The length of the silent lag time before elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) differs between individual pancreatic β-cells. One important question is whether these differences reflect a random phenomenon or whether the length of lag time is inherent in the individual β-cell. We compared the lag times, initial dips, and initial peak heights for [Ca2+]i from two consecutive glucose stimulations (with either 10 or 20 mM glucose) in individualob/ob mouse β-cells with the fura 2 technique in a microfluorimetric system. There was a strong correlation between the lengths of the lag times in each β-cell (10 mM glucose:r = 0.94, P < 0.001; 20 mM glucose:r = 0.96, P < 0.001) as well as between the initial dips in [Ca2+]i (10 mM glucose:r = 0.93, P < 0.001; 20 mM glucose:r = 0.79, P < 0.001) and between the initial peak heights (10 mM glucose: r = 0.51,P < 0.01; 20 mM glucose: r = 0.77,P < 0.001). These data provide evidence that the response pattern, including both the length of the lag time and the dynamics of the subsequent [Ca2+]i, is specific for the individual β-cell.
- cytoplasmic calcium
- repetitive glucose stimulation
pancreatic islets are composed mainly of insulin-producing β-cells, which in most mammals are located in the central part of the islets and surrounded by other hormone-producing cells (for review, see Ref. 30). There are close junctional contacts between different β-cells as well as between β-cells and other endocrine islet cells, and there is evidence that the β-cells in intact islets are functionally coupled and show synchronized activity (for review, see Ref. 29). However, both structural and functional differences have been found between individual β-cells within the islets. Regional heterogeneity and subpopulations within the pancreatic β-cell mass have been suggested on the basis of their ability to respond to stimulation by insulin secretagogues (8, 19, 30, 31, 36, 37, 38). Closely located β-cells show different amounts of secretory granules and rough endoplasmic reticulum (37), nuclear size (14), and number of gap junctions (26). Marked differences in the pattern of electrical activity among β-cells (22) as well as in the lag times between stimulation and onset of membrane electrical activity (6) have been noted. Also, large cell-cell variations in the patterns of cytosolic free Ca2+ concentration ([Ca2+]i) oscillations have been demonstrated, and the length of the lag time before the onset of [Ca2+]i response in individual β-cells (12, 18, 23, 33) shows a wide variation. This is of interest in view of the fact that early stages of type II diabetes mellitus are associated with an impairment particularly of the first phase of insulin release (4, 32). The question arises whether the initial response patterns in individual β-cells represent stable characteristics of the individual cells or merely random phenomena. [Ca2+]i is a key signal component regulating the insulin secretion under physiological conditions (for review, see Refs. 16, 39). Using the fura 2 technique, which monitors changes in [Ca2+]i, we have systematically investigated the lengths of lag times and the patterns of the early [Ca2+]i responses from isolated individualob/ob mouse β-cells exposed to repeated glucose stimulations.
MATERIALS AND METHODS
Non-inbred, 7- to 8-mo-old female ob/ob mice (Umeå-ob/ob ) were used throughout. Islets from these mice contain a high proportion of β-cells (>90%; Ref. 15), which makes it highly probable that the present data on isolated islet cells are representative of this cell type. Although these mice are metabolically abnormal, with mild hyperglycemia and hyperinsulinemia, their islets respond adequately to both stimulators and inhibitors of insulin release in vitro (13).
Preparation of islets.
Islets of Langerhans were isolated by collagenase digestion (24) and hand picked under a stereomicroscope. The medium used was a Krebs-Ringer medium (KRH) with the following salt composition (mM): 130 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, and 2.56 CaCl2. Bovine serum albumin (BSA) at 10 mg/ml and 3 mMd-glucose were added. The medium was buffered with 20 mM HEPES and NaOH to a final pH of 7.4, and the gas phase was the ambient air. After digestion, the BSA concentration in the KRH medium was 1 mg/ml throughout except during culture.
Preparation of β-cells.
The islets were dissociated into isolated cells by incubation in a Ca2+-free KRH medium supplemented with 1 mM EGTA and 1 mg/ml DNase (24), centrifuged through a 4% (wt/vol) BSA column, and resuspended in KRH medium. Portions of the cell suspension were plated on cover glasses, coated with polylysine (0.01% wt/vol), and allowed to attach for 15 min. The cells were maintained in a humidified incubator (5% CO2 in 95% air) for 1–2 days in 3 ml of culture medium RPMI 1640 containing 10% (wt/vol) heat-inactivated fetal calf serum, 11.1 mM glucose, 20 mM HEPES, 2 mMl-glutamine, 60 μg/ml Garamycin, and 60 μg/ml benzylpenicillin.
Cytoplasmic Ca2+ measurements.
