Departments of 1 Pharmacology and 2 Pathology, Institute of Physiological Sciences, University of Lund, S-223 62 Lund, Sweden
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ABSTRACT |
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We investigated, by a
combined in vivo and in vitro approach, the temporal changes of islet
nitric oxide synthase (NOS)-derived nitric oxide (NO) and heme
oxygenase (HO)-derived carbon monoxide (CO) production in relation to
insulin and glucagon secretion during acute endotoxemia induced by
lipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP and
cGMP content after in vitro incubation, the insulin response to glucose
in vivo and in vitro, and the insulin and glucagon responses to the
adenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) in
islet tissue. Immunocytochemistry revealed a marked expression of iNOS
in many -cells, but only in single
-cells after LPS. Moreover,
biochemical analysis showed a time dependent and markedly increased
production of NO and CO in these islets. Addition of a NOS inhibitor to
such islets evoked a marked potentiation of glucose-stimulated insulin
release. Finally, after incubation in vitro, a marked suppression of NO
production by both exogenous CO and glucagon was observed in control
islets. This effect occurred independently of a concomitant inhibition
of guanylyl cyclase. We suggest that the impairing effect of increased
production of islet NO on insulin secretion during acute endotoxemia is
antagonized by increased activities of the islet cAMP and HO-CO
systems, constituting important compensatory mechanisms against the
noxious and diabetogenic actions of NO in endocrine pancreas.
lipopolysaccharide; pancreatic islets; nitric oxide; carbon monoxide; cyclic nucleotides; insulin; glucagon secretion
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INTRODUCTION |
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NITRIC OXIDE (NO) is formed from L-arginine under the influence of the nitric oxide synthase (NOS) (29). This enzyme appears in two major isoforms: constitutive Ca2+/calmodulin dependent (constitutive NOS; cNOS) and inducible Ca2+/calmodulin independent (inducible NOS; iNOS) (23). Both isoforms have been shown to occur in the islets of Langerhans (2, 10, 28, 35, 42). Carbon monoxide (CO) (50) is formed from heme under the influence of the heme oxygenase (HO) enzyme (27), which like NOS, also appears in two isoforms, one inducible HO (HO-1) and one constitutive HO (HO-2) (27). Both these isoforms also exist in the islets of Langerhans (2, 19, 20, 51). It should be added, with regard to cNOS, that this enzyme has two isoforms, a neuronal form (ncNOS) and an endothelial form (ecNOS) (23). These isoforms can be differentiated by immunocytochemistry and immunoblotting (2).
Although NO, produced in small amounts by the cNOS enzyme, is regarded
as a putative physiological modulator of islet hormone release
(22, 24, 33, 35, 39, 42), there seems to be a general
agreement that the large amounts of NO produced by iNOS may have an
important role in the pathogenesis of insulin-dependent diabetes
mellitus (IDDM) via a noxious influence on -cells (8, 10, 11,
14, 28, 48). The expression of iNOS in islet
-cells is
mediated by the cytokines interleukin (IL)-1
, tumor necrosis
factor-
, and interferon-
, produced by lymphocytes and macrophages
that are known to infiltrate the islets during the development of IDDM
(8, 12, 14, 48). Furthermore, IL-1
can also induce the
expression of heat shock protein 70 and HO-1 in
-cells (47,
51). These proteins, in contrast, are thought to be implicated
in islet defense against oxidative stress (29, 47).
NO, when produced by iNOS, is thought to contribute to islet
dysfunction and destruction, impairing at several vital sites in the
-cell, such as nuclear DNA, Krebs cycle aconitase, mitochondrial electron transfer chain, and membrane ion channels (13, 14, 28,
48, 49). Moreover, NO has also been shown to induce apoptosis in pancreatic
-cell lines by a cGMP-mediated
effect (25).
