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Unité d’Endocrinologie et Métabolisme, University of Louvain Faculty of Medicine, B-1200 Brussels, Belgium
Address all correspondence and requests for reprints to: J. C. Henquin, Unité d’Endocrinologie et Metabolisme, UCL 55.30, avenue Hippocrate 55, B-1200 Brussels, Belgium. E-mail: henquin{at}endo.ucl.ac.be
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This elaborated stimulus-secretion coupling implicates a large number of proteins. Many of these proteins are identified and mutations of the gene coding for some of them, e.g. glucokinase (10, 11, 12) or the sulfonylurea receptor of the K+-ATP channel (13, 14, 15) profoundly perturb ß cell function. In contrast, virtually nothing is known about the stability of islet proteins. This is, however, a critical issue because changes in the synthesis or degradation rates of proteins with a short half-life might contribute to the ability or failure of ß cells to adapt to various pathophysiological conditions. Thus, distinct cell proteins may have vastly different half-lives, from minutes to days. Proteins with a long half-life usually are structural components or perform housekeeping functions, whereas proteins with a short half-life play more specialized, often regulatory, roles.
In the present study, we examined the consequences of a blockade of
islet protein synthesis on the regulation of insulin secretion. A
similar approach was already used 30 yr ago. Several groups showed that
high concentrations of cycloheximide (CHX) and puromycin, two
inhibitors of translation, caused a delayed (
60 min) inhibition of
glucose-induced insulin secretion from the perfused rat pancreas
(16, 17, 18) and isolated rat islets (19). The
initial interpretation that insulin biosynthesis was required for
sustained secretion (second phase) was rapidly refuted (20, 21), but no explanation for the inhibition of insulin secretion
by protein synthesis inhibition was ever provided. Assuming that those
steps of stimulus-secretion coupling involving the proteins with the
smallest pool size and shortest half-life would be first affected, we
evaluated the impact of different durations of protein synthesis
inhibition on the major events regulating insulin secretion.
Remarkably, glucose metabolism and the generation of the triggering
signal were only slightly and slowly affected whereas the efficiency of
Ca2+ on the secretory process was rapidly
impaired.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cycloheximide treatment
CHX was added to the culture medium or the experimental
solutions from a 5 mM stock solution prepared daily in
sterile water. The treatment with CHX was applied for different periods
of time which, for practical reasons (batching of the islets, transfer
from culture to preincubation medium) slightly varied: 2 h
(120–135 min), 5 h (4.5–5 h) and 20 h (19–20 h).
Except when indicated otherwise, CHX was not present during the acute
tests of islet functioning but was withdrawn just before the start of
these tests.
Measurements of insulin secretion and islet insulin content
Insulin secretion during the culture period was measured in
aliquots of medium taken after 18 h. When the reversibility of the
effects of CHX was studied, the culture medium was changed after
12 h and aliquots of the fresh medium were taken every 3 h.
At the end of the culture, the islets were recovered from the Petri
dishes, and their insulin content was determined after extraction in
acid-ethanol (23). Insulin was measured by a
double-antibody RIA (24) with rat insulin as the
standard.
Insulin secretion was also measured in acute experiments using islets cultured with or without CHX. At the end of the culture, the islets were first washed and preincubated in control medium containing CHX or not as appropriate. Batches of 20 islets were then placed in small chambers of a perifusion system (25). Effluent samples were collected every 2 or 6 min for insulin measurement. In one series of experiments batches of 3 islets were incubated for 1 h in 1 ml medium containing 15 mM glucose with or without 0.5 mM dibutyryl cAMP or 25 nM phorbol myristic acid (PMA) (Sigma, St Louis, MO). At the end of the incubation, a portion of medium was taken for insulin measurement.
