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The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104
Address all correspondence and requests for reprints to: Dr. John A. Corbett, Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, Saint Louis, Missouri 63104. E-mail: corbettj{at}wpogate.slu.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN)
results in a potent inhibition of mitochondrial aconitase activity.
NMMA, when cocultured with IL-1ß + IFN, completely prevents
cytokine-induced inhibition of human islet aconitase activity. NMMA,
when added to human islets pretreated for 18 h with IL-1ß +
IFN, stimulates the recovery of mitochondrial aconitase activity
after an additional 8 h incubation. These findings indicate that
nitric oxide-induced islet damage is reversible; however, prolonged
production of nitric oxide (after a 36-h exposure to IL-1ß) results
in the irreversible inhibition of islet metabolic and secretory
function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although IL-1 stimulates islet and ß-cell damage, the destructive effects of this cytokine on islet metabolic function are reversible. Rat islets incubated with IL-1ß for 15 h require a 4-day incubation in cytokine-free media to restore normal glucose-stimulated insulin secretion (12, 13). This window of functional recovery can be reduced to 8 h by the inhibition of iNOS (14). The addition of NMMA to islets preincubated for 18 h with IL-1ß, followed by an 8-h incubation in the presence of both NMMA and IL-1, results in a complete recovery of insulin secretion (14). Recovery of insulin secretion function is paralleled by a simultaneous recovery of mitochondrial aconitase activity (14). The aim of this study was to identify the length of exposure of islets to IL-1 that leads to irreversible inhibition of insulin secretion and to determine whether the irreversible inhibition of insulin secretion correlates with an irreversible inhibition of islet metabolic function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN) were from GIBCO Laboratories (Grand Island,
NY). FCS was obtained from Hyclone (Logan, UT). Human recombinant
IL-1ß was obtained from Cistron Biotechnology (Pine Brook, NJ). NMMA
was purchased from Calbiochem (San Diego, CA).
[35S]methionine was obtained from New England Nuclear
(Boston, MA). All other reagents were from commercially available
sources.
Rat islet isolation and culture
Islets were isolated from male Sprague-Dawley rats by
collagenase digestion as described previously (15). After isolation,
islets were cultured overnight in complete CMRL-1066 culture medium
(CMRL-1066 media supplemented with 2 mM glutamine, 10%
heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml
streptomycin) under an atmosphere of 95% air and 5% CO2
at 37 C. Experiments were initiated by the addition of 5 U/ml (20
pM) IL-1ß and/or iNOS inhibitors (0.5 mM NMMA
or AG) for the indicated times.
Glucose-stimulated insulin secretion
Islets (200 islets/ml of complete CMRL-1066) were cultured with
5 U/ml IL-1ß and/or the iNOS inhibitor, AG, for the indicated times.
After treatment, the islets were washed three times with Krebs-Ringer
bicarbonate buffer (25 mM HEPES, 115 mM NaCl,
24 mM NaHCO3, 5 mM KCl, 1
mM MgCl2, 2.5 mM CaCl2,
pH 7.4) containing 3 mM D-glucose and 0.1%
BSA. Groups of 20 islets were counted into 10 mm x 75 mm
borosilicate tubes and preincubated for 30 min in 200 µl of the same
buffer at 37 C with shaking. The preincubation solution was removed,
and glucose-stimulated insulin secretion was initiated by the addition
of 200 µl Krebs-Ringer bicarbonate buffer containing either 3
mM or 20 mM D-glucose. Islets
were then incubated for 30 min, incubation media were removed, and
insulin content was determined by RIA (16).
Aconitase activity
Before experiments, human islets were cultured for 3 days in
complete CMRL-1066 medium under an atmosphere of 95% air and 5%
CO2 at 37 C. Hand-picked human (7000 islets) or rat (6000
islets) islets were dispersed into single cells by treatment with
trypsin (1.0 mg/ml) in Ca2+ and Mg2+ free
Hanks’ solution for 3 min at 37 C as described previously (6, 17).
Dispersed islet cells (1 x 106 cells/2 ml complete
CMRL-1066) or RINm5F cells (5 x 106 cells/2 ml
complete CMRL-1066) were incubated for 3 h before experiments were
initiated. Rat islet cells or RINm5F cells were cultured in complete
CMRL-1066 with 5 U/ml IL-1ß and/or 0.5 mM NMMA for the
indicated times. Human islet cells were incubated in the presence or
absence of 75 U/ml IL-1ß and 750 U/ml IFN for 18 h followed
by an 8-h recovery period in the presence of NMMA. After culture, islet
cells or RINm5F cells were isolated by centrifugation, and
mitochondrial aconitase activity was measured using a modified version
of a method previously developed for rat islet cells (6, 14). In brief,
islet cells were prepared for aconitase activity measurements as
described previously (6), and aconitase was assayed at 340 nm in a
reaction containing 20 mM citrate, 0.2 mM
NADP+, 6 mM MnCl2, 50
mM Tris-Cl, pH 7.4, 0.6 U isocitrate dehydrogenase, and 50
µg islet cell extract in a total volume of 200 µl using a Thermo
max microtiter plate reader (Molecular Devices, Menlo Park, CA) at room
temperature. One unit of aconitase activity is defined as 1 pmol NADPH
formed/min per mg of protein.
