From the Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain
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ABSTRACT |
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A number of cytokines have been shown to alter
the function of pancreatic -cells and thus might be involved in the
development of type 1 diabetes. Interferon-
(IFN-
) expression is
induced in epithelial cells by several viruses, and it has been
detected in islets of type 1 diabetic patients. Here we show that
treatment of isolated mouse islets with this cytokine was able to alter insulin secretion in vitro. To study whether IFN-
alters
-cell function in vivo and leads to diabetes, we have
developed transgenic mice (C57BL6/SJL) expressing IFN-
in
-cells.
These mice showed functional alterations in islets and impaired
glucose-stimulated insulin secretion. Transgenic animals presented mild
hyperglycemia, hypoinsulinemia, hypertriglyceridemia, and altered
glucose tolerance test, all features of a prediabetic state. However,
they developed overt diabetes, with lymphocytic infiltration of the
islets, when treated with low doses of streptozotocin, which did not
induce diabetes in control mice. In addition, about 9% of the
transgenic mice obtained from the N3 back-cross to outbred albino CD-1
mice spontaneously developed severe hyperglycemia and hypoinsulinemia and showed mononuclear infiltration of the islets. These results suggest that IFN-
may be involved in the onset of type 1 diabetes when combined with either an additional factor or a susceptible genetic
background.
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INTRODUCTION |
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Despite an immense research effort, the etiology of type 1 diabetes has not been elucidated. It has been proposed that type 1 diabetes is caused by nongenetic factors, probably environmental, operating in a genetically susceptible host to initiate a
-cell-destructive immune process (1-3). These environmental
factors, such as viral infections, may operate over a limited period to
induce the immune process (4). Thereafter, there is a long prodrome
before the onset of clinical diabetes during which clinical,
immunulogic, and metabolic changes can be detected in the
-cells
(4). During the prediabetic period, a decline in the insulin secretion
as well as impaired glucose tolerance can be detected several years before the clinical onset of type 1 diabetes (5, 6). Thus, alterations
in
-cell function might be a previous step to the development of
diabetes mellitus.
Cytokines are hormone-like peptides mainly used by immune system cells
to control local and systemic events of immune and inflammatory
responses (7-9). Studies in vitro have demonstrated that
certain cytokines, such as
IL-1,1 tumor necrosis
factor-, or IFN-
(10-12), can be cytotoxic to pancreatic
-cells, inhibiting insulin secretion. Furthermore, when combined,
these cytokines can destroy
-cells (12). Moreover, these cytokines
have been found in the pancreatic insulitis lesion of NOD mice (13) and
BB rats (14), and they may thus be considered mediators of
-cell
damage in type 1 diabetes. Furthermore, transgenic mice expressing
IFN-
in pancreatic
-cells show lymphocytic infiltration of the
islets by mononuclear cells,
-cell destruction, and diabetes (15,
16). However, of all the cytokines that may be involved in the
development of the diabetic process, only a few can be produced by
normal epithelial cells, such as pancreatic
-cells. Type I
interferons (IFNs) are pleiotropic cytokines involved in host defenses
against viral infections that can be produced by most cell types in
response to a virus (17). There are three families of type I IFNs,
,
, and
, that are closely related structurally. These IFNs also
bind to a common receptor and have potent antiviral activities (17,
18). Several studies have implicated IFN-
in the development of type
1 diabetes. This cytokine can be detected in the islets of newly
diagnosed patients of type 1 diabetes (19, 20). In addition, IFN-
can induce the expression of MHC class I antigens in pancreatic
-cells (21), and it can lead to diabetes when expressed in islets of
transgenic mice (22). Similar to IFN-
, IFN-
has immunomodulatory
properties, it induces MHC class I antigen expression (23, 24), and it
has also been detected in newly diagnosed patients of type 1 diabetes
(19). However, little is known about the role of IFN-
in the
development of type 1 diabetes.
Here we studied whether IFN- may lead to type 1 diabetes. To this
end, we developed transgenic mice expressing IFN-
in
-cells. These mice presented impaired
-cell function, hypoinsulinemia, and
altered glucose tolerance test, but they did not develop overt type 1 diabetes. However, transgenic mice treated with multiple low doses of
streptozotocin or back-crossed to a CD-1 strain developed overt
diabetes, with marked hyperglycemia and lymphocytic infiltration of the
islets.
