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Diabetes Research Center, Faculty of Medicine, Vrije Universiteit Brussel, Brussels, Belgium B-1090
Address all correspondence and requests for reprints to: D. G. Pipeleers, Department of Metabolism and Endocrinology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium.
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Abstract |
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Introduction |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
The two ß-cell subpopulations were suspended in Lux dishes
(Miles, Naperville, IL) containing 2 ml culture medium, reaggregated
for 3 h in a rotatory shaking incubator (Braun, Melsugen,
Germany), and then further cultured under static conditions. Culture
medium was Ham’s F10 containing either 5 or 20 mmol/liter glucose
supplemented with 0.075 mg/ml penicillin, 0.1 mg/ml streptomycin, 2
mmol/liter L-glutamine, 2% (vol/vol) heat-inactivated FCS
(GIBCO, Paisley, Scotland), 1% (wt/vol) BSA pretreated with charcoal
(BSA, fraction V, RIA grade, Sigma, St. Louis, MO), and 50 µmol/liter
3-isobutyl-1-methylxanthine (IBMX, Janssen Chimica, Beerse, Belgium)
(9). The presence of IBMX maintains ß-cell survival in culture (9) at
a concentration with negligible effect on cAMP production in the
absence of adenyl cyclase activators (10). In the first series of
experiments, ß-cell subpopulations were preincubated for 3 h and
then cultured for 20 h with different concentrations of
recombinant human interleukin-1ß (IL-1ß, >98% pure, 13 U/ng,
Janssen Pharmaceutica, Brussels, Belgium). Under these conditions,
IL-1ß (0.2 ng/ml) was previously found to suppress glucose-induced
insulin synthesis in the total ß cell populations without decreasing
the synthesis of noninsulin proteins (11). The IL-1 receptor antagonist
protein (IL-1ra, kindly provided by Dr. D. E Tracey, then at the Upjohn
Company, Kalamazoo, MI) was tested at 1 or 10 ng/ml and added 15 min
before IL-1ß (12). To examine the role of IL-1ß-induced nitric
oxide production, cells were exposed for 24 h to IL-1ß (0.1
ng/ml; >95% pure, 280 U/ng, Genzyme Corp., Cambridge, MA) in the
presence or absence of 0.5 mmol/liter
NG-methyl-L-arginine, which is known to inhibit
NOS. At the end of culture, medium was collected for measurements of
nitrite, and cells were analyzed for functional activities and protein
expression. In all conditions, cell viability exceeded 90% at the end
of culture as evaluated by vital staining with neutral red (4).
Measurement of protein synthesis, insulin release, and nitrite
production
Protein synthesis and insulin release were measured during 2-h
incubations at 37 C in 200 µl Ham’s F10 containing 10 mmol/liter
glucose, 1% BSA, 2 mmol/liter glutamine, 50 µmol/liter IBMX and 50
µCi L-[3,5-3H]tyrosine (TRK200, Amersham,
Buckinghamshire, UK). In one series of experiments the
3H-labeled ß-cell aggregates were further incubated for
2 h at 2.5 or 20 mmol/liter glucose, after the extracellular
tracer had been washed out. Media were collected and centrifuged, and
supernatants were assayed for insulin. Cells were extracted in 1 ml
acetic acid (2 mol with 0.25% BSA) before their content in
3H-labeled protein, 3H-labeled insulin, and
immunoreactive insulin was measured (13).
The nitrite content in the culture media was determined by a modified Griess assay (14). Duplicate 100-µl aliquots were mixed with 5 µl naphtyletylene diamine dihydrochloride (1%) and 5 µl sulfanilamide (10% in 50% H3PO4) and incubated for 10 min at 60 C. The absorbance at 546 nm was measured spectrophotometrically (NY-160A, Shimazu, Kyoto, Japan), and nitrite concentrations were calculated against a sodium nitrite standard curve, with the lower limit of sensitivity at 1 µmol.