Nitrite formation
Nitrite formation was measured by mixing 50 µl culture media
with 50 µl Griess reagent (18). The absorbance was measured at 540
nm, and nitrite concentrations were calculated from a sodium nitrite
standard curve.
Islet degeneration
Islets (25 islets/500 µl of complete CMRL-1066) were cultured
for a total of 96 h in the presence or absence of 5 U/ml IL-1ß.
NMMA (0.5 mM) was added at the indicated times after
IL-1ß addition, and the islets were cultured for a total of 96
h. Islet degeneration was determined in a double-blinded manner by
phase-contrast microscopic analysis. Islet degeneration is
characterized by the loss of islet integrity, disintegration, and
partial dispersion of islets as described previously (14, 19).
Total protein synthesis
Total protein synthesis was determined by the method of Hughes
et al. (20). Briefly, islets were washed with
methionine-deficient media (9 parts MEM minus methionine: 1 part MEM
containing methionine), counted (50 islets per well), and incubated for
18 or 36 h with 5 U/ml IL-1ß and/or 0.5 mM NMMA as
indicated. After the initial incubation, NMMA was added and islets were
allowed to recover for an 8-h period. [35S]methionine (33
µCi/ml) was added for the final 2 h of each incubation. Islets
were isolated by centrifugation and washed once with complete CMRL-1066
and twice with PBS, and then islet protein was precipitated by the
addition of ice-cold trichloroacetic acid (10%). Total protein was
isolated by centrifugation, and [35S]methionine
incorporation was determined by liquid scintillation counting.
Statistical analysis
Statistical comparisons were made between groups using a one-way
ANOVA. Significant differences between groups were determined by
Scheffe’s F test post hoc analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The inhibitory effects of IL-1ß
are completely prevented if islets are coincubated with AG in addition
to IL-1ß (data not shown and Ref.21). Islets treated with IL-1ß
for 24 h, followed by an 8-h incubation with AG, results in the
recovery of glucose-stimulated insulin secretion to levels similar to
untreated control islets (Fig. 1a). In contrast, incubation of islets
for 36 or 48 h with IL-1ß results in the irreversible inhibition
of insulin secretion, as an additional 8-h incubation with AG does not
result in the recovery of islet-secretory function (Fig. 1a).
Furthermore, insulin secretion is not restored in islets incubated for
an additional 24 h with NMMA after the 36-h pretreatment with
IL-1ß (Fig. 1b). In these experiments, control islets were cultured
in the absence of IL-1ß and AG for the entire length of the
experiment (e.g. 24-h IL-1 incubation + 8-h AG recovery
incubation for a total incubation of 32 h). In addition, an 8-,
24-, 36-, or 48-h incubation with AG (in the absence of IL-1ß), and
incubation of islets for 24, 36, or 48 h in media followed by an
8 h incubation with AG does not modulate glucose-stimulated
insulin secretion by rat islets (data not shown, and Ref.21). These
findings indicate that islet secretory function is irreversibly
inhibited after a 36-h exposure to IL-1ß.
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, treatment of RINm5F cells with
IL-1ß for 18 or 36 h results in 
80% and 60%
inhibition of mitochondrial aconitase activity. The inhibitory effects
of IL-1ß on aconitase activity are completely prevented by NMMA when
cocultured with IL-1ß. Importantly, the addition of NMMA to RINm5F
cells after an 18-h incubation with IL-1ß, and continued culture for
8 h, results in the recovery of mitochondrial aconitase activity.
However, RINm5F cell mitochondrial aconitase activity is irreversibly
inhibited after a 36-h exposure to IL-1ß, as an additional 8-h
incubation with NMMA does not stimulate the recovery of aconitase
activity.
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).
However, the recovery of aconitase activity after a 36-h incubation
with IL-1ß is significantly attenuated. Rat islet cells recover
approximately 90% of islet aconitase activity after an 18-h incubation
with IL-1ß and an additional 8 h with NMMA, while a 36-h
exposure to IL-1ß followed by an additional 8 h with NMMA
results in the recovery of only 35% of islet aconitase activity. In
these experiments, control islets and RINm5F cells were cultured in the
absence of IL-1ß and NMMA for the entire length of the experiment
(e.g. 18-h IL-1ß incubation + 8-h NMMA incubation for a
total of 26 h). Also, NMMA does not affect islet or RINm5F cell
aconitase activity after an 8-, 18-, or 36-h incubation in the absence
of IL-1ß (data not shown). Similar to the effects of IL-1ß on
glucose-stimulated insulin secretion, these findings indicate that
exposure of rat islets to IL-1ß for 36 h results in irreversible
inhibition of mitochondrial aconitase activity.
