(Received for publication, April 2, 1997)
From the Mass Spectrometry Resource, Divisions of Endocrinology, Diabetes, and Metabolism and Laboratory Medicine, Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110
Culture of rat pancreatic islets with interleukin-1 (IL-1) results in up-regulation of the inducible isoform of nitric oxide synthase and overproduction of nitric oxide (NO). This is associated with reversible inhibition of both glucose-induced insulin secretion and islet glucose oxidation, and these effects are prevented by the inducible nitric oxide synthase inhibitor NG-monomethylarginine. IL-1 also induces accumulation of nonesterified arachidonic acid in islets by an NO-dependent mechanism, and one potential explanation for that effect would involve an IL-1-induced enhancement of islet glycolytic flux. We have therefore examined effects of IL-1 on islet glycolytic utilization of glucose and find that culture of islets with IL-1 in medium containing 5.5 mM glucose results in suppression of islet glucose utilization subsequently measured at glucose concentrations between 6 and 18 mM. The IL-1-induced suppression of islet glucose utilization is associated with a decline in islet glucokinase mRNA content, as determined by competitive reverse transcriptase-polymerase chain reaction, and in glucokinase protein synthesis, as determined by immuoprecipitation experiments, and all of these effects are prevented by NG-monomethylarginine. These findings suggest that IL-1 can down-regulate islet glucokinase, which is the primary component of the islet glucose-sensor apparatus, by an NO-dependent mechanism. Because reductions in islet glucokinase levels are known to cause a form of type II diabetes mellitus, these observations raise the possibility that factors which increase islet NO levels might contribute to development of glucose intolerance.
Culture of rat pancreatic islets with interleukin-1 (IL-1)1 induces islet expression of the inducible isoform of nitric oxide synthase and overproduction of nitric oxide (NO) (1-6). This is associated with inhibition of glucose-induced insulin secretion (7-10) and impaired islet oxidation of glucose (8-12), and both of these effects are prevented by the inducible nitric oxide synthase inhibitor NG-monomethylarginine (NMMA) (1, 2, 4), indicating that they occur through NO-dependent mechanisms.
We have recently reported that IL-1 also induces accumulation of nonesterified arachidonic acid in islets by an NO-dependent mechanism (6). Our findings suggested that this reflected suppression of re-esterification of arachidonic acid released during phospholipid turnover, but others have found that NO stimulates arachidonic acid release from macrophage-like cells by a mechanism involving accelerated glycolytic flux (13). This has been attributed to activation of a macrophage phospholipase A2 enzyme that is regulated by an isoform of the glycolytic enzyme phosphofructokinase (13-16). Because islets express a similar phospholipase A2 enzyme (17-19), it seemed possible that NO-induced acceleration of glycolytic flux might also contribute to IL-1 induced accumulation of nonesterified arachidonic acid in islets. We have therefore examined effects of culturing islets with IL-1 on islet glycolytic utilization of glucose, as reflected by production of [3H]OH from [5-3H]glucose (20-28), and on expression of glucokinase mRNA by competitive PCR.
Male Sprague-Dawley rats (180-220 g body weight)
were purchased from Sasco (O'Fallon, MO); collagenase from Boehringer
Mannheim; tissue culture medium (CMRL-1066), penicillin, streptomycin,
Hanks' balanced salt solution, heat-inactivated fetal bovine serum,
and L-glutamine from Life Technologies, Inc. (Grand Island,
NY); Pentex bovine serum albumin (fatty acid free, fraction V) from
Miles Laboratories (Elkhart, IN); Rodent Chow 5001 from Ralston
Purina (St. Louis, MO); D-glucose from the National
Bureau of Standards (Washington, D.C.); IL-1 from Cistron
Biotechnology (Pine Brook, NJ);
NG-monomethyl-L-arginine
acetate from Calbiochem (San Diego, CA); and
Trans35S-labeled methionine (1117 Ci/mmol) from ICN (Costa
Mesa, CA).
Media included KRB (Krebs-Ringer bicarbonate buffer: 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2), nKRB (KRB supplemented with 3 mM D-glucose), cCMRL-1066 (CMRL-1066 supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine and 1% (w/v) each of penicillin and streptomycin), and Hank's balanced salt solution supplemented with 0.5% penicillin-streptomycin.
Isolation of Pancreatic IsletsIslets were isolated aseptically from male Sprague-Dawley rats by a described procedure (29) involving collagenase digestion of excised, minced pancreas, density gradient isolation, and manual selection under microscopic visualization. Isolated islets were transferred into Falcon Petri dishes containing 2.5 ml of cCMRL-1066, placed under an atmosphere of 95% air, 5% CO2, and cultured at 37 °C with or without IL-1 or other additives.