Immunoblot analysis of iNOS, superoxide dismutase, heat shock
protein 70, and heme oxygenase
Groups of 105 cells were sonicated in 50 µl
SDS-gel buffer [5% SDS, 5% ß-mercaptoethanol, 80 M
Tris-Cl (pH 6.8), 5 mol/liter EDTA, 10% glycerol, and 1 mmol/liter
phenymethylsulfonyl fluoride], and samples were run on 10%
SDS-polyacrylamide gel. After electrophoresis, proteins were
electrically transferred to nitrocellulose filters and then incubated
with rabbit anti-rat iNOS antibody (1:10000; Transduction Laboratories,
Lexington, KY), rabbit anti-rat Mn superoxide dismutase (MnSOD)
antibody (1:3000; kindly provided by Dr. Kohtaro Asayama, Yamanashi
Medical University, Yamanashi, Japan), mouse anti-rat inducible form of
heat shock protein 70 (HSP70), or rabbit anti-human heme oxygenase
antibody (1:1000; Stressgen, Victoria, B.C., Canada). Horseradish
peroxidase-linked goat anti-rabbit, anti-mouse, or anti-rat Ig were
used as second antibodies. Peroxidase activity was detected by enhanced
chemiluminescence (Amersham, Buckinghamshire, UK), and the band
intensity was quantified on an Ultroscan XL Enhanced Laser Densitometer
(LKB, Bromma, Sweden) and expressed in arbitrary OD units.
Competitive RT-PCR analysis of iNOS mRNA expression
Isolation of cellular mRNA was performed immediately after
culture using oligo(dT)25-coated polystyrene beads
(Dynabeads) according to the manufacturer’s instructions (DYNAL, Oslo,
Norway). Reverse transcription mixture was prepared with the GeneAmp
RNA PCR Kit (Perkin-Elmer, Norwalk, CT) which consisted of 1 x
buffer (10 mmol/liter Tris-HCl, pH 8.3, 50 mmol/liter KCl), 5
mmol/liter MgCl2, 1 mmol/liter of each deoxynucleoside
triphosphate, 2.5 µmol/liter random hexamer primer, 1 U/µl
ribonuclease inhibitor, and 2.5 U/µl murine leukemia virus reverse
transcriptase in a final volume of 10 µl. The reaction mixture was
incubated at room temperature for 10 min, then at 42 C for 1 h,
and finally heated at 99 C for 5 min and immediately chilled on
ice.
iNOS mRNA levels were quantified by competitive RT-PCR (15). Competitor DNA, used as internal standards for iNOS cDNA, were constructed using a PCR MIMIC Construction Kit (Catalog no. 1700–1, Clontech, Palo Alto, CA). Neutral DNA, a BamHI/EcoRI fragment of the v-erbB oncogene, was first amplified with a composite primer pair each composed of a target gene-specific sequence linked to a sequence that anneals to the neutral DNA templates (5'-GACTGCACAGAATGTTCCAGCAAGTTTCGTGAGCTGATTG-3' and 5'-TGGCCAGATGTTCCTCTATTTTGAGTCCATGGGGAGCTTT-3'). The resulting fragments were then diluted 1:100 and reamplified with target-specific primer pair NOS-F 5'-GACTGCACAGAATGTTCCAG-3' and NOS-R 5'-TGGCCAGATGTTCCTCTATT-3'. The competitor DNA contains the same primer-annealing sequences as the target cDNA, but its PCR product is longer than that of the target cDNA. Competitor DNA was diluted in 50 µg/ml glycogen. Different concentrations of competitor DNA were added to the PCR reaction mixtures containing cDNA samples. PCR reactions were carried out in a TC 9600 thermocycler (Perkin Elmer-Cetus). PCR mixture contained 5 µl cDNA, 2 µl competitor DNA, 1 x buffer (10 mmol/liter Tris-HCl, pH 8.3, 50 mmol/liter KCl), 2 mmol/liter MgCl2, 0.4 mmol/liter of target-specific primers, 0.2 mmol/liter of each deoxynucleoside triphosphate, and 0.625 U AmpliTaq DNA polymerase (Perkin Elmer-Cetus), to a final volume of 25 µl. PCR conditions were as follows: 35 cycles each consisting of denaturation for 45 sec at 94 C, annealing for 45 sec at 58 C, and extension for 90 sec at 72 C, preceded by an initial denaturation of 2.5 min at 94 C. The last PCR step was a 10-min extension at 72 C. In parellel an cDNA aliquot was used to amplify the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase to confirm equal mRNA loading.
