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Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Hospital, Karolinska Institute, Stockholm, Sweden; and the Department of Internal Medicine, University of Trondheim, Trondheim, Norway
Address all correspondence and requests for reprints to: Dr. Valdemar Grill, Department of Molecular Medicine, Endocrine and Diabetes Unit, Karolinska Hospital, S-17176 Stockholm, Sweden.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms by which chronic hyperglycemia causes diabetic complications, such as retinopathy, neuropathy, and nephropathy, are in part related to nonenzymatic glycation and its sequelae. Hyperglycemia gives rise to nonenzymatic glycation of proteins and other molecules. Nonenzymatic glycation alters the physical properties of key molecules inside and outside of cells. This could lead to dysregulation of important cellular processes. Glycated molecules can be altered further by the formation of advanced glycosylation end products (AGEs). This formation may have serious negative effects. AGEs have thus been associated with cellular damage in microvascular complications of diabetes (7).
The effects of aminoguanidine (AG) constitute major evidence for the importance of AGEs in diabetic complications. AG is a nucleophilic hydrazine compound that potently inhibits AGE formation (8, 9). Beneficial effects of AG on diabetic complications have been reported in vivo and in vitro (10, 11, 12, 13) and are associated with inhibitory effects on AGE formation (14, 15, 16).
It is not known whether AGEs are formed in pancreatic islets or to which extent AGEs influence pancreatic B cells. Evidence against an important role for AGEs comes from observations that AG adversely affects insulin release in rat pancreatic islets (14, 17). However, in the previous studies islets were either acutely exposed to AG (14) or cultured for a only short period (2 days) with AG (17). From current knowledge of the time course of AGE formation, a much longer exposure time is needed to demonstrate AGE effects and reversal thereof by AG (14, 15, 16). Also, a beneficial effect of AG related to its effect on AGEs could be overshadowed by a negative effect related to the drug per se.
We aimed to test whether the formation of islet AGEs is of importance for negative effects of high glucose on B cell function. Specifically, we wished to test 1) whether high glucose induces the formation of AGEs in pancreatic islets, 2) whether AG affects AGE formation in pancreatic islets, and 3) whether long term exposure to AG in high glucose-cultured islets is beneficial for B cell function.
For these purposes, we assessed AGE formation in islets by fluorescence and measured the effects of AG on this parameter. Furthermore, we evaluated the effects of AG on insulin secretion, insulin biosynthesis, and insulin messenger RNA (mRNA) from high glucose-cultured rat islets. We reasoned that AGE-related effects of AG would affect that part of B cell dysfunction that is not rapidly reversible; therefore, B cell functional parameters were evaluated after an overnight wash-out period. A wash-out period also served to eliminate short term negative effects of AG per se. To evaluate the participation of a recently demonstrated effect of AG on NO synthase (18, 19), we also tested the effects of NG-methyl-L-arginine (L-NMA), a potent NO synthase inhibitor, on insulin secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Female Wistar rats were obtained from B&K Universal (Stockholm,
Sweden). At the time of experiments they weighed between 150–250 g.
They had free access to tap water and a standard pelleted diet. They
were exposed to a 12-h light (0600–1800 h), 12-h dark cycle. The
experimental protocols were approved by the Stockholm ethical committee
for research on animals.
Isolation of islets
Animals were killed by decapitation, and pancreatic islets were
isolated by collagenase digestion as previously described (20).
Collagenase was injected into the common bile duct at a concentration
of 2 mg/ml in 10 ml Hanks’ solution containing 5.5 mM
glucose. The pancreas was digested at 37 C for 20 min. The islets were
partly separated from exocrine tissue using gradient centrifugation
(700 x g, 20 min, 4 C) in Histopaque. Islets were
transferred to RPMI 1640 medium containing 11 mM glucose,
antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) and 10%
FCS. Islets were cultured free floating at 37 C with an atmosphere of
5% CO2-95% air for 1–2 days (primary culture) to remove
exocrine or other tissues.
Islet culture
After the period of primary culture, islets were selected under
a stereomicroscope and transferred into 260-ml tissue culture flasks
containing RPMI 1640 with the glucose concentrations described below
and with or without AG (0.1 or 1 mM). Before being added to
the culture medium, AG was dissolved in redistilled water at acidic
condition (pH 3.0) and brought to the same pH as the culture medium
(14). In one series of experiments, 0.1 mM L-NMA was used
instead of AG. Islets were cultured for 1–6 weeks. Culture medium was
exchanged every second day. At the end of a 1- or 6-week culture
period, some islets of each group were cultured for an additional
24 h in RPMI with 11 mM glucose but without AG or
L-NMA (wash-out period).