Incubation of Islets with IL-1 and Measurement of Insulin SecretionIslets (400 per condition) were placed in Petri dishes (10 × 35 mm); suspended in cCMRL medium (1 ml) containing no additives, IL-1 (5 units/ml) alone, or both IL-1 and NMMA (0.5 mM); and incubated (2-48 h, 37 °C). In some experiments, islets were then removed from the incubation medium, and their secretion of insulin, oxidation of [U-14C]glucose to [14C]O2, or production of [3H]OH from [5-3H]glucose was examined in a subsequent incubation. In such experiments, islets were washed 3 times in nKRB, transferred to siliconized test tubes (12 × 75 mm), and preincubated (30 min, 37 °C) in nKRB (0.2 ml). For insulin secretion experiments, islets were placed in fresh in KRB medium containing various concentrations (3-18 mM) of D-glucose and incubated (30 min, 37 °C, under 95% air, 5% CO2). Aliquots of medium were then removed for measurement of insulin by radioimmunoassay.
Islet Glucose UtilizationAs in previously described procedures (20, 25), triplicate batches of 10 islets per incubation condition were placed into Microfuge tubes (0.5 ml). Medium was then removed, and radioactive mixture (15 µl) was added. This mixture was prepared by placing [5-3H]glucose (7 µl, Amersham, specific activity 1 mCi/ml) into silanized glass test tubes and concentrating the solution to dryness under nitrogen to evaporate any [3H]OH. The desired final glucose concentrations (3-18 mM) of the radioactive mixtures were achieved by adding various volumes (0-167 µl) of glucose-free KRB containing 1% bovine serum albumin and an appropriate corresponding volume (33-200 µl) of KRB containing 18 mM glucose and 1% bovine serum albumin. The total initial amount of [3H] added to each condition was determined by adding 15 µl of each radioactive mixture directly to scintillation vials containing water (0.5 ml). For blank incubations, radioactive mixture was added, but no islets were present. After all additions were complete, tubes containing the islets were placed in scintillation vials (20 ml) containing water (0.5 ml). The vials were then flushed with 95% air, 5% CO2, capped with Teflon/silicone septum lids, and incubated (1 h, 37 °C, shaking water bath). A Hamilton syringe was then used to introduce 1 N HCl (20 µl) into the islet-containing tubes to prevent further catabolism of [5-3H]glucose. Vials containing these tubes were then incubated (24 h, 37 °C) to permit [3H]OH formed by the islets to evaporate and equilibrate with water in the vials. Vials were then cooled to room temperature. The Microfuge tubes were removed and their exterior surfaces rinsed with scintillation fluid (12 ml), which was placed in the vial containing the water in which the Microfuge tube had been immersed. The scintillation vial was then capped with the original septum-lid and mixed. The vials were then equilibrated in the dark, and their 3H-content was measured by liquid scintillation spectrometry. Average disintegrations/min in blank tubes was subtracted from experimental measurements, and glucose utilization was calculated as: [{[3H]OH formed (dpm)}/{(specific radioactivity of [5-3H]glucose (dpm/pmol)}].
Measurement of Islet Oxidation of [14C]Glucose to [14C]O2As in previously described
procedures (30, 31), islets (30 from each incubation condition) were
placed into Beckman polyallomer tubes. After centrifugation (Beckman
Microfuge, 5 s, 10,000 × g), supernatant was
discarded, and the islets were resuspended in fresh medium (nKRB, 0.2 ml) and preincubated (30 min, 37 °C). Islets were then collected by
centrifugation, supernatant discarded, and KRB medium (0.15 ml)
containing various concentrations (3-18 mM) of
[U-14C]glucose was added. After resuspension of the
islets, the polyallomer tubes were placed in scintillation vials
containing filter paper covering the bottom of the vial. The vials were
then equilibrated with 95% air, 5% CO2, sealed with lids
containing gas-tight Teflon/silicone septa, and incubated (2 h,
37 °C, shaking water bath) to permit islet metabolism of
[14C]glucose to [14C]O2.
Hyamine base (0.2 ml) was then applied to the filter paper in the vials
with a Hamilton syringe by penetrating the septa. Islet metabolism of
[14C]glucose was then terminated and dissolved
H[14C]O3 converted to
[14C]O2 by acidifying (0.2 N HCl,
0.2 ml) the medium inside the polyallomer tube. The sealed vials were
then incubated (overnight, room temperature, with shaking) to allow
[14C]O2 to escape from the incubation
solution and react with hyamine in the filter paper. The polyallomer
tubes were then removed from the vials and their exterior surfaces
rinsed with scintillation fluid (1 ml, ACS, Amersham), which was placed
inside the scintillation vial. Additional scintillation fluid (9 ml)
was added to each vial, and the 14C-content was measured by
liquid scintillation spectrometry. Total 14C- content of
the stock [U-14C]glucose solution and blank conversion of
[U-14C]glucose to [14C]O2
without islets were also determined.