In the present study, a primary aim was to elucidate possible effects
on islet NO production in relation to the function of -cells and
-cells after in vivo injection of the cytokine-producing endotoxin
lipopolysaccharide (LPS). It should be recalled that there is
reportedly no effect on iNOS expression in islets directly incubated in
vitro with LPS itself (9). Different time points after LPS
administration were studied both in vivo and in vitro, because several
earlier in vitro investigations on cytokine effects in cultured islets
have described both acute stimulatory and later inhibitory effects of
different cytokines on insulin secretion (cf. 16, 47, 52). The in vivo
route of administration of endotoxin was chosen as a model of islet
cell reaction during the "acute phase response," but also in an
attempt to mimic a putative, acute in vivo cytokine attack on the
-cells, an event assumed to be one of several important pathogenic
mechanisms in the development of IDDM (16). It should,
however, be recalled that the prediabetic state, in many cases,
involves a cell-to-cell cytokine delivery over prolonged periods of
time and thus might not always be strictly comparable with the present
experimental situation. Since we have very recently (19,
20) discovered that the pancreatic islets contain an HO-2 that
can produce large amounts of CO, which in turn might be able to inhibit
islet NO production (19), we found it of interest to
elucidate whether LPS injection would have any influence on islet CO
production. Therefore, biochemical analyses were performed to reveal
time-dependent changes in the production of NO and CO in islets from
LPS-injected animals in relation to islet hormone secretory capacity
both in vitro and in vivo. In certain experiments, we also measured
perturbations in islet cAMP and cGMP levels as well as changes in
protein expression of the different isoforms of the NOS and HO proteins
with the use of immunoblotting and immunocytochemistry.
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MATERIALS AND METHODS |
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Animals. Female mice of the NMRI strain (B&K, Sollentuna, Sweden) weighing 28-37 g were used in all studies. They were fed a standard pellet diet (B&K) and tap water ad libitum. All animals used for preparation of pancreatic islets were killed by cervical dislocation, and isolation of the islets was performed by retrograde injection of a collagenase solution via the bile-pancreatic duct (15). Animals treated with LPS were used at different time points after injection. Their body weights were thereby checked at 0, 24, and 48 h after LPS administration. There was a modest decrease in body weight (significant at 48 h) for LPS-injected mice compared with saline-injected controls during the experimental period; controls were 33.3 ± 1.2, 33.4 ± 0.9, and 33.6 ± 0.7 g at 0, 24, and 48 h vs. 34.6 ± 1.0, 31.7 ± 0.9, and 29.3 ± 1.0 g for LPS-treated animals. The animal experiments were approved by the local animal welfare committee (Lund, Sweden).
Chemicals. LPS from Salmonella typhimurium was from Sigma Chemical (St. Louis, MO). The radioimmunoassay kits for insulin and glucagon determination were obtained from Diagnostika (Falkenberg, Sweden) and Euro-Diagnostica (Malmö, Sweden), respectively. Collagenase was obtained from Worthington Biochemicals (Freehold, NJ). Bovine serum albumin was from ICN Biomedicals (High Wycombe, UK). NG-nitro-L-arginine methyl ester (L-NAME), hemin-HCl, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and forskolin, as well as all other chemicals, were from Sigma Chemical.
Immunocytochemistry. LPS-treated and control mice were killed by a blow to the neck, whereupon the pancreatic glands were dissected, divided into pieces, and further processed for immunocytochemistry as previously described in detail (19).
For the demonstration of HO-2, HO-1, or ncNOS, cryostat sections were incubated overnight with rabbit antisera to HO-2 (code OSA 200, lot 709422; 1:1,000), HO-1 (code OSA 100, lot 611416; 1:500; both antisera were purchased from StressGen Biotechnol, Victoria, BC, Canada), or ncNOS (1:2,400) (2, 3). The selectivity of the presently employed HO-1 and HO-2 antisera as well as appropriate control experiments were previously reported (2, 20). After being rinsed, the sections were incubated in Texas red-conjugated donkey anti-rabbit immunoglobulins (IgG) for 90 min, rinsed, and mounted. For the simultaneous demonstration of two antigens, sections were incubated overnight with antisera raised in guinea pigs to insulin (1:1,600) or glucagon (1:4,000; both antisera were purchased from Linco, St Louis, MO). After being rinsed, the sections were incubated for 90 min with fluorescein isothiocyanate (FITC)-conjugated goat anti-guinea pig IgG and rinsed. Immediately after that, some of the sections were incubated with Texas red-conjugated IgG (1:125), rinsed, and mounted (see above). For the demonstration of iNOS, other sections were mounted, and the insulin and glucagon immunoreactivities were documented by microphotography. After being rinsed overnight in phosphate-buffered saline (PBS) with elimination of the coverslips, the sections were incubated for 10 min in 5% swine serum in PBS and then overnight in rabbit antiserum to iNOS (1:2,000) (17, 26). After being rinsed, the sections were incubated with biotinylated swine anti-rabbit IgG (1:200 for 30 min) and then with peroxidase-coupled avidin (1:1,000 for 30 min). After being rinsed, the immunoreactive products were detected by incubation for 5 min in a solution containing 25 µg of 3,3'-diaminobenzidine, 100 ml of PBS, and 50 µl of hydrogen peroxide. After being rinsed in running tap water, the sections were dehydrated and mounted. An Olympus 3 × 50 fluorescence microscope, equipped with epi-illumination and appropriate filter settings for Texas red and FITC immunofluorescence, was used for the examinations of the sections (31). The primary and secondary antisera were diluted in PBS. In control experiments, no immunoreactivity could be detected in sections incubated in the absence of the primary antisera or with antisera absorbed with excess of the corresponding immunizing antigen (100 µg/ml). No absorption controls could be performed with the iNOS antiserum because antigenic substances were not available. The characteristics of the iNOS and the ncNOS antisera have been presented previously (3, 26, 45).In vivo experiments. LPS and D-glucose were dissolved in 0.9% NaCl (saline). Forskolin was dissolved in DMSO and then diluted in saline. LPS was dissolved in saline and then injected intraperitoneally (10 mg/kg), whereas glucose (3.3 mmol/kg) and forskolin (7.3 µmol/kg) were injected intravenously in a tail vein (volume load: 5-10 µl/g mouse). Controls received either saline (glucose) or vehicle (forskolin). All animals were fed freely during the whole study. Blood sampling was performed as described previously (37). The concentrations of insulin and glucagon in plasma were determined by radioimmunoassay (1, 18, 36). Plasma glucose concentrations were determined enzymatically (7).
