Glucose-induced pulsatile insulin release from single islets
at stable and oscillatory cytoplasmic
Ca2+
Peter
Bergsten
Department of Medical Cell Biology, Biomedicum, Uppsala University,
751 23 Uppsala, Sweden
 |
ABSTRACT |
The cytoplasmic
Ca2+ concentration
([Ca2+]i)
and insulin release were measured simultaneously in mouse pancreatic
islets cultured overnight.
[Ca2+]i
was 105 nM and insulin release 3 pmol · g
1 · s
1
at 3 mM glucose. An increase to 7 mM glucose reduced
[Ca2+]i
transiently, whereas insulin release doubled and was pulsatile with a
frequency of 0.47 min
1.
[Ca2+]i oscillations with similar frequency
appeared at 11 mM glucose associated with increased amplitude of the
insulin oscillations, raising the secretory rate 10-fold. In the
presence of 16 and 20 mM glucose
[Ca2+]i
was >300 nM and showed no oscillations apart from two islets, which
demonstrated
[Ca2+]i
oscillations with small amplitude at 16 mM glucose. Insulin release
with maintained frequency increased by 46 and 31%, respectively. When
the glucose concentration was increased from 3 to 11 mM, [Ca2+]i
decreased with a nadir that appeared significantly earlier than when
the glucose concentration was raised from 3 to 7 mM. Glucose-induced
insulin release from the isolated islet is pulsatile both at stable and
oscillatory
[Ca2+]i,
with changes in secretory rate caused by the sugar also when [Ca2+]i
is unchanged.
insulin secretion; cytoplasmic calcium concentration; oscillations; islets of Langerhans; endocrine pancreas
 |
INTRODUCTION |
THE REGULATORY ROLE of the cytoplasmic
Ca2+ concentration
([Ca2+]i)
for the release of insulin is well documented (19, 36). When
glucose-induced
[Ca2+]i
oscillations with periods of 2-5 min were discovered in the individual
-cell (13) and the isolated islet (31), the importance of
the ion for the dynamics of insulin release was indicated. Insulin
release from the isolated perifused pancreas and plasma insulin
describe oscillations with similar durations (26, 29). In combined
measurements from individual islets the oscillations in
[Ca2+]i
were synchronous with pulses of insulin release, suggesting a role of
[Ca2+]i
for the generation of the hormonal pulses (3, 4). However, insulin
release from the isolated islet is pulsatile in a wide range of glucose
concentrations with amplitude regulation of the pulses by the sugar (7,
35), whereas
[Ca2+]i
becomes oscillatory only when a certain glucose concentration is
reached and is sustained and elevated in the presence of high glucose
concentrations (3, 4, 12-14, 18, 20, 24, 34, 35). The results
suggest that there may be a dissociation between the dynamics in
insulin release and
[Ca2+]i
both at nonstimulatory and high glucose concentrations. In the present
study individual islets were perifused with varying glucose
concentrations and insulin release, and
[Ca2+]i
was monitored simultaneously. Evidence for a permissive role of
[Ca2+]i
oscillation for the generation of insulin oscillations is supplied.
 |
MATERIAL AND METHODS |
Materials.
Reagents of analytic grade and deionized water were used. Collagenase,
HEPES, and BSA (fraction V) were obtained from Boehringer Mannheim
(Mannheim, Germany). Tetramethylbenzidine,
insulin-peroxidase, and
poly-L-lysine (P-5899) were
supplied by Sigma Chemical (St. Louis, MO). FCS was provided by GIBCO
(Paisley, UK). The acetoxymethyl ester of fura 2 was from Molecular
Probes (Eugene, OR) and IgG-certified microtiter plates were from Nunc
(Roskilde, Denmark).
Preparation and culture of islets.