After incubation of islets under
various conditions, total RNA was isolated after solubilization in
guanidinium thiocyanate by phenol/chloroform/isoamyl alcohol extraction
and isopropyl alcohol precipitation (32). First strand cDNA was
transcribed from total RNA with avian myeloblastosis virus reverse
transcriptase (reverse transcriptase, Boehringer Mannheim). Polymerase
chain reactions (PCR) were performed on a Perkin-Elmer DNA Thermal
Cycler 480. Primer pairs used to amplify fragments of cDNA encoding
glucokinase (33) in competitive PCR reactions are described below.
Primer pairs used for other gene products were: inducible nitric oxide synthase (34), sense 5-TGCTTTGTGCGGAGTGTCAG and antisense
5
-AGATGCTGTAACTCTTCTGG (expected fragment 650 bp); and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (35), sense
5
-TAGACAAGATGGTGAAGG and antisense 5
-TCCTTGGAGGCCATGTAG (expected fragment length 1006 bp). Amplification steps (30 cycles) included denaturation (95 °C, 1 min), annealing (50 to
60 °C, 1 min), and extension (72 °C, 2 min) and were performed in
Taq-polymerase buffer (Life Technologies, Inc.) containing
1.5 mM MgCl2, 1 µM of each
primer, 200 µM each of dATP, dGTP, dCTP, and dTTP, and 25 units/ml Taq DNA polymerase (Life Technologies, Inc.). PCR products were analyzed by 1% agarose gel electrophoresis and
visualized by ethidium bromide staining (32). Band intensities of PCR
products were measured with an IS-1000 Digital Imaging System (Alpha
Innotech Corp.). Fluorescence was recorded, and the areas of the peaks were measured. Increased levels of inducible nitric oxide synthase mRNA in islets could be detected by RT-PCR after 4 h exposure to IL-1. Levels of islet GAPDH mRNA, as detected by RT-PCR, were constant in control and in IL-1-treated islets for up to 48 h in
culture. To examine the potential amplification of genomic DNA in PCR
reactions, a control amplification was performed in which the reverse
transcriptase step was omitted. In no case was a PCR product of the
expected length obtained under these conditions.
Competitive RT-PCR (72, 73) was used to determine the abundance of glucokinase mRNA. In this approach, a competitor DNA species is prepared which contains the same primer template sequences as the target cDNA but which contains an intervening sequence which differs from the target in size or in restriction sites so that PCR products from the target and competitor can be distinguished (72, 73). Using the competitor as an internal control, amounts of target cDNA can be determined by allowing known amounts of the competitor to compete with the target for primer binding during amplification (72, 73).
To prepare the competitor DNA, two composite primers were synthesized
(sense 5-TCACAAGTGGAGAGCGACTCACTGGCATGGCCTTCCG-3
and antisense 5
-ATTTGTGGTGTGTGGAGTCCTTGGAGGCCATGTAGGC-3
).
These primers contain the glucokinase primer sequence
(underlined) attached to sequences which hybridize to rat GAPDH
cDNA (35). This pair of primers was then used in PCR reactions
with rat GAPDH cDNA as template. In these reactions, the
glucokinase primer sequences are incorporated into the PCR product
during amplification, and the intervening sequence derives from GAPDH.
The resultant PCR product (360 bp in length) was analyzed by agarose
gel electrophoresis, isolated with a QIAEX gel extraction kit (QIAGEN),
and used as the competitor DNA species in subsequent PCR experiments.
In these experiments, the primer pair (sense
5
-TCACAAGTGGAGAGCGACTC-3
and antisense
5
-ATTTGTGGTGTGTGGAGTCC-3
) was used. These primers hybridize to the glucokinase cDNA sequence and to the competitor DNA sequence. The PCR product derived from the glucokinase cDNA is
450 bp in length, and that from the competitor DNA is 360 bp in length.
The products were then analyzed by 1% agarose gel electrophoresis and
visualized with ethidium bromide. Product band intensity was then
determined with an IS-1000 Digital Imaging System.