Islet β-cells were loaded with 1 μM fura 2-AM for 40 min at 37°C in KRH containing 3 mM d-glucose and 1 mg/ml BSA. Each cover glass was rinsed (KRH) and transferred to a temperature-controlled (37°C) perifusion chamber (170 μl) to form the bottom of the chamber. The chamber was mounted on the stage of an inverted microscope (Nikon Diaphot-TMD) and perifused at a flow rate of 0.6 ml/min.
The time for new medium to reach the chamber was determined before the start of the experiments (23). Fura 2 was excited alternately at 340 and 380 nm with band-pass filters in front of a 100-W xenon lamp. The resulting emitted fluorescence was measured with a photomultiplier at 510 nm. The time interval between successive cycles of 340- and 380-nm excitations was 0.77 s. All cells were initially preperifused at 3 mM glucose for 15 min with registration during the last 3 min. After change to a stimulatory glucose concentration, the individual β-cell was perifused for 10–15 min. The ultraviolet light was immediately closed, and the cell was kept in the chamber for 30 min at 3 mM glucose without change in the flow rate. After this rest period the cell was stimulated a second time with the same glucose concentration as during the first stimulation. The experiments were performed on individual β-cells not in contact with other cells. The apparent concentration of cytoplasmic Ca2+ was calculated from the 340-to-380-nm ratio, and calibration was performed using the equation by Grynkiewicz et al. (11), with a dissociation constant (K d) of 224 nM.
Student's t-test for paired analysis was used for comparison of the groups. Results are presented as means ± SE. The intercomparisons between first and second lag times were evaluated by calculating the correlation coefficient with Statworks software (Computer Associates International, Islandia, NY).
Comparison of lag times.
Each β-cell was stimulated twice by either 10 or 20 mM glucose over 10-min periods. For cells that were very slow in reacting, i.e., showing a lag time of ≥7 min, the stimulation period was prolonged by another 5–15 min. Before the second stimulation, the exposed β-cell was checked under the microscope as to whether it remained attached to the glass bottom of the chamber and/or for its viability (e.g., rarely occurring shape alterations or blebs). Figure1 A illustrates the correlation between lag times in experiments with two consecutive glucose stimulations (10 mM) of individual β-cells [r = 0.94, P < 0.001; average 169 ± 17 vs. 201 ± 21 s (P < 0.001), 1st and 2nd stimulations, respectively]. Figure 1 B shows the correlation between lag times for β-cells exposed twice to 20 mM glucose (r = 0.96, P < 0.001; 170 ± 18 vs. 183 ± 20 s, P < 0.025). Thus, although there was a clear-cut correlation between the lengths of the first and second lag periods, the second lag period was slightly but reproducibly longer (19% at 10 mM glucose and 8% at 20 mM glucose). The length of each lag time was calculated as the time from glucose entry into the flow chamber until the first increased [Ca2+]ivalue, representing the upstroke of the initial [Ca2+]i rise (23). This increase occurred, in most cases, after a slight decrease in [Ca2+]i (16). However, for comparison we also calculated each lag time as the first [Ca2+]i value above basal average (last 3 min in the presence of 3 mM glucose). This calculation led to almost the same result (r = 0.88, P < 0.001 for 10 mM glucose and r = 0.95, P < 0.001 for 20 mM glucose). The smallest difference in lag time comparing two consecutive exposures was 0.8 s, which also was the interval between the cycles of the 340-/380-nm excitations, and the largest difference was 166 s (cell stimulated twice with 20 mM glucose).
The majority of the β-cells exposed to either 10 or 20 mM glucose reacted with a rapid [Ca2+]i rise within 3 min from the start of stimulation (Fig. 1). However, some cells showed a longer lag time, up to almost 10 min. To be included in the comparative studies, the cells had to respond to both stimulation periods. Of the 155 cells tested, 12 cells (8% of total) responded neither to the first nor to the second glucose stimulation (measurement time was 12–15 min). Although we used islets from ob/obmice, which are known for their large proportion of β-cells (15), in the experimental process we cannot rule out that some of the cells chosen for measurement could be hormone-producing islets cells other than β-cells. Also, it is reasonable to assume that, although cultured for 1–2 days to recover from the isolation procedure, some of the β-cells were not completely functionally intact. Five cells (3% of total) responded during the first stimulation but were silent during the second stimulation, and four cells (2% of total) responded to the second but not to the first stimulation. This asymmetric behavior is difficult to explain, but, because for technical reasons each stimulation period was restricted to 12–15 min and the results show a wide range of lag times, it seems difficult to exclude the possibility that the lack of response in these few cells can be due to unusually long lag times.