Islets were collagenase isolated from
ob/ob-mice
and cultured overnight in the presence of 5.5 mM glucose in RPMI 1640 medium supplemented with 10% FCS. Subsequent experimental handling was performed in a medium containing (in mM) 125 NaCl, 5.9 KCl, 1.2 MgCl2, 2.56 CaCl2, and 25 HEPES, titrated to
pH 7.4 with NaOH. The medium was supplemented with 3, 7, 11, 16, or 20 mM glucose. BSA (0.5 mg/ml) was present in the medium except when the
islets were loaded with the Ca2+
indicator fura 2.
Measurements of cytoplasmic
Ca2+.
Individual islets were loaded with fura 2 during 40 min of incubation
at 37°C with 2 µM of fura 2-acetoxymethyl ester in the presence
of 3 mM glucose. After rinsing, each islet was placed in a 15-µl
perifusion chamber as previously described (3). The chamber was placed
on the stage of an inverted microscope (Nikon Diaphot) within a climate
box maintained at 37°C and perifused at a rate of 150-200
µl/min. The microscope was equipped for
[Ca2+]i
measurements by dual wavelength fluorometry with excitation at 340 and
380 nm and emission measured at 510 nm from which
[Ca2+]i
was calculated (17).
Measurements of insulin release.
Insulin secretion from individual islets was determined from
measurements of the hormone in 5-s fractions of the perifusate (3).
After perifusion the islets were freeze-dried overnight and weighed on
a quartz fiber balance, and secretion was expressed as picomole insulin
per gram dry islet weight per second. The dry weights of the islets
were 8-15 µg. Insulin was assayed by competitive ELISA as
previously described (7).
Statistical analysis.
Differences between paired and unpaired observations were evaluated
with Student's t-test.
 |
RESULTS |
The
[Ca2+]i
and insulin release were measured simultaneously in individual mouse
pancreatic islets cultured overnight in 5.5 mM glucose. In the presence
of 3 mM glucose
[Ca2+]i
was 105 nM and insulin release 3 pmol · g
1 · s
1
(Fig. 1 and Table 1).
Increase of the glucose concentration to 7 mM reduced
[Ca2+]i
transiently with a nadir after 2.1 min followed by return to levels
observed in the presence of 3 mM glucose. In parallel measurements of
insulin release the secretory rate was doubled and showed pulses with a
frequency of 0.47 min
1.
When the glucose concentration was changed to 11 mM,
[Ca2+]i
oscillations appeared after 0.3 min with a frequency of 0.49 min
1 and with a doubling of
the mean
[Ca2+]i.
The measurements of insulin release showed oscillations with a similar
frequency and a 10-fold increase of the secretory rate. In the presence
of 16 mM glucose the mean
[Ca2+]i
increased by ~50%. In two of five islets the
[Ca2+]i
oscillations persisted with the same frequency but appeared from a
higher
[Ca2+]i
level and with decreased amplitude. In the remaining three islets
[Ca2+]i
was stable and elevated. Insulin release was pulsatile in all five
islets with maintained frequency and a 46% increase of the secretory
rate. When the glucose concentration was raised to 20 mM, all islets
displayed a stable and elevated
[Ca2+]i
level with no further increase of the mean
[Ca2+]i
(Fig. 1 and Table 1). The corresponding insulin release measurements rose by 31% with maintained frequency of the insulin oscillations.

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Fig. 1.
Simultaneous recordings of cytoplasmic
Ca2+
(top) and insulin release
(middle and bottom) from individual
islet in response to different glucose concentrations. Islet was
cultured overnight in presence of 5.5 mM glucose. After fura 2 was
loaded at 3 mM glucose the islet was transferred to a microscope
chamber and perifused in the presence of 3, 7, 11, 16, and 20 mM
glucose. Periods indicated in the
middle (A-D) are expanded in the
bottom (A-D). A representative of 5 experiments.
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Table 1.