The primer set selected for amplification of glucokinase cDNA will not yield a product of the appropriate size with genomic DNA as template. In the rat glucokinase gene (75), the sequence recognized by the sense primer is interrupted by the intron between exons 8 and 9, and the sequence recognized by the antisense primer occurs in exon 10. The amplified sequence of the cDNA therefore includes 11 bp of exon 8, the entirety of exon 9, and a fragment of exon 10. The intervening intron sequences would cause any product amplified from glucokinase genomic DNA to be far larger than that from glucokinase cDNA. In addition, no products of the expected size were observed in control competitive PCR reactions in which the the reverse transcriptase step was omitted. The target glucokinase RT-PCR product was also subcloned and sequenced. The sequence corresponded exactly to the appropriate region of the glucokinase cDNA.
With a fixed amount of target and competitor, varying the PCR cycle number from 19 to 31 was found to yield a constant relative intensity of the target and competitor PCR product bands. In subsequent experiments, 28 PCR cycles were used. To examine the relationship between the amount of input DNA and the ratio of the signals for the target and competitor, in one set of experiments the competitor DNA solution was serially diluted, and aliquots of each dilution were added to a reaction mixture containing a fixed amount of glucokinase cDNA. In a second set of experiments, a solution of target glucokinase cDNA was serially diluted, and aliquots of each dilution were added to a reaction mixture containing a fixed amount of competitor DNA. After PCR amplification, products were analyzed by agarose gel electrophoresis, and product band intensity was determined as above. The ratio of target to competitor product band intensities was found to correspond to the amount of input DNA over a wide range of concentrations. To determine the relative abundance of glucokinase mRNA in islets after incubation under various conditions, total RNA was isolated as described above, and its concentration was determined spectrophotometrically (260 nm). Equal measured amounts of RNA from each incubation condition were then used in competitive RT-PCR reactions with a fixed amount of competitor DNA, and the target to competitor ratio was determined as described above.
Nitrite MeasurementMedium nitrite content was measured spectrophotometrically (540 nm, Titertek Multiskan MCC/340 microtiter plate reader) after mixing medium (0.1 ml) with Griess reagent (0.1 ml of a solution of 1 part of 1.32% sulfanilamide in 60% acetic acid and 1 part of 0.1% naphthylethylenediamine-HCl) and incubation (10 min, room temperature), as described previously (1, 2).
Immunochemical Analyses of Islet GlucokinaseTo generate an
anti-glucokinase antiserum, a fusion protein was prepared which
contained the sequence of Schistosoma japonicum glutathione
S-transferase joined to that of human islet glucokinase. The
fusion protein was prepared in Escherichia coli transformed with pGEX vector and purified from E. coli homogenates by
affinity chromatography on glutathione-agarose (36). The protein was injected subcutaneously on multiple occasions into a female New Zealand
White rabbit as an emulsion in Freund's adjuvant. Immunoprecipitation experiments with this antiserum were performed essentially as described
previously (37, 38). After incubation under various conditions, islets
were washed 3 times in methionine-deficient MEM (9 parts MEM without
methionine per 1 part MEM with methionine) and incubated (1 h,
37 °C) in methionine-deficient MEM. [35S]Methionine
(100 µCi/ml) was then added, and the islets were incubated (3 h,
37 °C), harvested by centrifugation, washed (3 times, 0.1 M PBS), and lysed (1 h, 4 °C) in PBS (1 ml) containing 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 1 µg/ml aprotinin, 100 µg/ml phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, and 1 mM iodoacetamide. Cell debris was removed (centrifugation,
15 min, 10,000 × g, 4 °C), and the protein content
of the supernatants was measured. Aliquots of supernatants from each
condition containing identical measured amounts of protein were then
diluted with lysis buffer to achieve a final volume of 800 µl. These
solutions were then preincubated (2 h) with preimmune serum (10 µl)
and precleared by treatment (1 h) with 100 µl of stapylococcal
protein A (Immunoprecipitin, Life Technologies, Inc.) and
centrifugation (1 min, 4 °C, 200 × g).
Anti-glucokinase antiserum (5 µl) was then added to the supernatants,
and the mixture was incubated (overnight, 4 °C, with shaking).
Staphylococcal protein A (100 µl) was then added, and the mixture was
incubated (2 h, 4 °C, with shaking). Staphylococcal protein
A-antibody complexes were then isolated by centrifugation and washed
four times with PBS (1 ml) containing 0.5% Triton X-100 and 0.05%
SDS. The immunoprecipitates were then washed twice with 10 mM PBS, reconstituted in SDS sample mixture (30 µl, 0.25 M Tris-HCl, 20% -mercaptoethanol, 4% SDS), and boiled
(5 min). After centrifugation, proteins in the supernatants were
analyzed on 10% SDS-polyacrylamide gels and visualized by
autoradiography (39).