Assay of islet NOS activity.
Isolated, handpicked, and thoroughly washed islets were collected in
ice-cold buffer (840 µl) containing 20 mmol/l HEPES, 0.5 mmol/l EDTA,
and 1 mmol/l dithiothreitol, pH 7.2, and immediately frozen at
20°C. On the day of assay, the islets were sonicated on ice, and
the buffer solution containing the islet homogenate was supplemented to
also contain 0.45 mmol/l CaCl2, 2 mmol/l NADPH, 25 units of
calmodulin, and 0.2 mmol/l L-arginine in a total volume of
1 ml as previously described (39). The buffer composition is essentially the same as previously described for assay of NOS in
brain tissue using radiolabeled L-arginine
(6). The homogenate was then incubated at 37°C under
constant air bubbling, 1.0 ml/min, for 3 h. It was ascertained
that under these conditions, the reaction velocity was linear for at
least 6 h. Aliquots of the incubated homogenate (200 µl) were
then passed through a 1-ml Amprep CBA cation-exchange column for
high-performance liquid chromatography analysis. The amount of
L-citrulline formed was then measured in a Hitachi F-1000
fluorescence spectrophotometer (Merck) as previously described
(39). NO and L-citrulline are produced in
equimolar concentrations. The nitrogen atom of the guanidino group of
L-arginine is released as NO, which is highly reactive, and
therefore the simultaneous liberated and stable
L-citrulline is preferably measured (6, 29).
The methodology has been described in detail earlier (21,
39). Protein was determined according to Bradford
(5) on samples from the original homogenate.
Western blot analysis. Approximately 200 islets were handpicked in Hanks' buffer under a stereomicroscope and sonicated on ice (3 × 10 s). The protein content was determined according to Bradford (5). Homogenate samples representing 20 µg of total protein from islet tissue were run on 10% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes by electrotransfer (10- to 15 V, 60 min; semi-dry transfer cell; Bio-Rad, Richmond, CA). The membranes were blocked in 9 mM Tris · HCl (pH 7.4) containing 5% nonfat milk powder for 40 min at 37°C. Immunoblotting with rabbit anti-mouse ncNOS (N-7155), iNOS (N-7782; 1:2,000; Sigma), HO-2, and HO-1 (1:2,000) antibodies (StressGen Biotechnol) was performed for 16 h at room temperature. The membranes were washed twice and then incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10,000; Sigma) for 90 min. Antibody binding to ncNOS, iNOS, HO-2, and HO-1 was detected using 0.25 mM CDP-Star (Boehringer Mannheim, Mannheim, Germany) and the signal enhancer Nitro Block II (Tropix, Bedford, MA) for 5 min at room temperature. The chemiluminescence signal was visualized by exposing the membranes to DuPont Cronex X-ray films for 1- to 5 min.
Hormone secretion in vitro. Freshly isolated islets were preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, supplemented with 10 mmol/l HEPES, 0.1% bovine serum albumin, and 1 mmol/l glucose. Each incubation vial contained 10 islets in 1.0 ml buffer solution, and, unless otherwise stated, was gassed with 95% O2-5% CO2 to obtain constant pH and oxygenation. After preincubation, the buffer was changed to a medium containing either 1.0, 7.0, or 16.7 mmol/l of glucose together with the different test agents, and the islets were incubated for 60 min. Aliquots of the medium were then removed and frozen for subsequent assays of insulin (18) and glucagon (1, 36).