Effects of actinomycin D on the recovery of aconitase activity by
RINm5F cells
To examine the potential role of new gene transcription in the
recovery of islet metabolic function, RINm5F cells were treated for
18 h with IL-1 or treated for 5 h with IL-1ß followed by
the addition of actinomycin D and continued cultured for an additional
13 h. NMMA was then added, and the cells were incubated for 8
h additional to allow for recovery of aconitase activity. As shown in
Fig. 3, IL-1ß stimulates a potent
inhibition of RINm5F cell aconitase activity that is restored to
control levels by an additional 8 h incubation with NMMA. Actinomycin
D, added to RINm5F cells 5 h after the addition of IL-1ß,
completely prevents NMMA-induced recovery of aconitase activity. The
inhibitory effects of actinomycin D on the recovery of aconitase
activity do not appear to be mediated by the inhibition of normal
protein turnover. Actinomycin D alone does not inhibit RINm5F cell
aconitase activity (data not shown) and nearly completely prevents
IL-1-induced inhibition of aconitase activity (Fig. 3). Also, the
addition of actinomycin D to RINm5F cells 5 h after the addition
of IL-1ß does not inhibit IL-1ß-induced nitrite production (Fig. 3), presumably because maximal IL-1ß-induced iNOS mRNA accumulation
occurs within the first 5–6 h of treatment (4, 22). These results
indicate that the recovery of mitochondrial function appears to require
the expression of proteins that may participate in the repair of nitric
oxide-mediated cellular damage.
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). The inhibitory effects are
completely prevented by the coincubation of islets with NMMA in
addition to IL-1ß. Preincubation of islets for 18 h with IL-1ß
followed by the addition of NMMA and continued culture for 8 h (in
the presence of IL-1ß and NMMA) results in a complete restoration of
[35S]methionine incorporation. This finding is consistent
with the ability of islets to completely recover secretory function and
aconitase activity under similar conditions. Importantly, protein
synthesis is irreversibly inhibited in islets treated for 36 h
with IL-1ß. The amount of [35S]methionine incorporation
is nearly identical in islets exposed for 36 h to IL-1ß as
compared with islets preincubated for 36 h with IL-1ß and then
cultured for an additional 8 h with IL-1ß and NMMA. These
results indicate that a 36-h exposure of rat islets to IL-1ß results
in an irreversible inhibition of protein synthesis.
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). However, treatment of rat islets for
36 or 48 h with IL-1ß, followed by the addition of NMMA and
continued culture for a total of 96 h, does not prevent islet
disintegration. These findings indicate that a 36-h exposure to IL-1ß
commits islets to destruction; however, if iNOS activity is inhibited
by NMMA at times before 36 h, islets recover from IL-1-induced
damage.
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(23, 24, 25). Treatment of human islets for 18 h with
75 U/ml IL-1ß and 750 U/ml IFN results in a 4-fold increase in
nitrite production and 50% inhibition of mitochondrial aconitase
activity (Fig. 6). NMMA completely
prevents cytokine-induced nitrite production and the inhibition of
aconitase activity by human islets. Treatment of human islets for
18 h with IL-1ß + IFN, followed by the addition of NMMA and
continued culture for 8 h, results in the recovery of aconitase
activity. These findings provide the first evidence that IL-1ß +
IFN induce an inhibition of human islet aconitase activity that is
mediated by nitric oxide and that human islets have the capacity to
recover from nitric oxide-mediated damage.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recovery of islet mitochondrial aconitase activity after IL-1ß-induced damage requires mRNA transcription as actinomycin D prevents NMMA-induced recovery. Consistent with the ability of actinomycin D to inhibit the recovery of insulin secretion under similar conditions (14), this result suggests that islets express recovery proteins in response to IL-1ß-induced damage. The identity of potential recovery factors and the stimuli that induce the expression of these factors are unknown. Recent studies by Kolb and co-workers (26, 27) suggest that heat shock proteins may participate in the recovery of islet function. This group has shown that preincubation of rat islets at 42 C attenuates the destructive effects of nitric oxide on islet viability (26), and that overexpression of heat shock protein 70 confers resistance to nitric oxide-mediated damage (27). In preliminary studies, we have shown that heat shock treatment attenuates the inhibitory effects of IL-1ß on RINm5F cell mitochondrial aconitase activity (our unpublished observation). The stimuli for expression of potential recovery proteins are unknown. Lancaster and co-workers (28) have shown that pretreatment of rat hepatocytes with nitric oxide attenuates cytokine-induced hepatocyte injury. The protective actions of nitric oxide are dependent on new protein synthesis, suggesting that nitric oxide may be the stimulus for the expression of potential recovery factors. We are currently investigating these possibilities.