Isolated islets were incubated under conditions described above with no additions (control), with IL-1 (5 units/ml) alone, or with IL-1 plus NMMA (0.5 mM) for 24 h. The islets were then washed three times with MEM without methionine containing 5% fetal bovine serum and, after 1 h of incubation in methionine-deficient medium, were incubated (3 h) with [35S]methionine under conditions described above. The islets were then placed in 15-ml conical test tubes, washed three times with PBS to remove unincorporated [35S]methionine, and homogenized by sonication (Vibracell probe sonicator, 0.5-s bursts at 12% amplitude for 20 s, Sonics & Materials, Inc., Danbury, CT) in buffer A (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM phenylmethanesulfonyl fluoride). The homogenates were transferred to 1.5-ml Microfuge tubes and centrifuged (15,000 rpm, 4 °C, 15 min, Beckman Microfuge). Aliquots (10 µl) of the supernatants were then placed on 2-mm square sections of Whatman filter paper and allowed to dry. The filter paper pieces were then boiled in 10% trichloroacetic acid for 10 min, rinsed twice with ice-cold 5% trichloroacetic acid, rinsed once with ice-cold absolute ethanol, allowed to dry, and then placed in scintillation vials to which 12 ml of ACS scintillation mixture (Amersham) was added. The 35S-content was then determined by liquid scintillation spectrometry. Other aliquots of the supernatants were used to determine total protein content or were subjected to SDS-PAGE and autoradiography under conditions described above.
The initial question that motivated this study was whether treatment of islets with IL-1 would enhance glycolytic flux. Because culture of islets with IL-1 has been reported not to affect glycolytic utilization of 16.7 mM glucose (10, 11), we measured glucose utilization at several concentrations between 3 and 18 mM to determine whether enhanced utilization occurred at lower concentrations. Freshly isolated islets were incubated for 24 h in medium containing 5.5 mM glucose and no other additions, IL-1 alone, or both IL-1 and NMMA. The islets were then placed in fresh medium and incubated with various concentrations of [5-3H]glucose. Glycolytic utilization of this substrate was determined by measurement of [3H]OH production, which is generated at the triose-phosphate isomerase and enolase reactions reactions (20-28) and provides a quantitative measure of islet glycolytic flux (27).
With control islets, glucose utilization increased with the medium
glucose concentration until a maximal rate was achieved at 15-18
mM glucose (Fig. 1). About half of that rate
was achieved at 8 mM glucose, as previously reported (20,
21, 23). In contrast, islets that had been incubated with IL-1
exhibited little increase in glucose utilization as the medium glucose
concentration was increased within the range of 3-18 mM.
With islets that had been incubated with both IL-1 and the nitric oxide
synthase inhibitor NMMA, glucose utilization was similar to that of
control islets at all tested glucose concentrations (Fig. 1).
Incubation of islets with NMMA alone was found not to influence islet
glucose utilization significantly at 3 mM glucose
(9.94 ± 0.87 pmol/islet × h versus control value
of 11.59 ± 1.59, p value 0.332) or at 18 mM glucose (40.04 ± 4.74 pmol/islet × h
versus control value of 44.10 ± 6.73, p
value 0.646). These findings suggested that IL-1 induced a reduction in
islet glucose utilization by an NO-dependent mechanism.
Because these findings differed from the reported lack of effect of
IL-1-treatment on islet utilization of 16.7 mM glucose (10,
11), we examined effects of IL-1 on insulin secretion and glucose
oxidation to determine whether these parameters were affected under our
incubation conditions in a manner similar to that previously reported.
These experiments were conducted at multiple glucose concentrations to
supplement information from previous studies performed at basal and
near-maximally stimulatory glucose concentrations but not at
intermediate concentrations (1, 2, 4, 7, 8, 10-12, 31, 40).
With control islets that had been incubated for 24 h at 5.5 mM glucose without IL-1, subsequent incubation with various
concentrations of glucose induced a progessive rise in insulin
secretion as the medium glucose concentration increased (Fig.
2). With islets that had incubated with IL-1, there was
little rise in insulin secretion as the medium glucose concentration
increased within the range of 3-18 mM, in agreement with
previous reports (7, 31, 40). With islets that had been incubated with
IL-1 and NMMA, insulin secretion was similar to that of control islets,
as previously reported (2, 4). Incubation of islets with NMMA alone did not influence islet insulin secretion significantly at 3 mM
glucose (1.19 ± 0.83 fmol/islet × min versus
control value of 1.04 ± 0.13, p value 0.61) or at 18 mM glucose (3.71 ± 0.83 fmol/islet × min versus control value of 3.58 + 0.53, p value
0.90).
