⫾ 54 369 ⫾ 75 3.8 ⫾ 0.8 1.9 ⫾ 0.5 5.0 ⫾ 0.8* 2 7 2.7 ⫾ 0.2 6 7 5.4 ⫾ 0.7 2 9 3.0 ⫾ 0.4 1.51 ⫾ 0.06 1.49 ⫾ 0.08 1.49 ⫾ 0.06 10.2 ⫾ 1.1 8.5 ⫾ 2.2 12.7 ⫾ 2.2* 15 9 12 11 243 ⫾ 27 293 ⫾ 30 281 ⫾ 22 132 ⫾ 16 2.4 ⫾ 0.5 1.5 ⫾ 0.3 4.8 ⫾ 1.0* 1.1 ⫾ 0.4 3.1 ⫾ 0.6 5 8 6.0 ⫾ 1.4 9 6 4.3 ⫾ 1.2 0 9 5.4 ⫾ 1.3 8 4 3.3 ⫾ 1.0 2 9 3.8 ⫾ 0.6 1.43 ⫾ 0.05 1.44 ⫾ 0.09 1.47 ⫾ 0.07 13.1 ⫾ 1.1* 4.1 ⫾ 1.5 9.8 ⫾ 2.2 1.42 ⫾ 0.05 1.30 ⫾ 0.03 8.6 ⫾ 1.6 4.1 ⫾ 1.0 Data are means ⫾ SE unless indicated otherwise. The experiments using a single islet at a time were performed according to the protocol illustrated in Fig. 2. The islet was stimulated with 30 mmol/l K⫹ for 35 min in a medium containing 12 mmol/l glucose and 0.1 mmol/l diazoxide. The data from last 20 min of the individual insulin secretion profiles were subjected to cluster analysis. Peak interval was calculated only for those experiments in which more than one peak was detected. *P ⬍ 0.05 or less vs. 7-month-old lean or young NMRI islets (Newman-Keuls test). observed in 7 of 106 islets (two ob/ob islets, four lean islets, and one NMRI islet). These experiments were not further analyzed. In 93 islets, the time course of insulin secretion was biphasic, as illustrated in Fig. 2A– C, but the size of the initial peak (relative to the sustained phase) was not always as large as that shown here for ob/ob islets cultured in 5.5 mmol/l glucose (see references 11 and 12 for comparison). All these insulin profiles were subjected to cluster analysis for the period 20 – 40 min, i.e., after the initial peak and the following trough. In experiments performed without islets but with a medium containing a constant concentration of insulin (in the range 22–310 pg/ml, covering the range of insulin concentrations measured in real experiments), the assay detected no peaks (6 of 15), one peak (7 of 15), and two peaks (2 of 15)— on average 0.7 ⫾ 0.2 peaks. The calculated “pseudo-pulsatility” amounted to 3.8 ⫾ 1.1%. Figure 3 shows examples of insulin secretion profiles obtained during the last 20 min of stimulation of different types of islets in experiments similar to those in Fig. 2. Significant peaks were detected in a number of experiments. They displayed very different shapes (amplitude, duration, and frequency), however, as can be seen from the comparison of the four peaks in a 10-month-old ob/ob islet (Fig. 3A) with those of a 10-month-old lean islet (Fig. 3B). Pulsatility sometimes characterized the whole period of measurement (Fig. 3C) or only a portion of it (Fig. 3A). The incidence of insulin peaks and some of their characteristics are presented in Table 3 for the different groups of islets. In our usual model of young NMRI mice, two thirds of the profiles showed no peaks or only one insulin peak during stimulation by stable [Ca2⫹]i. In three islets, two peaks were observed, and in one islet, four peaks were detected. This resulted in a mean of 1.1 peak/20 min and a percentage of pulsatility of 4.1% for total insulin secretion (Table 3). These values are not statistically different from those obtained by analysis of insulin concentration profiles without islets. We confirm, therefore, DIABETES, VOL. 51, SUPPLEMENT 1, FEBRUARY 2002 that in islets from young adult normoglycemic NMRI mice, insulin secretion does not significantly oscillate when [Ca2⫹]i is stably elevated by high K⫹ in the presence of diazoxide (11). Similar conclusions can be drawn for islets isolated from 7-month-old lean littermates of ob/ob mice (Table 3). However, in both old NMRI mice and 10-month-old lean mice, all or nearly all insulin secretion profiles presented more than one peak during steady-state stimulation with 30 mmol/l K⫹, but the amplitude of the peaks was small, much smaller than that in the presence of [Ca2⫹]i oscillations (compare Tables 2 and 3). Therefore, the fraction of insulin secretion that occurred in a pulsatile manner did not exceed 10 –13%. The situation was somewhat different in islets from ob/ob mice (Table 3). Two or more significant peaks of insulin secretion were observed in most islets from 7-month-old ob/ob mice, producing 10 –13% of pulsatility in the whole response. In contrast to what happened in lean mouse islets, further aging did not increase the pulsatility of the secretory response in ob/ob islets (Table 3). CONCLUSIONS The present results support our previous conclusions that no significant oscillations of insulin secretion occur in islets from young (4-month-old) normal NMRI mice when [Ca2⫹]i does not oscillate in ␤-cells. The same holds true for 7-month-old lean littermates of ob/ob mice. With aging, however, the stability of insulin secretion decreased. We agree with a previous report (14) of oscillations in insulin secretion occurring in the absence of [Ca2⫹]i oscillations in islets from aged (8- to 11-month-old), hyperglycemic ob/ob mice from the Swedish colony. However, we find it important to qualify this conclusion. First, the fraction of total insulin secretion that occurs in a pulsatile manner is small (maximum 13%), much less than that during oscillations of [Ca2⫹]i. Oscillations of insulin secreS181 Ca2ⴙ OSCILLATIONS IN ␤-CELLS AND INSULIN SECRETION tion in the absence of [Ca2⫹]i oscillations are thought to be mediated by oscillations of a metabolic amplifying signal (25). We have previously shown that the proposed mechanism is plausible, but that its efficiency is poor, much less than that of [Ca2⫹]i oscillations (12). The present experiments are in keeping with our previous conclusions. Second, the recorded pulsatility of insulin secretion reflects irregularity more than true oscillations. The possibility that these irregularities are simply due to the large size of ob/ob islets seems unlikely because they also occurred in islets from older nonobese mice, which were not larger than the islets of young mice. Aging and hyperstimulation of ␤-cells, rather than the genetic defect of the ob/ob mice (deficiency in leptin), may be responsible for this change in the characteristics of insulin secretion. Our observations may be relevant to the detection of alterations of plasma insulin oscillations in aged subjects (26) and in patients with type 2 diabetes (27–29). 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