The average lag times for the first glucose stimulation induced by either 10 or 20 mM glucose were quite similar [169 (n= 34) vs. 170 s (n = 46); see Fig. 1], suggesting a lack of dose dependence. Thus we stimulated individual β-cells first with 10 mM glucose and then, after the resting period, with 20 mM glucose. The average lag times found were 210 ± 29 vs. 176 ± 20 s (r = 0.80, P < 0.001,n = 27). In the reverse situation (first 20 mM and then 10 mM glucose), the average lag times were 136 ± 17 and 200 ± 27 s (r = 0.78, P < 0.001,n = 27). The Ca2+ response patterns within the pairs in this series of experiments did not express the same convincing similarities as those of β-cells stimulated twice with the same glucose concentration (data not shown).
Comparison of initial slight reductions and peak heights.
To obtain further information about possible similarities in the Ca2+ response pattern, we compared the depths of the initial slight reductions before the first peak within the pairs. The correlation coefficient for the initial reduction was r= 0.93, P < 0.001 for β-cells exposed twice to 10 mM glucose (51 ± 3 and 64 ± 4 nM, 1st and 2nd stimulations, respectively; P > 0.05) and r = 0.79,P < 0.001 for β-cells exposed twice to 20 mM glucose (48 ± 2 and 63 ± 2 nM, 1st and 2nd stimulations, respectively; P < 0.005) (Fig.2). Because the basal Ca2+ level was somewhat higher before the second stimulation compared with the first stimulation, we also calculated the relative maximal decrease compared with the average preceding basal level (3 final min at 3 mM glucose). The decrease for β-cells exposed twice to 10 mM glucose was 23 ± 2 and 24 ± 2 nM (P > 0.05) and for β-cells exposed twice to 20 mM glucose 22 ± 2 and 30 ± 3 nM (P < 0.005) for first and second glucose treatments, respectively. At present we have no ready explanation for this more accentuated dip at the second stimulation with 20 mM glucose. It should be noted, however, that such a difference was not found with 10 mM glucose (see above) and that correlation analysis of the relative decrease in Ca2+ level and lag time for the first and second stimulations showed no significant correlation (data not shown).
The heights of the first peak within the pairs were also compared. It was not always the first peak that showed the highest value during the response. Oscillating β-cells could show both a second and a third peak that were higher then the first. However, because not all β-cells displayed oscillating response patterns within the time window used, we decided to compare the first peaks, which represent the Ca2+ signal upstroke from basal level. Correlation between two consecutive stimulations with 10 mM glucose showedr = 0.51, P < 0.01 (719 ± 32 and 716 ± 31 nM, 1st and 2nd stimulations, respectively) and between two stimulations with 20 mM glucose showed r = 0.77,P < 0.001 (743 ± 25 and 747 ± 27 nM, 1st and 2nd stimulations, respectively). When the increments above the average basal level were calculated, principally the same observations were made.
Comparison of [Ca2+]iresponse patterns.
Although the response patterns from different β-cells after glucose stimulation showed marked differences, for the majority of the individual β-cells it was obvious that the response patterns from the first and second glucose stimulations were quite similar within each pair. Figure 3 illustrates six representative traces of twin patterns from single β-cells exposed twice to glucose (A–C: β-cells exposed to 10 mM glucose; D–F: cells exposed to 20 mM glucose).
The present study reports on the early [Ca2+]i dynamics elicited by repeated glucose stimulations in ob/ob mouse β-cells. The depth of the initial slight reduction in [Ca2+]i before the first peak as well as the length of the lag period and the height of the first peak showed very strong correlation within the pairs when the conditions during the two stimulations were not altered. The results show that the early response patterns from single β-cells that are not in physical contact with each other have unique and reproducible characteristics. The clearly reproducible Ca2+response for each β-cell found in the present study is in line with observations on clonal HIT insulin-secreting cells (34) in which a cell-specific oscillating Ca2+ pattern was induced by the muscarinic agonist carbamylcholine. Ca2+oscillations occur in a variety of cell types. Cell-specific Ca2+ handling has been noted in several cell types but has only been mentioned briefly and not characterized in detail. For example, images of [Ca2+]i in airway epithelium cells have shown that the patterns of Ca2+oscillations differ greatly between cells but that the frequencies and amplitudes of oscillations are intrinsic to each cell (7). Similar findings have also been made in single hepatocytes, in which successive readditions of the same agonist elicited identical cell-specific patterns of Ca2+ oscillations (35).