The influence of IL-1 treatment on islet oxidation of
[U-14C]glucose to [14C]O2 was
examined next (Fig. 3). Control islets produced
increasing amounts of [14C]O2 as the medium
[U-14C]glucose concentration increased, with near maximal
levels at 15-18 mM glucose and half-maximal levels at
about 7 mM glucose, in agreement with previous reports
(22). Comparison of data in Figs. 1 and 3 indicated that control islets
oxidatively metabolized 30 ± 1% of the glucose that had been
utilized, and this value did not vary with the glucose concentration,
as previously reported (20, 27). IL-1-treated islets produced amounts
of [14C]O2 similar to that of control islets
at 3 mM glucose but produced smaller amounts than control
islets at glucose concentrations between 6 and 18 mM. The
46% suppression in [14C]O2 production at 15 mM glucose approximates the reported 49% suppression at
16.7 mM glucose (9). Islets incubated with both IL-1 and
NMMA exhibited rates of glucose oxidation that were statistically indistinguishable from those of control islets, consistent with previous reports (1, 2). Incubation of islets with NMMA alone had
little influence on glucose oxidation at 3 mM glucose (2.97 pmol/islet × 2 h versus control value of 3.79) or
at 18 mM glucose (23.37 pmol/islet × 2 h
versus control value of 26.67).
Data in Figs. 2 and 3 therefore conform to reported effects of IL-1 on
islet glucose oxidation and insulin secretion, but the effect of IL-1
to reduce islet glucose utilization in Fig. 1 differs from previous
studies (10, 11). This discrepancy may be attributable to differences
in conditions under which islets were exposed to IL-1. One difference
is that, in our study, islets were exposed to IL-1 in medium containing
5.5 mM glucose, while in previous studies of IL-1 effects
on islet glucose utilization, the medium glucose concentration during
IL-1 exposure was considerably higher (10, 11). As illustrated in Fig.
4, when islets were incubated for 24 h without IL-1
in medium containing 5.5, 11, or 23 mM glucose,
subsequently measured utilization of 15 mM
[5-3H]glucose was similar for all three groups of islets.
In contrast, suppression of glucose utilization was observed with
islets that had been incubated for 24 h with IL-1 at 5.5 mM glucose, but this effect was reduced or absent with
islets that had been incubated with IL-1 at 11 or 23 mM
glucose, respectively. The effect of IL-1 to reduce islet glucose
utilization therefore occurs when islets are cultured at a glucose
concentration corresponding to a euglycemic state, but this effect is
attenuated or prevented when IL-1 exposure occurs at higher glucose
concentrations.
The shape of the control curve in Fig. 1 for glucose utilization as a
function of glucose concentration over the range of 3-18
mM is highly characteristic of pancreatic islets (20, 21, 27) and is attributable to the kinetic properties of the enzyme glucokinase, which governs the overall rate of islet glycolytic flux
(26, 27, 41-43). The diminished rise in glucose utilization with
increasing glucose concentrations in IL-1-treated islets suggested that
IL-1 might reduce islet glucokinase expression under our culture
conditions. To evaluate this possibility, islet glucokinase mRNA
content was examined by competitive RT-PCR using cDNA prepared from
islet RNA as template, oligonucleotide primers designed from the rat
glucokinase cDNA sequence, and a competitor DNA. The competitor DNA
prepared for these experiments shares with glucokinase cDNA the
sequences recognized by the primers in the PCR reactions, but the
competitor yields a smaller product (360 bp) than that derived from
glucokinase cDNA (450 bp) (Fig. 5A). The
ratio of signals from the target and competitor was found to correspond
to the input DNA over a wide range of concentrations (Fig.
5B).
As illustrated in Fig. 6, when such competitive RT-PCR
reactions were performed with equal measured amounts of input RNA from islets that had been incubated with IL-1 for various periods, the
glucokinase target to competitor ratio declined substantially after
8 h of incubation and continued to fall at longer incubation intervals. In contrast, when equal measured amounts of input RNA from
control islets incubated without IL-1 were used as template in the
competitive RT-PCR reactions, the glucokinase target to competitor
ratio declined relatively little as a function of time in culture (Fig.
6). As illustrated in Fig. 7, when islets were incubated
with both IL-1 and NMMA for 24 h, RNA from the islets yielded a
glucokinase target to competitor ratio similar to that of control
islets and substantially greater than that of islets incubated with
IL-1 alone for 24 h.