Cytoplasmic Ca2+ and insulin release from isolated
islets in response to 4 different glucose concentrations
|
|
The
[Ca2+]i
was monitored alone in individual islets cultured overnight in 5.5 mM
glucose. After initial exposure to 3 mM glucose the sugar concentration
was increased to 11 mM, which decreased [Ca2+]i
transiently with the nadir appearing after 1.6 min, significantly earlier (P < 0.01) than in the
presence of 7 mM glucose (Figs. 1 and 2;
Tables 1 and 2). The magnitude of the lowering in
[Ca2+]i
was similar for both sugar concentrations, however. The first oscillation appeared after 2.8 min, significantly later
(P < 0.001) than when the sugar
concentration was raised from 7 to 11 mM. However, both the peak
[Ca2+]i
of the first oscillation as well as the frequency of the following oscillations and the mean
[Ca2+]i
were not affected by 7 mM glucose preceding 11 mM of the
sugar. In the presence of 16 or 20 mM glucose, neither the
oscillations nor the means in
[Ca2+]i
were affected by the omission of 7 mM glucose (Figs. 1 and 2; Tables 1
and 2).

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Fig. 2.
Recordings of cytoplasmic Ca2+
from 2 islets in response to different glucose concentrations. Islets
were cultured overnight in presence of 5.5 mM glucose. After fura 2 was
loaded at 3 mM glucose islets were transferred to a microscope chamber
and perifused in the presence of 3, 11, 16, and 20 mM glucose. A
representative experiment of 6 (top)
and 2 (bottom).
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 |
DISCUSSION |
Glucose-induced increase of the amplitude of insulin pulses has been
explained by recruitment of
-cells into the secretory phase by
transformation of
[Ca2+]i
from the low and stable to the oscillatory mode (18, 20, 23). Whereas
some cells show such a response already at 8 mM glucose (14), most
cells and islets require 10-11 mM glucose (3, 4, 12-14, 18,
20, 34, 35) and some need as much as 20 mM of the sugar to show the
[Ca2+]i
oscillations (18). These differences probably reflect variations between
-cells in the metabolism of the sugar (25). When the glucose
concentration is 7 mM or less,
[Ca2+]i
is low and stable (3, 4, 12-14, 18, 20, 24, 34, 35). A transient
decrease in
[Ca2+]i
is observed when the glucose concentration is increased above 5 mM
(20), which is due to sequestration of
Ca2+ into intracellular stores and
promotion of the outward transport of the ion (19). If the lowering
period of
[Ca2+]i
is extended by counteracting the entry of
Ca2+ into the
-cells (5, 19),
there is a concomitant decrease in the release of insulin (5, 6). In
the presence of 20 mM or higher concentrations of glucose
[Ca2+]i
is sustained and elevated in isolated islets and in some individual
-cells (3, 4, 13, 14, 24, 34). The majority of individual
-cells
maintain an oscillatory
[Ca2+]i
(14). The
[Ca2+]i
oscillations are the result of intermittent
Ca2+ influx linked to the rhythmic
variations in the permeability of ATP-sensitive
K+ channels reflecting
glucose-induced metabolic cyclic changes in the ATP/ADP ratio (1, 8,
30).
Glucose metabolism is oscillatory and modulated by alterations in the
sugar already at glucose concentrations below 3-4 mM (10, 22),
which could explain the pulsatile insulin release in the presence of
such low glucose concentrations, when there is no entry of
Ca2+ (35). The secretory activity
probably resides with a metabolically responsive subpopulation of the
-cells. When
-cells were sorted according to their metabolic
activity (32), glucose-induced stimulation of insulin release in the
presence of 4.2 mM was seen only in this subpopulation (33). The
metabolically recruited
-cells also show
[Ca2+]i
oscillations if the glucose concentration is further elevated (3, 4,
12-14, 18, 31, 34), which increases the metabolism leading to the
critical rise in the ATP/ADP ratio (1, 8, 9, 30). Although oscillations
in
[Ca2+]i
could be pacing the secretory pulses via the
Ca2+-sensitive mitochondrial
dehydrogenases (28), it seems more likely that
[Ca2+]i
oscillations are coupled to and act synergistically with metabolic oscillations via fluctuations in the ATP/ADP ratio to amplify the
insulin oscillations. Metabolic and secretory oscillations with similar
frequency in the absence of
[Ca2+]i
oscillations (10, 35) argue in favor of an oscillatory metabolism
generating the pulses in insulin release. Furthermore, when the glucose
concentration is raised to promote
[Ca2+]i
oscillations, changes in metabolic parameters precede the rise in
[Ca2+]i,
the metabolic parameters are not further altered by the rise in
[Ca2+]i
(27, 30), and no change in the frequency of the insulin oscillations is
observed with the onset of
[Ca2+]i
oscillations (34).