Measurement of AGE-associated fluorescence
Islets were cultured as described above in RPMI medium
containing 5.5, 11, or 38 mM glucose with or without AG.
After culture, islets (20 for each determination) were washed 3 times
in PBS, then sonicated in 300 µl redistilled water. The sonicates
were centrifuged at 10,000 x g for 10 min at 4 C, and
fluorescence was measured in the supernatants, using excitation at 370
nm and emission at 440 nm (21) by SPEX-1681 0.22-m spectrometer (SPEX
industries, Edison, NJ).
Islet volume
In some experiments islets cultured under different conditions
were randomly selected, and perpendicular diameters were measured under
a stereomicroscope. Diameters were averaged, and islet volume was
calculated by the formula: islet volume (nl) =
4
r3/3 x 10-6 (r = radian,
microns) (22).
Insulin release
After the culture period, islets were preincubated at 37 C for
30 min in Krebs-Ringer bicarbonate (KRB) medium (23) with the following
composition: 143 mM Na+, 5.8 mM
K+, 2.5 mM Ca2+, 1.2 mM
Mg2+, 124.1 mM Cl-, 1.2
mM PO43-, 1.2 mM
SO42-, and 25 mM
CO32-, pH 7.4, supplemented with 10
mM HEPES, 0.2% BSA (fraction IV, Sigma Chemical Co.), and
3.3 mM glucose. Islets were selected after preincubation in
batches of three islets in 300 µl KRB containing either 3.3 or 27
mM glucose and, in some experiments, other additions. Final
incubations were then carried out at 37 C for 60 min in a shaking water
bath and an atmosphere of 95% O2-5% CO2. At
the end of incubations, aliquots of the incubation medium were removed
for assay of insulin concentrations. Islets that had been exposed to
3.3 mM glucose in final incubations were retrieved for the
determination of islet insulin content.
Proinsulin and total protein biosynthesis
After culture, groups of 10 islets were incubated in 100 µl
KRB medium containing 50 µCi/ml
L-[4,5-3H]leucine and 27 mM
glucose. Incubations were carried out at 37 C for 2 h in 95%
O2-5% CO2 without shaking. The medium
containing radioactive leucine was then carefully removed, and the
islets were washed three times with Hanks’ solution containing 10
mM nonradioactive leucine. The washed islets were sonicated
in 200 µl redistilled water (twice for 10 sec using a model B-12
sonifier at setting 4, Branson Ultrasonics Corp., Danbury, CT). The
sonicates were centrifuged at 10,000 x g for 10 min at
4 C, and the supernatants were used for subsequent analysis.
Proinsulin-insulin biosynthesis was measured by immunoprecipitation
with undiluted antiinsulin serum in excess. Separation of bound from
free insulin was achieved with protein A-Sepharose CL-4B, as previously
described (24). Total protein synthesis was assessed from measurements
of the radioactivity in the 10% trichloroacetic acid-precipitable
fraction of the sonicates.
Total RNA and preproinsulin mRNA
Total RNA was prepared from 30 islets cultured in each condition
as described by Chomczynski and Nicoletta (25). The total RNA content
of the extracts was measured by spectrophotometry (1AOD260
= 40 µg/ml). The quantification of preproinsulin mRNA was achieved by
a solution hybridization assay using a RNA probe radiolabeled with
[35S]UTP (26). An in vitro synthesized 58-bp
oligonucleotide corresponding to the last part of exon 3 of the rat
preproinsulin II gene and flanked by BamHI and
KpnI restriction sites was inserted into pGEM-3Zf(+). The
resulting vector, prINS2, was linearized by EcoRI and
transcribed in vitro with SP6 RNA polymerase in the presence
of 3 µmol/L [35S]UTP for synthesis of the probe.