Measurement of islet HO activity.
CO production was determined with a sensitive gas chromatographic
method essentially as previously described (19, 20). Islets were isolated and handpicked under a stereomicroscope at room
temperature and then thoroughly washed and collected in ice-cold phosphate buffer (0.1 mol/l, pH 7.4; ~300 islets in 200 µl buffer) and thereafter immediately frozen at 20°C. On the day of assay, the
islets were sonicated on ice, and methemalbumin (30 µl),
-NADPH [100 µl; 4 mg dissolved in 1 ml phosphate buffer (0.1 mol/l)], and
hemoglobin (2 mg) were added with phosphate buffer up to a final volume
of 1 ml. Hemoglobin is added to trap the liberated CO produced in the
assay mixture (19, 20). Methemalbumin solution (substrate)
was prepared by dissolving 25 mg hemin, 82.5 mg NaCl, and 12.1 mg Tris
base in 5 ml of 0.1 mol/l NaOH, followed by the addition of 5 ml
albumin solution (20 g/l) and 5 ml distilled water. The homogenate was
then incubated in a water bath at 37°C and protected from light.
Aliquots (330 µl) were taken after 6 min of incubation, which was
terminated by placing the tubes on ice. The samples were then injected
into reaction tubes containing ferricyanide-citric acid (100 µl).
Nitrogen was used as a carrier gas as well as to purge the reaction
vessels for 4 min before the samples were injected into them. After a
reaction time of 4 min, the liberated CO was brought to a nickel
catalyst and mixed with H2, thus giving methane, which was
brought further to the detector. CO (99.9%) was used as a
standard. The amount of CO produced was calculated from the area under
the curve. Protein was determined according to Bradford
(5) on samples from the original homogenate.
Measurement of islet cAMP and cGMP.
Incubation of isolated islets in the presence of 0.2 mM IBMX was
stopped by removal of the buffer and addition of 0.5 ml of ice-cold
10% trichloroacetic acid, followed by immediate freezing in a 70°C
ethanol bath (34). Before assay, 0.5 ml of H2O
was added, and the samples were sonicated for 3 × 5 s,
followed by centrifugation at 1,100 g for 15 min. The
supernatants were collected and extracted with 4 × 2 ml of
water-saturated diethyl ether. The aqueous phase was removed and
freeze-dried with a Lyovac GT-2 freeze dryer. The residue was then
dissolved in 450 µl of 50 mmol/l sodium acetate buffer (pH 6.2). The
amounts of cAMP and cGMP were quantified with a
125I-labeled cAMP and 125I-labeled cGMP
radioimmunoassay kit (RIANEN; DuPont, Boston, MA).
Statistics. Statistical significance between sets of data was assessed using unpaired Student's t-test, or, where applicable, analysis of variance followed by the Tukey-Kramer multiple comparisons test. Results are expressed as means ± SE.
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RESULTS |
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Effect of LPS on basal plasma levels of insulin, glucagon, and
glucose.
To investigate putative effects of LPS on plasma levels of insulin,
glucagon, and glucose in relation to time, the plasma concentrations of
these parameters were determined at different time points (1, 2, 4, 6, and 24 h after intraperitoneal injection of LPS). Figure
1 shows that from 4 h and onward
after LPS injection, the plasma glucose levels were suppressed
compared with controls. The plasma glucagon levels started to rise
already at 2 h after LPS injection and remained elevated
throughout. No apparent changes in the plasma insulin levels were
observed. To investigate the possibility of a long-lasting, LPS-induced
impairment of basal glucose homeostasis, the plasma glucose levels were
also determined at 11 days after LPS injection. In LPS-injected
animals, the basal plasma glucose concentration was 9.0 ± 0.3 mmol/l (n = 8) vs. 9.7 ± 0.4 mmol/l
(n = 8) in saline-injected animals, indicating no
apparent impairment of basal glucose homeostasis at this time point
following a single injection of LPS.
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In vivo effects of glucose and forskolin on insulin and glucagon
responses at 3 and 6 h after LPS injection.