Measurements of total protein synthesis
The islets were cultured for 1–18 h in 1.25–2.5 ml RPMI medium
supplemented with L-[3,5-3H] tyrosine (20–40
µCi/ml depending on the duration of the experiment), and with or
without CHX. At the end of the culture period with the tracer, the
medium was removed and the islets were collected and washed 4 times
with control medium containing 1 mM nonradioactive
tyrosine. They were then distributed in batches of 15 in polyethylene
conical tubes, to which 500 µl ice-cold trichloracetic acid (10%)
was added to precipitate proteins. The tubes were centrifuged, the
supernatant was discarded and the pellet was rinsed again with
trichloracetic acid (3 times). The pellet was eventually solubilized in
200 µl 0.1 M NaOH, and its radioactive content measured
by liquid scintillation spectrometry, using Flow-Safe F (Lumac,
Groningen, The Netherlands) as scintillator and counting at an
efficiency of approximately 60%. The results (cpm/islet) obtained in
test groups were expressed as a percentage of those in control islets
treated in the same way within the same experiment.
Measurements of glucose oxidation
After culture and preincubation with or without CHX, batches of
10 islets were incubated for 2 h in 50 µl control medium
supplemented with 1 µCi [U-14C] glucose.
Oxidation of glucose was calculated from the production of
[14C]CO2. Technical
aspects of the method have been described previously (26).
In these experiments CHX was still present during the incubation.
Measurements of [Ca2+]i and NAD(P)H
After culture, the islets to be used for
[Ca2+]i measurements were
loaded with fura-PE3 during 2 h of preincubation in the presence
of 2 µM fura-PE3 acetoxymethylester. The islets to be
used for NAD(P)H measurements were preincubated without dye. The medium
contained CHX (test islets) or not (control islets). After
preincubation, the islets were transferred into the perifusion chamber
of a microspectrofluorimeter system that has previously been described
in detail (27, 28). NAD(P)H measurements were obtained
from one test and one CHX-treated islet side by side.
Measurements of 86Rb+ efflux
After culture, the islets were loaded with
86Rb+ (used as a tracer for
K+) during 90 min of preincubation in control
medium containing 80 µCi 86RbCl (0.37
mM). The medium contained CHX (test islets) or not (control
islets). The islets were then washed with nonradioactive medium and
placed in batches of 30 in the same perifusion system as that used to
study insulin secretion. The radioactivity lost by the islets was
measured in effluent fractions collected at 2 min intervals, and the
fractional efflux rate was calculated for each period
(25).
Electrophysiological experiments
After isolation, the islets were dispersed into single cells
(29) and cultured on glass coverslips in RPMI 1640 medium
as described above. The cells were allowed to attach to the glass
during 16–18 h before addition of CHX to the culture medium for 19–20
h.
The electrical recordings were made using the amphotericine-perforated
whole-cell mode of the patch-clamp technique at 21–23 C
(30, 31). Perforation required a few min and the
voltage-clamp was considered satisfactory when the series resistance
had fallen below 20 M
. Membrane currents were measured
using an EPC-9 patch-clamp amplifier (Heka Electronics,
Lambrecht/Pfalz, Germany) and the software Pulsefit (version
8–31).
To measure K+-ATP currents, 100 ms depo- and hyperpolarizing pulses of 20 mV were applied alternatively from a holding potential of -70 mV. The pipette solution was (mM): K2SO4 70, NaCl 10, KCl 10, MgCl2 3.7, HEPES 5 (adjusted to pH 7.1), and the bath solution was (mM): NaCl 120, KCl 4.8, CaCl2 2.5, MgCl2 1.2, NaHCO3 24, HEPES 5 (adjusted to pH 7.4). To measure Ca2+ currents, the holding potential was -80 mV, and 100 ms depolarizing pulses were applied to various potentials to establish current-voltage (I/V) relationships. The pipette solution was (mM): Cs2SO4 76, NaCl 10, KCl 10, MgCl2 1, HEPES 5 (adjusted to pH 7.1), and the bath solution was (mM): NaCl 125, KCl 4.8, CaCl2 2.5, MgCl2 1.2, tetraethylammonium-Cl 10, HEPES 5 (adjusted to pH 7.4).