We also show that a combination of IL-1ß + IFN stimulates a 4-fold
increase in the production of nitric oxide and a potent inhibition of
human islet mitochondrial aconitase activity. NMMA completely prevents
the inhibitory effects of IL-1ß + IFN on aconitase activity. NMMA
also stimulates the recovery of aconitase activity when added to human
islets after an 18-h incubation with this combination of cytokines.
This result is consistent with previous studies showing that an 18-h
incubation with IL-1ß + IFN + tumor necrosis factor-
(TNF)
stimulates the formation of iron-nitrosyl complexes (detected by
electron paramagentic resonance spectroscopy) and inhibits insulin
secretion by human islets (23). NMMA prevents iron-nitrosyl complex
formation and attenuates the inhibitory effects on insulin secretion
(23). These results indicate that cytokine-induced inhibition of
insulin secretion by human islets may be mediated, in part, by the
ability of nitric oxide to target and inhibit the function of
mitochondrial iron-sulfur-containing enzymes, specifically
aconitase.
It has been suggested that the damaging effects of cytokines on human
islet function can be disassociated from the production of nitric oxide
(29, 30, 31). However, in one of these studies, AG (5 mM) alone
inhibited insulin secretion by human islets to levels comparable to the
effects of IL-1ß + TNF + IFN (29). In addition, AG has been
shown to attenuate cytokine-induced media insulin accumulation after a
2-day culture of human islets with IL-1ß + TNF + IFN (29), and
nicotinamide attenuates IL-1ß + TNF + IFN-induced nitrite
production and inhibition of insulin secretion by human islets (30).
Human islets appear to be highly sensitive to peroxynitrite (32), a
compound that is produced by the interactions of nitric oxide and
oxygen radicals. The lack of complete protection afforded by NMMA (23)
suggests that other effector molecules (in addition to nitric oxide)
may also participate in cytokine-induced inhibition of human islet
function. Potential mediators include oxygen radicals (33) and
peroxynitrite, or a nitrogen dioxide species formed by the reaction of
nitric oxide and superoxide (34, 35). However, it should be noted that
the endogenous production of peroxynitrite by human islets has yet to
be demonstrated.
A critical question concerning the development of autoimmune diabetes is the mechanism of ß-cell destruction. A number of studies have suggested that ß-cell destruction induced by cytokines in vitro, or during the development of autoimmune diabetes in the nonobese diabetic mouse (NOD), is by apoptotic mechanisms (36, 37, 38). These studies are primarily based on the detection of DNA damage, either by TUNEL staining or DNA fragmentation by agarose gel electrophoresis. Apoptosis is characterized by both morphological and biochemical changes that include cell shrinking, membrane blebbing, nuclear condensation, and formation of apoptotic bodies (39). The net outcome of apoptotic death is the packaging of the cellular contents into membrane-bound vesicles that are ingested by phagocytes in the absence of an inflammatory response (39, 40, 41). Since ß-cell destruction during the development of autoimmune diabetes is characterized by a local inflammatory response, and apoptosis is a mechanism to remove unwanted cells in the absence of inflammation (39, 40, 41), it is difficult to rationalize an apoptotic mechanism of ß-cell death during the development of autoimmune diabetes. In this study we show that IL-1ß-induced islet damage is associated with the inhibition of islet mitochondrial function and reduced cellular levels of ATP (10, 11). Recently, Keust et al. (42) have provided evidence that implicates ATP as an intracellular switch that determines whether a cell dies by apoptosis or necrosis. T cell death (stimulated by staurosporin or CD95) occurs by apoptosis in the presence of ATP and by necrosis in the absence of ATP (39). Cytokines, particularly IL-1ß, have been shown to stimulate islet and ß-cell DNA damage in a nitric oxide-dependent manner (36, 37, 43); however, the presence of DNA damage in IL-1ß-treated islets does not necessarily mean that ß-cells die by apoptotic mechanisms. Our findings, along with results of others (36, 37, 43), indicate that the demise of ß-cells is most likely a combination of cytokine-induced DNA damage and the irreversible inhibition of islet metabolic function.
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Acknowledgments |
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Footnotes |
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2 Supported by a Career Development Award from the Juvenile Diabetes
Foundation International.
Received June 3, 1997.
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References |
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via an L-arginine-dependent nitric oxide generation mechanism. FEBS
Lett 276:42–44[CrossRef][Medline]
B in the signaling mechanism. Endocrinology 136:4790–4795[Abstract]
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) are as indicated.