The data in Figs. 6 and 7 therefore suggest that culture of islets with
IL-1 in medium containing 5.5 mM glucose induces a decline
in islet content of glucokinase mRNA by an NO-dependent mechanism. Consistent with this possibility, IL-1 was found to induce
an increase in islet production of NO, as reflected by nitrite
accumulation in the incubation medium, with a time course similar to
that of the IL-1-induced decline in islet glucokinase mRNA content
(Fig. 8). Nitrite is produced from NO by spontaneous oxidation in aqueous solutions, and its production by islets has previously been demonstrated to correlate with IL-1-induced synthesis of the inducible isoform of nitric oxide synthase and to be prevented by NMMA (37).
To determine whether the IL-1-induced reduction in islet glucokinase
mRNA was associated with a reduction in synthesis of glucokinase
protein, islets were incubated at 5.5 mM glucose for 24 h without IL-1, with IL-1 alone, or with both IL-1 and NMMA, and the islets were then incubated with [35S]methionine.
Immunoprecipitation was then performed with anti-glucokinase antiserum
or with preimmune serum, and the immunoprecipitates were analyzed by
SDS-PAGE and autoradiography. Immunoprecipitates obtained from control
islets with anti-glucokinase antibody but not with preimmune serum
contained a radiolabeled band of the 50-kDa size expected for
glucokinase (44) (Fig. 9). The intensity of the
glucokinase band was greatly reduced in immunoprecipitates from islets
that had been incubated with IL-1, but the intensity of the band from
islets that had been incubated with both IL-1 and NMMA was similar to
that from control islets, suggesting that IL-1 induces a decline in
islet synthesis of glucokinase protein by an NO-dependent
mechanism.
As illustrated in Fig. 10, SDS-PAGE and
autoradiographic analysis of identical loaded amounts of total protein
from [35S]methionine-labeled islets that had been
incubated under control conditions (first lane), with IL-1
alone (second lane), or with IL-1 plus NMMA (third
lane) did not indicate a global reduction in the synthesis of
proteins induced by incubation with IL-1. Under these
[35S]methionine-labeling conditions, total incorporation
of [35S]methionine into trichloroacetic acid-precipitable
protein in islets that had been incubated under control conditions
(4.22 ± 0.24 × 104 dpm/µg) for 24 h was
similar to that of islets that had been incubated with IL-1 alone
(3.68 ± 0.11 × 104 dpm/µg, p value
0.12) or with IL-1 plus NMMA (5.12 ± 0.42 × 104
dpm/µg, p value 0.12). The total cytosolic protein content
of islets incubated under control conditions (0.291 ± 0.035 µg/islet) for 24 h was also similar to that of islets incubated
with IL-1 alone (0.243 ± 0.032 µg/islet, p
value 0.36) or with IL-1 plus NMMA (0.272 ± 0.035 µg/islet, p value 0.75).
Stimulation of pancreatic islets with concentrations of glucose
exceeding 5 mM induces insulin secretion (27), and this requires that glucose be transported into islet -cells by GLUT-2 facilitative transporters (45-47) and metabolized (27, 48). The first
step in glycolytic utilization of glucose is its conversion to glucose
6-phosphate, and in
-cells this step is catalyzed predominantly by
glucokinase (26, 27, 41, 42, 49-51). Glucokinase is expressed only in
glucose-sensing cells, such as hepatocytes and
-cells (52), and its
kinetic properties cause the rate of glucose entry into glycolysis
within
-cells to rise with extracellular glucose concentrations in
the physiologic range (27, 42, 52). Because glucokinase activity in
-cells is substantially lower than that of other glycolytic enzymes,
it governs glycolytic flux (26, 27, 42). Overwhelming evidence
indicates that glucokinase is the primary component of the
-cell
glucose-sensor apparatus and accounts for the characteristic glucose
concentration dependence of insulin secretion (27, 42, 52).
This is supported by the fact that genetic mutations in glucokinase can
cause a form of type II diabetes mellitus designated maturity-onset
diabetes of the young (MODY) and that catalytic activities of mutant
glucokinases encoded by MODY genes are reduced (53-55). MODY is an
autosomal dominant disorder and occurs despite the presence of one
normal glucokinase allele (53). Because glucokinase is active as a
monomer, dominant negative effects of a mutant gene product are
unlikely (54), suggesting that 50% reduction of -cell glucokinase
levels can produce diabetes (42, 54). Similarly,
-cell-specific
targeted disruption of the glucokinase gene in mice produces diabetes
in heterozygotes, and islets isolated from such mice exhibit about 48%
of normal glucokinase activity (56). Nearly normal levels of
-cell
glucokinase therefore appear to be required to maintain euglycemia, and
relatively modest reductions in glucokinase may result in diabetes
(42).