The acute in vivo effects of saline (basal values), glucose, or
forskolin administration on insulin and glucagon responses after LPS
treatment vs. controls were investigated at 3 and 6 h after
injection of LPS, i.e., at time points when initial changes in the
basal plasma levels of glucagon and glucose were observed. Figure
2 (the four bars to the left) shows that
the basal plasma glucagon levels were greatly enhanced at both time
points (3 and 6 h) in LPS-treated mice, whereas the insulin levels
were unaffected. Furthermore, the basal plasma glucose concentrations
were suppressed in the LPS mice. Figure 2 (the four bars in the middle)
shows that in the LPS mice, the acute insulin response (at 2 min) after an intravenous challenge of glucose was increased at both 3 and 6 h after LPS treatment, compared with glucose-injected controls. Glucose
injection, however, did not acutely suppress the elevated levels of
glucagon. Since activation of the cAMP system in the -cell is known
to potentiate glucose-stimulated insulin release, influence of LPS
administration on this system was studied by measuring the acute
insulin and glucagon response following an intravenous injection of the
adenylate cyclase activator forskolin. Figure 2 (the four bars to the
right) shows that LPS-treated animals displayed a greatly increased
acute insulin and glucagon response to forskolin at 3 h after LPS.
This increased response was further exaggerated when forskolin was
injected at 6 h after LPS administration.
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Islet NOS activity after LPS injection.
Figure 3A shows the production
of NO in islets isolated at 3, 6, 16, 24, and 48 h after LPS
injection. At 6 h after LPS administration, there was a clear
increase in NO production that was further increased with a peak value
at 16 h, representing a threefold increase compared with controls
(75.2 ± 2.0 vs. 22.8 ± 1.3 pmol · min1 · mg protein
1).
The NO production was still increased at 2 days after LPS
administration (Fig. 3A).
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Western blot of NOS isoforms. In islets from mice treated with LPS (20 h after LPS administration), there was an exclusive expression of iNOS protein (Fig. 3B), whereas none could be detected in islets from controls given saline. In contrast, expression of ncNOS protein could be detected in islets from both control and LPS-treated animals (Fig. 3B).
Effects of LPS injection on insulin and glucagon secretion from
isolated islets in the presence of low and high concentrations of
glucose.
Insulin secretion stimulated by a high concentration (16.7 mmol/l) of
glucose was greater from islets of LPS-treated animals than from
control islets at 3, 6, and 16 h after LPS administration (Fig.
4A). No appreciable
differences in insulin secretion between the two groups could be seen
in the presence of the low concentration of glucose. In comparison,
with regard to glucagon release, no apparent differences were noted at
either low (glucagon stimulating) or high glucose concentration after
LPS treatment (Fig. 4B).
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Effects of in vitro NOS inhibition on hormone release from islets
isolated from LPS- or saline-treated mice.
In the presence of 16.7 mmol/l glucose, the NOS inhibitor
L-NAME (1.0 and 5.0 mmol/l) potentiated the secretion of
insulin from islets of both control and LPS-treated mice (Fig.
5A). At a concentration of 5 mmol/l of L-NAME, the rate of insulin secretion from
islets of LPS-treated mice was markedly enhanced; i.e., 18.0 ± 0.87 nmol · islet1 · h
1
compared with 9.71 ± 0.89 nmol · islet
1 · h
1 in
control islets. Glucagon secretion from control islets, but not from
LPS islets, was inhibited by L-NAME (Fig. 5B).
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Effect of LPS injection on hormone release from isolated islets
stimulated by the adenylate cyclase activator forskolin.
The insulin secretory response to forskolin in the presence of a basal
concentration of glucose (7 mmol/l) was four- to sevenfold greater from
incubated islets isolated from LPS-treated mice compared with islets
from control mice (Fig. 6). This
difference could be seen at all time points tested (3, 6, 16, and
24 h after LPS treatment), with the peak value at 24 h. The
glucagon secretion was also greater in islets from LPS-treated animals
than from controls. In contrast to the insulin release, the
potentiation of the glucagon release was more pronounced at the early
time points (3 and 6 h) and declined with time.
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Immunocytochemical findings.
ncNOS immunoreactivity was observed in the majority of islet cells.
Double immunolabeling showed that most ncNOS immunoreactive cells also expressed insulin immunoreactivity (Fig.
7, A and B). Glucagon immunoreactivity occurred in a smaller number of islet cells,
mostly located in the periphery, and some of these also displayed ncNOS
immunoreactivity (Fig. 7, C and D). Note the
absence of ncNOS immunoreactivity in the surrounding exocrine tissue. After LPS administration, no overt change of ncNOS immunoreactivity in
islet cells could be seen (data not shown).
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Islet HO activity after LPS injection.
HO activity measured as production of CO in isolated islets at 3, 6, 20, and 48 h after intraperitoneal LPS administration is
shown in Fig. 9A. The
production of CO was significantly increased at 6 h (50%) after
LPS treatment and then further elevated after 20 (120%) and 48 (130%)
h compared with controls.