Presentation of results
Results are presented as means ± SEM. The
statistical significance of differences between means was assessed by
ANOVA followed by a Newman-Keuls test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). At the end of the culture period,
the insulin content of control islets (cultured without CHX) averaged
100 ± 6 ng, which was 47% less than that of fresh, noncultured
islets (P < 0.001), but the sum of insulin content and
secretion (233 ± 6 ng) was 23% larger (P <
0.05) (Fig. 1b). Insulin synthesis has thus partially compensated for
insulin secretion. This was not the case after culture with CHX: the
insulin content was slightly lower than that of fresh islets
(P < 0.01), but the sum of insulin content and
secretion was not different. Importantly, the insulin content of islets
cultured with CHX was higher (P < 0.05 or less) than
that of control islets (Fig. 1b). This rules out the trivial
possibility that the defects of insulin secretion caused by CHX are
simply due to insufficient insulin stores. Because maximum effects were
obtained with 10 µM CHX, this concentration was
used in all subsequent experiments.
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). The islets
were then transferred to fresh culture medium. When CHX remained
present, both insulin secretion and protein synthesis were strongly
depressed as compared with controls. When CHX was withdrawn, a
significant synthesis of proteins was noted after 3 h (Fig. 2b),
whereas insulin secretion did not significantly increase before 6
h (Fig. 2a).
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). There was thus a clear
temporal sequence in the effects of CHX: upon addition of the drug, the
inhibition of insulin secretion was delayed and progressive compared
with that of protein synthesis; upon withdrawal of CHX, recovery of
insulin secretion followed resumption of protein synthesis.
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). By opening
K+-ATP channels and preventing ß cell
depolarization (32, 33), diazoxide abolished
glucose-induced insulin secretion, but a rise in extracellular
K+ to 30 mM triggered a large
monophasic response (Fig. 4b) (7). After 20 h of CHX
treatment, the stimulatory effects of glucose, tolbutamide, and high
K+ were markedly (60–80%) impaired. The
inhibition increased with the duration of CHX pretreatment, but the
major impairment (P < 0.001) occurred early, during
the first two hours, regardless of the stimulus (Fig. 4, c–e).
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Glucose metabolism by islets pretreated with cycloheximide
The effect of glucose on ß cell metabolism was first measured by
recording the NAD(P)H fluorescence of the islets (34, 35).
Raising the concentration of glucose from 3 to 15 mM
markedly and reversibly increased the signal (Fig. 5a). The response was not different from
that of controls after 5 h of treatment with 10 µM
CHX, but it was partially impaired (P < 0.01) after
20 h of treatment. This slight alteration of glucose metabolism
was confirmed by measurements of glucose oxidation (Fig. 5b). In both
low and high glucose, CO2 production from glucose
was slightly reduced by 20 h CHX treatment (20 and 16%,
respectively, P < 0.01). The inhibition was not larger
when the concentration of CHX was raised to 100
µM during the 20 h of treatment (not
shown), indicating that the small change in glucose metabolism is
secondary to inhibition of protein synthesis (already maximal with 10
µM CHX) rather than to a side effect of the
drug.
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).
Addition of 3 and then 15 mM glucose to the medium (Fig. 6a) or addition of 25 and then 100 µM tolbutamide to the
glucose-free medium (Fig. 6b) induced a graded and reversible
inhibition of 86Rb+ efflux
that is known to reflect closure of K+-ATP
channels (4, 36). Similar changes were produced by glucose
and tolbutamide when islets had been pretreated with 10
µM CHX for 5 h. After 20 h of treatment,
however, the basal rate of
86Rb+ efflux was lower than
in controls (P < 0.01), but glucose and tolbutamide
still decreased it to a similar minimum rate as in control islets. The
magnitude of the inhibitory effect was thus smaller, but the absolute
final rate was not different.
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. K+-ATP currents
were initially small because of the presence of 10 mM
glucose in the medium. Lowering ß cell ATP with 3 mM
azide and direct opening of the channels with diazoxide caused a marked
increase in K+-ATP currents, which was reversed
by tolbutamide. In 10 mM glucose alone,
K+-ATP currents were not significantly different
in ß cells pretreated with 10 µM CHX for 20 h and
in controls (Fig. 7b). However, the increase produced by azide and
diazoxide was smaller after CHX (P < 0.01), which
suggests that the total number of functional
K+-ATP channels was decreased. The relative
inhibitory action of tolbutamide was similar in control and CHX treated
ß cells, so that a significant difference (P < 0.01)
in total current persisted in the presence of the three drugs.