Five microliters of PCR products were separated by electrophoresis in 2% agarose gel and stained with ethidium bromide. The stained gels were photographed under UV transillumination using Polaroid type 665 film (Polaroid, Cambridge, MA). The fragment intensities were scanned by Ultroscan XL Enhanced Laser Densitometer (LKB) of the negative films and were expressed in arbitrary OD units. The ratios of iNOS cDNA to competitor DNA PCR products were plotted against the reciprocal molar amount of competitor DNA.
Data analysis
Data are presented as means ± SEM. Statistical
significance of differences were calculated by Student’s t
test. For multiple comparison, data were examined by ANOVA, followed by
Fisher’s Least Significant Difference Test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The presence of IL-1 (0.1 ng/ml)
during culture suppressed both insulin and noninsulin protein synthesis
in the responsive subpopulation by more than 80% (Table 1); in the
unresponsive subpopulation, only insulin synthesis was inhibited, and
the degree of inhibition was less pronounced (50%) (Table 1). These
suppressive effects of IL-1ß were counteracted by an IL-1ra to which
both subpopulations exhibited a similar sensitivity (Fig. 1). At lower concentrations (0.02 ng/ml),
IL-1 exerted its inhibitory effects only in the glucose-responsive
subpopulation (9.3 ± 0.9 cpm/cell in control and 6.6 ± 0.8
cpm/cell in the presence of IL-1, Fig. 2). Similar observations were made for
the secretory activities of both subpopulations. After culture, the
glucose-induced insulin release from the glucose-responsive
subpopulation was higher than that from the glucose-unresponsive one
(Table 2). In the presence of IL-1 (0.1
ng/ml), hormone release was completely suppressed in the
glucose-responsive preparation while it was only 50% inhibited in the
glucose-unresponsive one (Table 2).
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). This condition also increased the
rates of non-insulin protein synthesis in both preparations (Table 1).
The presence of IL-1ß exerted now comparable suppressive effects in
the two subpopulations, with a marked inhibition in both insulin- and
noninsulin protein synthesis (Table 1). The glucose-unresponsive
subpopulation exhibited now the same IL-1 sensitivity as the
glucose-responsive subpopulation (Fig. 2).
Comparison of both ß-cell subpopulations for the IL-1ß-induced
expression of proteins
After 20 h culture at 5 mmol/liter glucose, both ß-cell
subpopulations exhibited a comparable expression of HSP70, heme
oxygenase, and MnSOD, while they both lacked detectable iNOS protein
(Fig 3). The presence of IL-1 (0.1 ng/ml)
in the culture medium increased the expression of HSP70, HO, and MnSOD
and induced the expression of iNOS in the two subpopulations (Fig. 3).
The expression of HO and MnSOD was stimulated to the same extent in the
two subpopulations, but that of HSP70 and iNOS was more strongly
stimulated in the glucose-responsive subpopulations (Fig. 3).
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Addition of N-methyl-L-arginine (0.5 mmol/liter)
to the culture medium prevented the IL-1ß-induced nitrite production
in the two subpopulations; the presence of the iNOS blocker also
prevented the IL-1ß-induced suppression of insulin synthesis in the
glucose-responsive subpopulation (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7.5 mmol/liter), up to 50% of the ß-cells are stimulated in their
glucose-regulated functions (2, 8), a state in which they are more
sensitive to the functional suppression by IL-1ß than the
glucose-unresponsive cells. Prolonged exposure to high glucose (20
mmol/liter) activates virtually all ß-cells (3, 19), which exhibit
now all the same high sensitivity to the IL-1ß-induced suppression.
