Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umeå University, SE-901 87 Umeå, Sweden
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ABSTRACT |
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The length of the silent lag time before
elevation of the cytosolic free Ca2+ concentration
([Ca2+]i) differs between individual
pancreatic -cells. One important question is whether these
differences reflect a random phenomenon or whether the length of lag
time is inherent in the individual
-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 individual
ob/ob mouse
-cells with the fura 2 technique in a
microfluorimetric system. There was a strong correlation between the
lengths of the lag times in each
-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
-cell.
delay; onset; cytoplasmic calcium; repetitive glucose stimulation
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INTRODUCTION |
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PANCREATIC ISLETS ARE
COMPOSED mainly of insulin-producing -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
-cells as well
as between
-cells and other endocrine islet cells, and there is
evidence that the
-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
-cells within the islets. Regional heterogeneity
and subpopulations within the pancreatic
-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
-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
-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
-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
-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 individual
ob/ob mouse
-cells exposed to repeated glucose stimulations.
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MATERIALS AND METHODS |
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Animals.
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 -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 mM D-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 -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 mM
L-glutamine, 60 µg/ml Garamycin, and 60 µg/ml benzylpenicillin.
Cytoplasmic Ca2+ measurements.
Islet -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.
Statistical analysis. 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).
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RESULTS |
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Comparison of lag times.
Each -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
-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). Figure
1A illustrates the correlation
between lag times in experiments with two consecutive glucose
stimulations (10 mM) of individual
-cells [r = 0.94, P < 0.001; average 169 ± 17 vs. 201 ± 21 s (P < 0.001), 1st and 2nd stimulations,
respectively]. Figure 1B shows the correlation between lag
times for
-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+]i
value, 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).
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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 -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
-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
-cells exposed
twice to 10 mM glucose was 23 ± 2 and 24 ± 2 nM
(P > 0.05) and for
-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).
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Comparison of [Ca2+]i
response patterns.
Although the response patterns from different -cells after glucose
stimulation showed marked differences, for the majority of the
individual
-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
-cells exposed
twice to glucose (A-C:
-cells exposed to 10 mM
glucose; D-F: cells exposed to 20 mM glucose).
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DISCUSSION |
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The present study reports on the early
[Ca2+]i dynamics elicited by repeated glucose
stimulations in ob/ob mouse -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
-cells
that are not in physical contact with each other have unique and
reproducible characteristics. The clearly reproducible Ca2+
response for each
-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 -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
-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
-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
-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
-cell response. Together, these findings indicate that
the length of the lag time for each
-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
-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
-cells (25, 27).
The present results conform with previous findings indicating that
individual -cells show considerable differences in their response to
stimulatory agents (8, 30, 36, 37) but also indicating
similarity in functional behavior of individual
-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
-cells. This suggests that each
-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 -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
-cells in this study react to a glucose challenge (Fig. 1). Thus the
cohort of the fast-reacting
-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
-cells
with the most prompt response may act as intraislet pacemakers. Some
-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 -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
-cells in situ in the intact pancreatic islets.
However, the fingerprints for each
-cell (34) might be
less visible in intact islets, where the
-cells are structurally and
functionally coupled to each other or to other endocrine cells in an
integrated functional system.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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: gerd.larsson-nyren{at}histocel.umu.se).
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
Received 31 August 2001; accepted in final form 21 December 2001.
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