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),
that increased in amplitude up to 0 mV and then decreased at more
positive potentials (Fig. 7d). After pretreatment of the cells with 10
µM CHX for 20 h, both peak and sustained currents
were inhibited by about 40% (P < 0.001). The percent
block was similar at all voltages between -30 and +50 mV.
Stimulation of control islets with a rise in the glucose concentration
from 3 to 15 mM induced
[Ca2+]i changes in three
phases: an initial small decrease (seen in 20/24 islets) was followed
by a large increase and eventually by oscillations. These oscillations,
illustrated for a representative islet in the inset, are masked by
averaging of the traces (Fig. 8a). After
treatment of the islets with 10 µM CHX for 20 h,
basal [Ca2+]i was not
significantly changed but 15 mM glucose only evoked a
monophasic rise: the initial drop was consistently abolished, the
increase in [Ca2+]i
started sooner than in controls, and no oscillations occurred in any of
the 16 tested islets. Average
[Ca2+]i was not
significantly decreased, but the difference between basal and
steady-state [Ca2+]i in
15 mM glucose was 35% smaller after 20 h CHX than in
controls (P < 0.001). After treatment of the islets
with CHX for 5 h, the initial
[Ca2+]i decrease
persisted in only 6/20 islets, and the subsequent rise was attenuated,
although the difference with controls was significant
(P < 0.05) only for the change above basal levels.
During steady-state stimulation, oscillations of
[Ca2+]i undistinguishable
from control ones were present in all islets (not shown). Treatment of
the islets with CHX for only 2 h had no quantitative or
qualitative impact on glucose-induced
[Ca2+]i changes.
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). This rise was smaller in islets treated with 10
µM CHX for 5 h (P < 0.05) and
20 h (P < 0.001), but it was unaltered after
2 h of CHX treatment (Fig. 8, a and d).
When diazoxide was present in the medium containing 15 mM
glucose, [Ca2+]i was low
and stable, but a large and sustained rise occurred when extracellular
K+ was increased to 30 mM. This rise
was inhibited (P < 0.001) by pretreatment with 10
µM CHX for 20 h (Fig. 8, b and e). Two
hours of treatment had no effect, and the small inhibition by 5 h
of treatment was significant (P < 0.05) only when
calculated above baseline.
Links between CHX-induced [Ca2+]i and
insulin secretion changes
The results presented in Figs. 4 and 8 showed that 2 h of CHX
treatment inhibited insulin secretion without significantly affecting
[Ca2+]i, whereas longer
treatment decreased the Ca2+ signal as well.
Figure 9 displays the relationship
between [Ca2+]i and
insulin secretion during stimulation by 15 mM glucose
alone, with tolbutamide or with high K+ and
diazoxide. For each stimulus are shown, linked by the broken lines, the
results obtained in control islets (open symbols) and in
islets treated with CHX for 2, 5 and 20 h (filled
symbols from right to left). Two excellent
correlations with distinct slopes were found for control and
CHX-treated islets. Fitting all data with a single regression line
resulted in a much weaker coefficient of correlation (R = 0.79).
For each stimulus, the inhibition of insulin secretion by CHX treatment
appeared to involve two successive changes: an initial decrease in the
efficiency of Ca2+ (after 2 h) followed by a
decrease in [Ca2+]i.