If IL-1ß is released in situ within the endocrine
pancreas, it is thus expected that the degree of hormone suppression
will depend on the number of glucose-activated cells, and hence, on the
prevailing glucose concentration. The inhibitory effects of IL-1ß
will therefore be more pronounced in hyperglycemia and, as a
consequence, aggravate the hyperglycemic state. Whether
IL-1ß-suppressed ß-cells are more vulnerable to cytodestruction by
other immune mediators has not yet been directly investigated, but
indirect support comes from the observation that increased glucose
concentrations amplify cell damage in IL-1ß-exposed islets (20). IL-1ß also exerts stimulatory effects on pancreatic ß-cells. It induces expression of iNOS mRNA and protein. The stimulation was 2- to 3-fold stronger in glucose-responsive ß-cells than in the unresponsive ones, which is in line with their higher sensitivity to the cytokine. Consequently, nitrite production was higher in the responsive subpopulation. Prevention of nitrite formation by N-methyl-L-arginine also prevented the IL-1ß-induced suppression of insulin synthesis, suggesting that NO is responsible for the cytokine’s suppressive action. That the medium nitrite content in the glucose-responsive subpopulation is only 30% higher than in the unresponsive one is not necessarily in conflict with this possibility. It is indeed conceivable that the medium nitrite accumulation over a 24-h culture period is a poor parameter for NO’s biological effects, which may be more dependent on the rapid concentration changes at its cellular targets.
The IL-1ß-exposed ß-cells also exhibited a higher content in other noninsulin proteins such as HSP70, heme oxygenase, and MnSOD. This was the case in both the glucose-responsive and unresponsive subpopulations. The effect on HSP70 was stronger in the responsive subpopulation, but that on the two other proteins was similar in both subpopulations. We have not examined whether it results from an increased rate of protein synthesis and/or a decreased rate of degradation. A higher cellular content in HSP70 and MnSOD may play a role in defense and/or repair mechanisms that can protect cells against injury (21). Therefore, it remains to be examined also whether IL-1ß-treated ß-cells exhibit a higher resistance to cytotoxic agents.
Previous studies on rat and human ß-cells have demonstrated the existence of intercellular differences in glucose responsiveness and, hence, in the rates of insulin synthesis and release (2, 3). This cellular heterogeneity determines the proportion of ß-cells that fulfils the acute needs of glucose homeostasis. Our study shows that exposure to IL-1 eliminates this potential by shifting the glucose-responsive ß-cells to a functional state with suppressed insulin synthesis and release. This shift results in a disappearance of the functional heterogeneity within the ß-cell population. After IL-1 treatment, ß-cells have shifted to activities that are more dependent on the synthesis of noninsulin proteins than of insulin. On the basis of the present functional data and previous morphological observations (11), it can be suggested that IL-1ß alters the phenotype of rat ß-cells. Further experiments should investigate the consequences of such phenotypic change for the survival and function of the ß-cells.
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Acknowledgments |
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Footnotes |
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2 Research fellow from the Belgian National Fund for Scientific
Research.
Received October 10, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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via an L-arginine-dependent nitric oxide generating mechanism. FEBS
Lett 276:42–44[CrossRef][Medline]
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) subpopulations. Both subpopulations were
cultured for 20 h at 5 mmol/liter glucose with IL-1ß (0.1 ng/ml)
and/or IL-1ra (1–10 ng/ml). Their biosynthetic activity after culture
was expressed as a percentage of that measured in control preparations,
cultured in parallel without IL-1ß and IL-1ra. The rates of protein
synthesis in control condition are 18 ± 1 and 14 ± 1
cpm/cell in responsive and unresponsive subpopulations, respectively.
Data represent means ± SEM of four independent
observations. Statistical significance of differences between
glucose-responsive and -unresponsive subpopulations was calculated by
Student’s t test. *, P < 0.01.