Glucokinase gene transcription is thought to occur constitutively in
-cells (42), and its regulation is incompletely understood, although
a 50-kDa factor that binds to the upstream promoter of the gene is
preferentially expressed in
-cells (57). No compensatory overexpression of the normal glucokinase gene appears to occur in MODY
patients or in mice heterozygous for a disrupted glucokinase gene (42,
56). This suggests that mechanisms to up-regulate glucokinase gene
transcription may not be available in
-cells, although insulin
enhances transcription of this gene in hepatocytes (42, 58, 59), which
employ a different promoter than that employed by
-cells (33, 57,
59). Some observations suggest that
-cell glucokinase mRNA
expression is not entirely constitutive and can be regulated: 1)
exercise training of rats reduces both islet glucokinase mRNA
content (60) and insulin secretion (61); and 2) individual
-cells in
rat islets differ in glucokinase mRNA content, with highly
glucose-responsive cells containing twice the amount of less responsive
cells (43).
Our findings provide another example of regulation of islet glucokinase mRNA content and indicate that a reduction in this mRNA species occurs by an NO-dependent mechanism when islets are incubated with IL-1 in the presence of a physiologic concentration of glucose. This is associated with corresponding reductions in glucokinase protein synthesis, glycolytic utilization of glucose, and glucose-induced insulin secretion. These observations raise the possibility that factors which increase islet NO production could contribute to the development of glucose intolerance by reducing islet glucokinase levels. Mutations in the glucokinase gene probably account for a minority of the type II diabetic population (42, 53-55, 62), and down-regulation of glucokinase levels in islets with normal glucokinase genes might be considered as a potential contributor to the evolution of diabetes. There has been considerable interest in the possibility that immunomodulatory cytokines such as IL-1 contribute to development of type I diabetes mellitus (63-65), but NO production can also be increased by non-immunologic stimuli in various target cells (66). Endogenous factors which increased islet NO production sufficiently to reduce glucokinase levels by 50% might impair glucose tolerance (42).
Previous reports that IL-1 does not affect islet glucokinase levels (9)
or glucose utilization (10, 11) employed culture conditions that
differed from those used here. We exposed freshly isolated islets to
IL-1 for 24 h in medium containing 5.5 mM glucose. Previous studies involved culture of islets for 5 days in medium containing 11.1 mM glucose, followed by exposure to IL-1
for two additional days at 11.1 mM glucose (9-11). When
islets are cultured for several days in medium containing glucose
concentrations exceeding 9 mM, glucokinase levels are
up-regulated to values exceeding those of freshly isolated islets,
although glucokinase mRNA levels are unaffected (41). It has been
proposed that glucose increases islet glucokinase expression
post-transcriptionally by stabilizing the enzyme and protecting it from
proteolysis (42). Any effect of IL-1 to reduce islet glucokinase levels
in previous studies may have been offset by the effect of prolonged
culture in medium containing high glucose concentrations to increase
glucokinase expression. Consistent with this possibility is our
observation that culture of islets at 11 or 23 mM glucose
attenuates or prevents, respectively, the effect of IL-1 to suppress
the low-affinity component of islet glucose utilization attributable to
glucokinase. Although glucokinase down-regulation may not be required
for IL-1 to inhibit glucose-induced insulin secretion (9-11), such
inhibition would be an expected consequence of glucokinase
down-regulation. It is of interest that culture of clonal HIT-T15 cells
with IL-1 has been reported to reduce glucose-induced insulin secretion and to reduce both the glycolytic utilization of glucose and
glucokinase activity in these cells (74), providing additional support
for the possibility that IL-1 can down-regulate -cell glucokinase levels under some conditions.
IL-1-induced NO-dependent down-regulation of islet glucokinase mRNA content could reflect a reduction in gene transcription or in mRNA stability. There are several precedents for effects of NO on gene transcription. NO amplifies both IL-1-induced transcription of the cyclooxygenase-2 gene in rat mesangial cells (67) and Ca2+-induced gene transcription in neuronal cells (68) through cGMP-dependent mechanisms that reflect activation of guanylate cyclase by NO (66). NO also exerts cGMP-independent effects to down-regulate early growth response-1 gene expression in macrophages (69) and to inhibit Epstein-Barr virus gene transcription in lymphocytes (70). These effects may reflect S-nitrosylation of critical thiol groups in transcription factors (70, 71), which appears to account for the ability of NO to reduce activity of the transcriptional factor AP-1 (71). Determination of whether similar mechanisms are involved in the IL-1-induced NO-dependent decline in islet glucokinase mRNA content will require further study.
The excellent technical assistance of Z. Hu, Dr. Mary Mueller, and Bingbing Li is gratefully acknowledged.