Unlabeled sense RNA was obtained by transcription with T7 RNA
polymerase after linearization with XbaI. The DNA template
was removed by RQ1 deoxyribonuclease I, and transcripts were separated
from unincorporated nucleotides on nick columns. Three serial dilutions
of each RNA sample in 20 µl 0.2 x SET [1 x SET contains
1.0% SDS, 20 mM Tris-HCl (pH 7.5), and 10 mM
EDTA] were mixed with 20 µl of 2 x hybridization solution
(20,000 cpm probe, 1.2 M NaCl, 8 mM EDTA, 1.5
mM dithiothreitol, 50% formamide, and 40 mM
Tris-HCl, pH 7.5). After hybridization at 70 C for 18 h, the
samples were treated with 40 µg RNase A and 100 U RNaseT1 in the
presence of 100 µg herring sperm DNA for 60 min at 37 C in a volume
of 1 ml. Protected probe was precipitated with 100 µl 100%
trichloroacetic acid. Precipitates were collected on glass fiber
filters (GF/C), and the radioactivity was counted in a scintillation
counter. Parallel hybridizations with increasing amounts of unlabeled
sense RNA allowed construction of a standard curve. The amount of
preproinsulin mRNA was calculated by comparison to the standard curve,
and results were expressed as picograms of preproinsulin mRNA per µg
total RNA.
Insulin assay
Insulin was measured by RIA using rat insulin as standard,
monoiodinated porcine insulin as tracer, and antibody raised in our
laboratory against porcine insulin. Antibody-bound insulin was
separated from free insulin using Dextran T70-coated charcoal (27). For
the determination of islet insulin contents, three islets were
transferred into 200 µl acid-ethanol (0.18 M HCl in 95%
ethanol). Insulin was extracted overnight at 4 C after sonication as
previously described (28).
Presentation of results
All results are expressed as the mean ± SEM.
Analysis between groups was carried out as appropriate by Student’s
unpaired t test or one-way ANOVA with Student-Newman-Keuls’
test. P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lines 1–8). The response in absolute
terms to 27 mM glucose postculture, however, was reduced in
islets cultured at 38 mM glucose relative to that in islets
cultured at 11 mM glucose. This difference was especially
apparent after an overnight wash-out period (Table 1, lines 4
vs. 2 and 8 vs. 6). Glucose-induced insulin
responses tended (nonsignificantly) to decrease between 1 and 6 weeks
of culture (Table 1, lines 5–8 vs. 1–4). Such a tendency
was seen when islets were cultured at both 11 and 38 mM
glucose.
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Islet volume was increased by high glucose during culture (Table 2). Although mean values of islet volumes were somewhat
larger after AG, there was not a significant effect (P
> 0.3).
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)
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). The effect seemed dose dependent, as 1
mM AG exerted a more pronounced effect than 0.1
mM. The inhibitory effect was not appreciably altered by
the time of exposure to AG (1, 3, or 6 weeks; Table 3).
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, upper graphs).
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).
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). Under the same conditions, AG tended to inhibit
arginine- and isobutylmethylxanthine-induced insulin secretion
(Fig. 3).
The effects of AG after wash-out are shown in Fig. 2, lower
graphs. When cocultured with 11 mM glucose, AG did not
affect basal or glucose-stimulated insulin release after wash-out. In
contrast, when AG was cocultured with 38 mM glucose, both
basal and glucose-stimulated insulin release were markedly and
significantly increased.
Effects of AG on proinsulin and total protein biosynthesis
Six weeks of culture with 38 mM glucose enhanced both
proinsulin and total protein synthesis relative to culture with 11
mM glucose (Fig. 4, upper
graphs). AG enhanced both proinsulin synthesis and total protein
synthesis in 38 mM, but not in 11 mM, cultured
islets. A wash-out period eliminated the enhancing effect of 38
vs. 11 mM glucose-cultured islets on
biosynthesis (Fig. 4, lower graphs). However, the effect of
previous exposure to AG persisted with regard to both proinsulin and
total protein synthesis.
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Preproinsulin mRNA was also measured at the end of a 6-week culture
period (Fig. 5). Preproinsulin mRNA was expectedly
higher after culture with 11 or 38 mM glucose than after
culture with 5.5 mM glucose. The coexistence of AG with 38
mM glucose enhanced levels of preproinsulin mRNA. The
increase was 53% with 0.1 mM AG and 94% with 1
mM AG.
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). The lack of
effect was seen after both 1 and 6 weeks of culture and in the presence
of both 11 and 38 mM glucose. The effect of a wash-out
period was to increase insulin content in islets previously cultured
with AG and 38 mM glucose.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nevertheless, our results indicate that B cell secretory potential was decreased after culture with high glucose. Hence, although a fold increase in secretion due to 27 mM glucose was not affected, the absolute insulin responses were decreased after culture with 38 vs. 11 mM glucose. Notably, this decrease in insulin response was most apparent after wash-out. This indicates that the decrease reflects a lasting negative effect of high glucose exposure.