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Western blot of HO isoforms. Expression of HO-2 at 20 h after LPS administration was seen in islets of both saline- and LPS-injected animals. Notably, the expression of HO-2 protein was stronger after LPS than in controls (Fig. 9B). Although the blots are only semiquantitative, the visual increase in HO-2 protein expression was in good accordance with the marked increase of CO production at 20 h (Fig. 9A). Expression of HO-1 protein could be detected only in LPS-treated animals (Fig. 9B).
Influence of LPS injection on islet cAMP and cGMP content.
Islets taken from mice 20 h after LPS treatment and then incubated
in the presence of 1 mmol/l glucose for 1 h showed a 75% higher
content of cAMP than control islets (Table
1). Incubation of islets at a high
concentration of glucose (16.7 mmol/l) increased cAMP levels almost
sixfold in control islets but only threefold in LPS-treated islets. At
this high concentration of glucose, no differences could be seen in
islet cAMP content after LPS treatment compared with controls. cGMP
levels were significantly greater (55%) in islets from LPS-treated
mice than in islets from control mice after incubation at both a low (1 mmol/l) and a high (16.7 mmol/l) concentration of glucose (Table 1).
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Effect of glucagon and exogenous CO in the absence and presence of
the guanylyl cyclase inhibitor ODQ on islet NOS activity and insulin
and glucagon secretion.
The most prominent effects recorded following LPS injection, apart from
the increase in islet NO evolution, were a marked increase in plasma
glucagon levels (Fig. 1B) as well as a great stimulation of
islet CO production (Fig. 9A). In view of these findings,
the effects of glucagon and CO on NOS activity in normal control islets
were tested. The production of NO in islets incubated at 16.7 mmol/l of
glucose was significantly decreased both by means of exogenously
applied CO (10 µmol/l) and glucagon (10 µmol/l; Fig.
10A). Preparation of CO
solutions and estimation of concentration were previously described
(32). CO was still able to inhibit NO production in the
presence of the guanylyl cyclase inhibitor ODQ (10 µmol/l). Both
glucagon and CO were stimulatory to insulin secretion (Fig.
10B), and CO also stimulated glucagon secretion (Fig.
10C). The stimulatory effects of CO on insulin and
glucagon secretion were inhibited in the presence of ODQ (Fig.
10, B and C).
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DISCUSSION |
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In this study, we have presented a model of islet cell reaction
during the acute phase response after LPS injection. A single high dose
of LPS had no appreciable effect on basal plasma levels of insulin
during the time period studied (1 to 24 h), whereas basal glucagon
levels started to increase at 2 h and remained elevated
throughout, probably to counterbalance an apparent decrease in plasma
glucose levels. The in vivo insulin response to glucose was already
markedly increased at 3 h after LPS injection. At this time point,
both the insulin and glucagon responses to the adenylate cyclase
activator forskolin were greatly exaggerated both in vivo and in vitro,
suggesting that an early event induced by the LPS-cytokine attack on
the pancreatic islets is manifested as a marked increase in the
sensitivity and capacity of the islet cAMP pathway. This enhanced
secretory capacity of the islet cAMP system increased with time in
insulin-producing -cells but decreased in glucagon-producing
-cells. In
-cells, such a temporal pattern may be brought about
as a compensatory mechanism against the accompanying gradual increase
in the production of islet NO, which is known to greatly impair
glucose-stimulated insulin release (9, 14). In contrast,
according to ancillary experiments in our laboratory, NO has no
inhibitory effect on insulin release stimulated by the phosphodiesterase inhibiting and thus cAMP-stabilizing agent IBMX. Moreover, NO itself is stimulatory to glucagon release (21, 33,
39-41) and thus there is probably no further need for
stimulation of the
-cell cAMP system during the latter part of the
time period studied. In addition, the production of islet CO, which in
contrast to NO is stimulatory to insulin release (19, 20),
was temporally increased, almost in parallel to the NO production and
thus similar to the cAMP system, possibly serving as a compensatory
mechanism against the impairing effect of NO on the
-cells. It is
notable that activation of the cAMP system seems to be more rapid in
onset than the activation of the HO-CO system. Islet NO production
slightly declined after 16 h but was still twofold elevated at
48 h, whereas CO production was maximally elevated at both 20 and
48 h. This pattern could be a reflection of our recent suggestion
(19) and our present demonstration (Fig. 10A)
that CO has a direct inhibitory action on islet NOS activity.