In the physiological range of glucose concentrations, when the sugar is
raised from ~4 to 11 mM glucose, the number of metabolically activated
-cells increases sigmoidally two- to threefold to ~70% (25). Although a similar sigmoidal increase is observed in insulin release the rise in secretion is at least 10-fold (2, 7, 34, 36),
manifested as an increase in the amplitude of the insulin oscillations
(7, 34). Within the same glucose range, [Ca2+]i
is transformed from the low and stable to the oscillatory mode in as
many as 85% of the
-cells (20). This demonstrates the important
role of
[Ca2+]i
in the amplitude regulation by glucose of the insulin oscillations in
the physiological range of the sugar. However, the almost doubling in
secretory rate when the glucose concentration is further raised from 11 to 20 mM glucose observed in this and other studies (3, 4, 7) excludes
a mere recruitment of
-cells accounting for the glucose-induced
amplification of insulin release.
The glucose-induced increase in insulin release at high glucose
concentrations could reflect elevation of the ATP content because the
exocytosis of insulin-containing granules is an ATP-dependent process
(11). In tolbutamide-stimulated islets a high glucose concentration
maintains the amplitude of the insulin oscillations, whereas a decline
in the amplitude is seen in the presence of a low glucose concentration
despite a sustained and elevated
[Ca2+]i
at both glucose concentrations (34). Changes in the ATP/ADP ratio are
probably not only important for the induction of
[Ca2+]i
oscillations (1, 8, 23, 30) and synchronized with insulin oscillations
(23) in the normal glucose range but also play an important role in
regulating insulin release at high glucose concentrations. In fact,
such changes in the ATP/ADP ratio caused by high glucose concentrations
have been observed (9). The cellular content of cAMP shows a similar
glucose-dependent increase in isolated islets (15) and may well
participate in the potentiation of the amplitudes of insulin pulses (7)
at a stage distal to the elevation of
[Ca2+]i
(16). The glucose-induced increase in cAMP could also be important for
the potentiation of the amplitudes of insulin pulses by affecting the
recruitment phenomenon per se because addition of glucagon decreases
the glucose concentration required for induction of
[Ca2+]i
oscillations in isolated
-cells (14). In this context it can be
argued that isolated islets could have a higher cellular content of
cAMP through paracrine secretion of glucagon than isolated
-cells.
Although no detailed comparisons have been made there is no evidence to
support that isolated islets require lower glucose concentrations for
the induction of
[Ca2+]i
oscillations (2, 4, 12-14, 18, 34, 35).
In conclusion, transformation from low and stable to oscillatory
[Ca2+]i
in the individual
-cell, which occurs at different glucose concentrations for different
-cells, is an important factor for explaining the glucose-modulated amplification of pulsatile insulin release. However, apart from inducing
[Ca2+]i
oscillations, increase in the glucose concentration may also be capable
of amplifying the secretory response of the individual
-cell for
glucose concentration below or above this threshold concentration (21).
To determine whether the individual
-cell can modulate its secretory
response at low and high glucose concentrations, when no changes in
[Ca2+]i
are observed, an assay capable of monitoring dynamically the secretory
activity of the pancreatic
-cell over sufficient time and with
enough sensitivity would be required.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Swedish Medical
Research Council (12X-11203), the Swedish Diabetes Association, the
Novo Nordisk Foundation, and the Family Ernfors Foundation.
 |
FOOTNOTES |
Address for reprint requests: P. Bergsten, Dept. of Medical Cell
Biology, Uppsala Univ., Box 571, 751 23 Uppsala, Sweden.
Received 4 November 1997; accepted in final form 28 January 1998.
 |
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