The main purpose of the present study was to systematically analyze the variations in the length of lag times between β-cells. Such variations have been observed previously (12, 18, 23, 33,34) but not extensively characterized. The high degree of correlation found between the lag times within the pairs of stimulation indicates that the length of the lag time, like the initial Ca2+ pattern, seems to be unique and intrinsic to each β-cell. It was noted that at both 10 and 20 mM glucose the second lag period was slightly longer than the first in each pair, which is interesting because the second stimulation also tended to cause a larger initial dip in the Ca2+ level. However, because this enlarged dip was only seen at 20 mM and not at 10 mM glucose and there was no correlation between the lag times and initial dips in Ca2+ level at either 10 or 20 mM glucose, there seems to be no clear-cut relationship between these two glucose effects. A slower response at the second glucose stimulation may seem somewhat astonishing in view of the well-known potentiating priming effect reported for glucose-stimulated insulin release (9, 10,28). However, this priming effect has been found in rat pancreas (9, 10, 28) but not in mouse pancreas (1), in which repeated treatments with glucose instead resulted in a reduced secretory response. Although there was a significant correlation between lag times from individual β-cells stimulated with different glucose concentrations, first 10 mM and then 20 mM glucose or vice versa, the correlation was not as strong as for the β-cells stimulated twice with the same glucose concentration. Also, we observed that change from 10 to 20 mM glucose did not result in the increase in lag time seen with 10 and 10 mM glucose. This is interesting, because it suggests that pretreatment for only 10–15 min can affect subsequent β-cell response. Together, these findings indicate that the length of the lag time for each β-cell also may be related to the glucose dose given, at least at the concentrations tested here. That the Ca2+ response pattern from these cells did not show the same degree of similarities within the pairs as the cells stimulated twice with the same glucose concentration points to the possibility that the Ca2+ response is also related to a specific Ca2+ pattern due to the degree of stimulation. The results in Figs. 1 and 2 suggest that all β-cells studied belong to the same population. Thus, as concerns the functional parameters tested here, there is no indication of distinctly different subpopulations among the β-cells (25, 27).
The present results conform with previous findings indicating that individual β-cells show considerable differences in their response to stimulatory agents (8, 30, 36, 37) but also indicating similarity in functional behavior of individual β-cells studied in repeated stimulations (3, 8, 38). The present data show a very strong reproducibility of both initial dynamics and signal pattern of the [Ca2+]i responses in individual β-cells. This suggests that each β-cell has its individual [Ca2+]i response pattern. These results are in consonance with earlier observations describing a cell-specific oscillating Ca2+ pattern (34) and the notion that the increase in [Ca2+]i shows large reproducibility during repetitive depolarizations (2).
If the results in isolated β-cells are representative of the function in intact islets, we may speculate that the distributions of lag times found here could mirror the characteristics of the insulin secretion dynamics in ob/ob mouse islets, because cytosolic free Ca2+ represents a major signal for insulin release in response to glucose (16, 17, 39). Normally, glucose-induced first-phase insulin release from ob/ob mouse islets is delivered within 1–3 min (21, 23). This time period seems to overlap the period in which the majority of the β-cells in this study react to a glucose challenge (Fig. 1). Thus the cohort of the fast-reacting β-cells may be the cells that are responsible for the first-phase insulin secretion. Another possibility is that the fast cells may represent a pacemaker function, which by cell-cell coupling initiates the total islets' secretory response. The role of pacemaker cells is well established in, for example, smooth muscle tissue (for review, see Ref. 20). Although a pacemaker function has also been discussed for pancreatic islets (cf. Ref. 5), the mechanisms underlying such a function are unclear. The present data point to the possibility that the β-cells with the most prompt response may act as intraislet pacemakers. Some β-cells showed a very slow response to glucose. It is quite remarkable that the response pattern in these cells still was very similar to that in the faster cells. The only obvious difference noticed was the prolonged silent lag time. This clearly indicates that the lag time behavior and signal pattern of [Ca2+]i are controlled by different mechanisms.
In conclusion, the present results suggest that each β-cell in a pancreatic islet has an individual capacity to respond to a glucose challenge within a certain reaction time and with a specific response pattern. These individual profiles may correspond to different functions of the β-cells in situ in the intact pancreatic islets. However, the fingerprints for each β-cell (34) might be less visible in intact islets, where the β-cells are structurally and functionally coupled to each other or to other endocrine cells in an integrated functional system.
This work was supported in part by grants from the Swedish Medical Research Council (Grant 12X-4756), the Swedish Diabetes Association, the Elsa and Folke Sahlberg Fund, and the Lars Hierta's Memorial Fund.
Address for reprint requests and other correspondence: G. Larsson-Nyrén, Dept. of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå Univ., S-901 87 Umeå, Sweden (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 9, 2002;10.1152/ajpcell.00009.2001
- Copyright © 2002 the American Physiological Society