Therefore, "normalization" of glucose-stimulated insulin release in
isolated islets at 24 h after LPS injection could represent a
balance between the inhibitory effect of NO and the stimulatory effects
of CO and cAMP, the cAMP system in
-cells being continuously
stimulated by the enhanced intra-islet glucagon concentrations
following the increased glucagon release and also in turn by the
long-lasting elevation of the plasma glucagon levels. It should be
noted that the in vivo glucagon release was much more pronounced than
in vitro, suggesting that neural stimulation is involved in the
LPS-injected animals. It seems conceivable that these animals are
subjected to acute stress during the initial stage of endotoxemia,
possibly involving adrenergic stimulation of glucagon release
(43). The very high plasma glucagon levels are also, most
probably, a defense against the LPS-induced hypoglycemia, which, in
turn, largely seems to be the result of an increased peripheral glucose
utilization being, at least partly, mediated by an increased NO
production in these tissues (38). Hence, during the
beginning of this initial LPS period, the increased sensitivity to
glucose stimulation in the
-cell most probably depends on an
increased cAMP activity and later on the additional activation of the
CO production. As discussed below, both glucagon and CO directly
suppress islet NO production, resulting in a beneficial effect on
glucose-stimulated insulin release. During the later stage of the
endotoxemic time period studied, i.e., at 24 to 48 h, it cannot be
excluded that a reduced food intake (manifested as a significant weight
loss at 48 h) could have influenced our results. However, because
fasting is known to greatly reduce basal and glucose-stimulated insulin
release, this possibly only has a marginal effect since insulin release
was normal or enhanced, even in the face of hypoglycemia.
Immunocytochemistry and immunoblots showed a significant expression of
iNOS in islet tissue at 16 h after LPS treatment, whereas no iNOS
activity was detected in islets of control mice. iNOS immunoreactivity
was most convincingly seen in many insulin-producing -cells, but
only in single glucagon-producing
-cells. This pattern is in perfect
accordance with our very recent immunocytochemical findings in the rat
endocrine pancreas, where we also used confocal microscopy
(2). Sixteen hours after LPS, we found that NO production was increased threefold. The inducible isoform of NOS is known to
produce much larger amounts of NO than cNOS. Thus NO production by iNOS
is continuous and elicited during long time periods, whereas cNOS-derived NO is manifested in small NO bursts (44). In
the present study, NO production was still doubled at 48 h after
in vivo LPS administration and probably derived mostly from iNOS. In
this context, it should be recalled, as mentioned earlier in this
paper, that direct addition of LPS to isolated islets reportedly has
only negligible effects on NO production (9) and thus the increase of islet NOS activity in the present study is most probably a
result of LPS-stimulated cytokine production in vivo.
In agreement with earlier studies in the rat (52), the present data indicate that the insulin secretory machinery is somehow sensitized to glucose after in vivo LPS treatment and that endotoxic shock can be accompanied by hypoglycemia (4). Most of the effects on islet hormone secretion were already manifested during the first 6 h after LPS injection. It is notable that glucose-stimulated insulin release was increased not only in the face of an increased islet NO production but also during a gradual decrease of the plasma glucose levels. In fact, islets of LPS-injected mice displayed a normal glucose-stimulated insulin release at 24 h, when the animals suffered weight reduction and a profound hypoglycemia. This underlines the importance of the regulatory and compensatory system(s) that could contribute to restoring secretion of insulin during the increased production of NO, which, as mentioned earlier in this paper, has been shown to be a powerful inhibitor of glucose-stimulated insulin secretion (14, 19, 40, 41).
Hemin has been shown to protect against IL-1-induced inhibition of
islet function in the rat, probably by scavenging NO and/or increasing
the resistance to NO production (49), and, like NO, CO is
able to bind to heme-containing enzymes (the different NOS isoforms)
and regulate the activity of these enzymes. Islet CO production after
LPS injection was time dependently increased, supporting the idea that
the activity of islet HO is increased as a consequence of the action of
increased levels of cytokines brought about by LPS administration.
Interestingly, this increased CO production seemed to be derived from
both the acutely expressed HO-1 protein as well as from an enhanced
amount of the HO-2 protein (cf. Fig. 9). It should be noted, however,
that the immunocytochemical techniques used did not seem to be
sensitive enough to detect these changes. A few reports indicate that
NO itself, at least in other tissues, is responsible for the induction
of HO-1 and that NO can increase CO production via binding to the heme
moiety of HO (30). In addition to CO, equimolar
concentrations of biliverdin is produced during the HO-mediated heme
degradation. Biliverdin is then degraded to bilirubin, also known as a
strong antioxidant. All these data indicate that the newly discovered
HO-CO pathway within the islets of Langerhans (19, 20)
might constitute an important defense mechanism against oxidative
stress and against the deleterious effects of NO.