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, a and b, 5 h of CHX washing
after 15 h pretreatment was followed by a greater reversibility of
insulin secretion than
[Ca2+]i during
stimulation with high K+. The values of these new
control and test experiments fell close to the regression lines
calculated from the first experimental series (Fig. 10c). The critical
observation was that the value for the reversibility group did not move
along the lower curve of CHX-treated islets, but jumped to the upper
curve of control islets. The recovery of secretion thus mainly involved
an increase in Ca2+ efficiency.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rapid and virtually complete (95% after 1 h) blockade of
protein synthesis was achieved by 10 µM CHX, an inhibitor
of translation. The sensitivity of our preparation of mouse islets to
CHX was similar to that of rat islets in which 1–10 µM
CHX inhibited protein synthesis by 75–95% (37, 38, 39). The
present results confirm the observations (16, 17, 18, 19) that the
inhibition of insulin secretion by CHX starts only after about 1 h
of drug application. They further show that there is a time lag between
the reversibility of the inhibition of protein synthesis and insulin
secretion. Thus, both onset and offset of the inhibition of secretion
are delayed compared with those of protein synthesis. Several
observations also indicate that the changes in ß cell function
produced by CHX are not secondary to a crude toxic effect: their
reversibility, the minor alteration of glucose metabolism even after
20 h, and the longer delay (2–3 days) before appearance of
apoptosis in CHX-treated ß cells (40). It seems
reasonable to propose that the time-dependent changes in
stimulus-secretion coupling produced by addition and removal of CHX
result from disappearance and reappearance of proteins with distinct
half-lives.
The inhibition of insulin secretion measured after 2 h of CHX treatment cannot be explained by insufficient total insulin stores, but could theoretically result from the depletion of a finite, small pool of insulin that cannot be replenished because of the suppression of insulin biosynthesis. Newly synthesized insulin can be released rapidly without the newly formed granules mixing with the older ones (20, 21, 41, 42). However, this only corresponds to a small (<10%) fraction of total insulin secretion (20, 21, 43), much smaller than the approximately 50% inhibition observed here already after 2 h. After longer treatment of the islets with CHX (18 h) insulin reserves were higher than in untreated controls. We, therefore, conclude that neither the initial nor the sustained effect of CHX on insulin secretion is mediated by the loss of a particular pool of insulin.
Insulin secretion is tightly dependent on glucose metabolism in ß
cells. A slight (15%) decrease in glucose-induced rise in NAD(P)H
fluorescence and in glucose oxidation was observed after 20 h of
protein synthesis inhibition. This inhibition of glucose metabolism may
contribute to the delayed inhibition of insulin secretion, but it is
too small to explain it entirely. In contrast, we did not observe any
impairment of the NAD(P)H signal after 5 h of CHX treatment, and
others have reported that glycolysis and glucose oxidation in mouse
islets are unaffected by 1 mM CHX after 2–3 h (44, 45). The inhibition of insulin secretion occurring during the
first hours of protein synthesis inhibition cannot be ascribed to any
major defect in glucose metabolism. It can also be concluded that the
proteins involved in glucose metabolism have a relatively long
half-life, which may explain why ß cells can survive a few days
without protein synthesis (40).
K+-ATP channels serve as transduction units between nutrient metabolism and biophysical events in ß cells, and are the direct target of sulfonylureas (4, 5, 6, 15). An inhibition of insulin secretion could result from an impairment of the ability of glucose or tolbutamide to close the channels. Neither 86Rb+ efflux measurements nor patch-clamp recordings suggested that this might be the case after 5 or 20 h of protein synthesis inhibition. The maximum K+-ATP current activated by metabolic poisoning and diazoxide was decreased by approximately 50% in ß cells treated with CHX for 20 h. Because over 90% K+-ATP channels must be closed before the ß cell membrane depolarizes (46, 47), a 50% decrease in available channels is not expected to cause major effects on ß cell function. It may explain, why basal 86Rb+ efflux was slightly decreased in test islets, but it is important to note that this decrease was less than that produced in control islets by 3 mM glucose, a concentration that is insufficient to depolarize ß cells (36).