Two lines of evidence support a role for AGE formation on B cell function. The first one pertains to fluorescence measurements. These measurements document a time- and glucose-dependent increase in islet fluorescence during conditions of measurement that have, in other tissues, been shown to reflect the contents of AGE. Furthermore, increased fluorescence could partly be inhibited by AG. The second line of evidence pertains to effects of AG on B cell function, and these effects are discussed below.
Both inhibitory and stimulatory effects of AG were observed. The inhibitory effects of AG on insulin release under some conditions are in line with previous reports. Inhibition by AG was thus found during short term incubation with the drug (14) or after 2 days of islet culture with AG (17). It has been proposed that an inhibitory effect of AG could be due to intracellular acidification by the drug (14). However, the precise mechanism behind inhibition has not been elucidated.
The stimulatory effects of AG on insulin secretion were apparent after a wash-out procedure that consisted of an additional 24-h culture period at 11 mM glucose in the absence of the drug. The stimulatory effect of AG on basal and glucose-induced insulin secretion was dependent on the period of high glucose exposure preceding wash-out. The stimulatory effect of previous AG treatment was thus apparent after 6 weeks, but not after 1 week, of culture. We interpret the wash-out effect to mean that a long term stimulatory effect by AG can be unmasked by removing a negative effect of ambient AG on secretion. Islets not subjected to a wash-out period were indeed washed in KRB before final incubations. However, this procedure most likely did not remove AG completely from islets due to a delay in diffusion of the drug from the interior of islets and cells.
The beneficial effects of AG on protein biosynthesis were also apparent in 6-week high glucose-cultured islets. In contrast to the effects of AG on insulin secretion, an enhancing effect on [3H]leucine accumulation into proinsulin and total protein synthesis was apparent both before and after wash-out. A beneficial effect of AG on insulin biosynthesis agrees well with our data on the islet content of preproinsulin mRNA obtained immediately after culture. Thus, previous exposure to AG markedly increased the islet content of preproinsulin mRNA.
The available evidence indicates that the beneficial effects of AG on insulin secretion and biosynthesis are due to prevention of AGE formation in islets. A multitude of studies have shown that AG prevents the formation of AGE in other tissues and that such effects prevent tissue damage (10, 11, 12, 13). Furthermore, our assessment of AGE formation in islets indicates that AG inhibits AGE formation in islets. Moreover, our data argue against the alternative explanation, that AG exerts its stimulatory effects through inhibition of nitric oxide (NO) synthase. The time dependency for the stimulatory effects of AG speaks against involvement of NO synthase, as inhibitory effects of AG on this enzyme are immediate and not time dependent (18, 19). Also, the effects of AG on insulin biosynthesis persisted after a 24-h wash-out period. Furthermore, a specific inhibitor of NO synthase, present during 6 weeks of culture, failed to reproduce the effects of AG.
Granted that the beneficial (stimulatory) effects of AG are linked to the prevention of AGE formation, it is not known which aspects of formation exert negative effects on B cell function. Multiple proteins of importance for the secretory process could be affected as well as molecules of importance for transcription and translation (30, 31). A role for altered transcription factors would be compatible with the influences on protein synthesis and insulin mRNA presently found. Further research is needed to elucidate these uncertainties.
Our results could be important for the future treatment of patients with noninsulin-dependent diabetes mellitus. Insulin secretion in these patients deteriorates with the duration of the disease (32); this could in part be due to glucotoxicity. Our results indicate a role for islet AGE formation in glucotoxicity. Theoretically, AGE formation could be even more important than indicated by our results, as a negative effect of circulating AGE on B cells was not tested and therefore cannot be ruled out. In this context it should be mentioned that we have also performed in vivo studies showing a beneficial effect of AG on islet graft function in a rat transplantation model (33). If AGE formation plays a role in glucotoxicity toward B cells, it may be possible to treat patients with drugs such as AG to avoid or retard the deterioration of B cell function.
In conclusion, our results provide evidence for glucose-induced formation of AGEs in pancreatic islets and for the inhibition of such formation by AG. Moreover, in high glucose-cultured islets, AG time-dependently increases B cell secretion and insulin biosynthesis. These observations indicate a negative role of AGEs in B cell function.
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Footnotes |
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Received July 3, 1996.
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, P < 0.05
vs. measurements at 38 mM glucose without AG
at each culture period; §, P < 0.01
vs. measurements at 38 mM glucose without AG
after 3 weeks.