In the search for other possible compensatory mechanisms of keeping
insulin secretion almost normal during endotoxemia, we found a
remarkable, high secretory response of insulin and glucagon to the
adenylate cyclase activator forskolin after LPS treatment. In islets of
LPS-treated mice, forskolin-induced insulin secretion was four to seven
times higher than in islets of untreated mice, depending on the time
interval after LPS administration. This, together with the observation
of an increased content of cAMP in islets of LPS-treated animals,
suggests that there might be an upregulation and/or increased
sensitivity of the islet cAMP protein kinase A system during the
development of such an endotoxin-derived type of islet dysfunction. In
fact, our present data suggest that this compensatory system is more
rapid in onset than is the compensatory mechanisms exerted by the HO-CO
system. It should be noted in this context that during in vitro
culture, IL-1 and NO donors have earlier been shown to decrease the
cAMP content in rat islets (16). In contrast, as shown by
our present data in vivo as well as in vitro using freshly isolated
islets, there seems to be an increased capacity of the secretory
pathway(s) mediated by the islet cAMP system, which together with the
HO-CO system could be at least partly responsible for islet
compensatory mechanisms against increased NO production.
cGMP levels were also significantly increased after LPS treatment. This
could be due to both an increased NO and an increased CO production,
since both gases are known as potent guanylyl cyclase activators, by
binding to the heme moiety of the enzyme. In this context, it should be
noted that CO is reportedly considerably less potent than NO as an
activator of guanylyl cyclase (12). Earlier studies
described both a large arginine dependent and a small
arginine-independent increase in cGMP in rat islets after in vitro
exposure to IL-1 (15). The function of a guanylyl cyclase-cGMP protein kinase G-activating system in the islets of
Langerhans, as well as the function of NO itself, are not clearly elucidated, although cGMP is reported as a potent mediator of long-term, NO-induced apoptosis in
-cells (25).
In contrast, we have recently suggested that cGMP might be at least
partly responsible for mediating acute CO-stimulated insulin and
glucagon secretion (19). Regarding NO, the stimulating
effect on the cGMP system, through its binding to the heme group of
guanylyl cyclase in the
-cell, is likely to be strongly counteracted
and overshadowed by its ability to induce formation of
S-nitrosothiols (46), which apparently
negatively modulates stimulus-secretion coupling of nutrient-induced
insulin secretion (21, 34, 39-41).
Islet NOS activity was markedly decreased after addition of either glucagon or CO to control islets (cf. Fig. 10). The NOS inhibitory effect exerted by CO does not seem to be mediated by its stimulatory effect on glucagon release, since guanylyl cyclase inhibition by ODQ, which extinguished the stimulation of glucagon secretion induced by CO, did not affect its ability to inhibit NO production. This finding might have a possible clinical application in the future. People developing islet dysfunction during acute or long-term, endotoxemia-induced cytokine production might be helped with glucagon and/or a putative "CO-promoting" therapy to suppress the deleterious effects of the increased NO evolution.
In summary, isolated islets from LPS-treated mice displayed expression
of iNOS and HO-1 proteins and an increased expression of HO-2 protein
concomitant with increased production of NO and CO. Immunocytochemistry
demonstrated that LPS-induced iNOS immunoreactivity could be
convincingly seen in many insulin-producing -cells, but only in
single glucagon-producing
-cells. ncNOS immunoreactivity could be
readily detected in both
- and
-cells but was seemingly unaffected by LPS. Moreover, glucose-stimulated insulin release was not
impaired despite the well-known negative influence of NO on
nutrient-induced insulin secretion. This could be explained by both an
increased activity and sensitivity of the cAMP system and an increased
CO production in islets from LPS-treated animals. The beneficial
(inhibiting) effects of glucagon and CO on islet NOS activity may have
important implications regarding possible future treatment of
endotoxemia-induced dysfunction of the pancreatic islets.
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ACKNOWLEDGEMENTS |
---|
The skillful technical assistance of Maj-Britt Johansson, Britt-Marie Nilsson, and Lillemor Thuresson is gratefully acknowledged.
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FOOTNOTES |
---|
This study was supported by the Swedish Medical Research Council (14X-4286 and 11205), the Swedish Diabetes Association, and the foundations of Magnus Bergvall, Crafoord, Anna-Lisa, and Sven-Erik Lundgren, Albert Påhlsson, Åke Wiberg, and Thelma Zoega.
Address for reprint requests and other correspondence: R. Henningsson, Dept. of Pharmacology, Sölvegatan 10, S-223 62 Lund, Sweden (E-mail: Ragnar.Henningsson{at}farm.lu.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.
Received 9 June 2000; accepted in final form 28 November 2000.
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