Upon membrane depolarization, voltage-dependent Ca2+ channels open and allow an influx of Ca2+ that raises [Ca2+]i in ß cells (3, 4, 5, 6, 27). Not only glucose and tolbutamide, but also high K+, that depolarizes independently of K+-ATP channels (by shifting the equilibrium potential for K+), were less effective in increasing [Ca2+]i after 20 h of protein synthesis inhibition. This reduced efficacy is entirely compatible with the commensurate decrease in Ca2+ current measured in similarly treated ß cells. Whether the actual number of Ca2+ channels is decreased by 40% under these conditions or whether the availability of the channels is reduced because of the loss of a regulatory protein cannot be answered by these experiments. Two other aspects of the specific [Ca2+]i response to glucose were perturbed after 20 h of CHX treatment: the small decrease preceding the initial rise, and the oscillations occurring during sustained stimulation were abrogated. The significance of these changes is difficult to establish because the inhibition of glucose-induced insulin secretion after 5 h of CHX was accompanied by an absence of the initial [Ca2+]i decrease in 70% of the islets but a persistence of [Ca2+]i oscillations in all islets. Anyhow it is intriguing that the small initial decrease, which reflects glucose-induced Ca2+ sequestration in the endoplasmic reticulum (48), is lost in ß cells from diabetic db/db mice (49). From our data, one may conclude that a lesser rise in ß cell [Ca2+]i, secondary to a decrease in functional Ca2+ channels, contributes to the inhibition of insulin secretion probably after 5 and certainly after 20 h of protein synthesis inhibition. However, no similar mechanism explains the inhibition of insulin secretion after 2 h.
A major observation of the present study was that short (2 h) treatment of the islets with CHX inhibited insulin secretion without impairing the rise in [Ca2+]i produced by three distinct agents, glucose, tolbutamide and high K+. This dissociation indicates that the efficacy of Ca2+ on secretion was decreased. Thus, the relationship between [Ca2+]i and insulin secretion in response to the three agents could be fitted by distinct, tight correlations corresponding to the absence and presence of CHX, respectively. Moreover, after resumption of protein synthesis (after CHX washing), the recovery of insulin secretion was more dependent on restoration of the action of Ca2+ than elevation of [Ca2+]i. Altogether our data indicate that protein synthesis inhibition in ß cells inhibits insulin secretion in sequential steps: a decrease in the efficiency of Ca2+, followed by a decrease in Ca2+ influx and glucose metabolism.
The synthesis of a distinct subset of islet proteins other than proinsulin is stimulated by glucose (50, 51, 52). Among these proteins, other secretory peptides and proteolytic enzymes involved in prohormone processing have been identified. Many others are unknown and might include proteins whose loss rapidly leads to impairment of insulin secretion. We have not identified these proteins that seem to be distinct from protein kinases A and C and their targets because activation of these kinases normally potentiated insulin secretion after short treatment with CHX. We speculate that these proteins might be implicated in the amplification pathway of glucose-induced insulin secretion (1, 7, 8, 9), the pathway through which glucose increases the efficiency of Ca2+ on exocytosis of insulin granules. It is, however, not necessary to postulate that these proteins with a short half-life are glucose dependent. They could also be important in other secretory systems.
In conclusion, islet protein inhibition impairs insulin secretion by a sequential alteration of distinct steps of stimulus-secretion coupling. Glucose metabolism is not readily affected, which indicates that the involved enzymes have a relatively long half-life. K+-ATP and Ca2+ channels may be similarly affected but only the decrease in Ca2+ channels exerts a negative impact on insulin secretion. Unexpectedly, among all ß cell proteins implicated in the regulation of insulin secretion, that or those with the shortest half-life appear to modulate (directly or indirectly) the action of Ca2+ on exocytosis. These characteristics make them the potential Achille’s heel of stimulus-secretion coupling in ß cells.
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Acknowledgments |
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Footnotes |
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2 Postdoctoral fellow on leave from the Department of Pharmacology,
Faculty of Medicine, University of Salamanca, Spain.
Received July 26, 2000.
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and •) or changed
from a CHX-containing to a control medium (
). Insulin secretion was
then monitored every 3 h between 12 and 21 h. Values are
means ± SEM for 21 batches of islets from three
experiments. b, Other batches of islets were similarly treated except
that [3,5-3H] tyrosine was added to the fresh culture
medium at 12 h, and its incorporation into newly synthesized
proteins was studied at 15 and 18 h. Values are means ±
SEM for eight batches of islets from two experiments.