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EXPERIMENTAL PROCEDURES |
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Construction of the RIP-I/IFN- Chimeric Gene and Generation of
Transgenic Mice--
To obtain the RIP-I/IFN-
chimeric gene a
SacI-BamHI fragment (
570 bp to +3 bp) of the
rat insulin I promoter (RIP-I) (25) was linked to a
TaqI-TaqI fragment (+302 bp to +1140 bp) of the human IFN-
gene, which contains the entire coding sequence and the
polyadenylation signal (26). The construction of the RIP-I/IFN-
chimeric gene was initiated by subcloning the
SacI-BamHI fragment of the RIP-I at the same
sites of the BluescriptR polylinker. Afterward, the
TaqI-TaqI fragment of the IFN-
was introduced
at the ClaI site of the polylinker. A 1.3-kb
SacI-XhoI fragment, containing the entire
RIP-I/IFN-
chimeric gene, was microinjected into fertilized mouse
eggs from a C57Bl6/SJL background. The general procedures used for
microinjection of the RIP-I/IFN-
chimeric gene were as described
elsewhere (27). At 3 weeks of age, the animals were tested for the
presence of the transgene by Southern blot analysis of tail DNA (10 µg) digested with BglII. The RIP-I/IFN-
construct
contains a unique BglII site at the end of the IFN-
gene.
Blots were hybridized with the 1.3-kb SacI- XhoI
fragment containing the entire RIP-I/IFN-
chimeric gene, radiolabeled with [
-32P]dCTP ((3000 Ci/mmol), Amersham
Pharmacia Biotech) by random oligopriming (Boehringer Mannheim,
Germany). A 1.3-kb fragment was produced by the restriction of
BglII at the site of an RIP-I/IFN-
molecule and at the
site of a second molecule of the chimeric gene, inserted in the genome
in a head-to-tail manner (data not shown). In the experiments described
below, heterozygous male mice aged 2 to 3 months were used. However,
similar results were obtained in animals more than 6 months old.
Transgenic mice expressing the chimeric gene RIP/IFN-
with a
C57Bl6/SJL genetic background were also back-crossed to CD-1 mice
(Charles River), and N1 to N3 generations were analyzed.
Isolation of Pancreatic Islets--
Islets were isolated from
the pancreas of control or transgenic mice fed a standard or a high
carbohydrate diet. Islets were released from pancreatic acinar tissues
by digestion with collagenase P (Boehringer Mannheim) (28). Islets were
collected by handpicking under a dissection microscope. For in
vitro studies, islets from control mice were cultured
free-floating in groups of 150-200 in plastic Petri dishes containing
RPMI 1640 medium with 5% fetal calf serum, maintained at 37 °C in
an atmosphere of 5% CO2 in air. Experiments were initiated
24 h after culture, by placing islets in RPMI 1640 with 5% fetal
calf serum in the absence or in the presence of either murine
(103 units/ml) or human (106 units/ml) IFN-.
After 3 days of treatment with the cytokines, insulin secretion by
these islets was determined.
RNA Analysis--
To study the expression of the transgene,
total RNA was obtained from pancreas by the guanidine isothiocyanate
method (29), and RNA samples (30 µg) were electrophoresed on a 1%
agarose gel containing 2.2 M formaldehyde. Northern blot
was hybridized to a 32P-labeled 0.8-kb
EcoRI-XhoI fragment of the IFN- gene. The
-actin probe corresponded to a 1.3-kb
EcoRI-EcoRI fragment of the rabbit cDNA. To
analyze the expression of the
2-microglobulin, RNA was obtained from islets cultured in the presence of IFN-
or from islets
of transgenic mice using the acid guanidinium
thiocyanate/phenol/chloroform procedure (30). RNA samples were treated
with DNase for 30 min at 37 °C, extracted with a phenol/chloroform
mixture, and precipitated with ethanol. cDNA synthesis was carried
out using 50 ng of total RNA at 42 °C for 60 min in a reaction
volume of 20 µl. The cDNA was then used for PCR co-amplification
of
2-microglobulin and
-actin with the following
temperature program: 30 s at 94 °C, 60 s at 60 °C, and
30 s at 72 °C for 40 cycles. The primer pair used for
2-microglobulin (sense, 5'-GTATACTCACGGATCCCACCGGAGA-3', and antisense 5'-CATGTCTCGATCCCAGTAGACGG-3') yielded a 272-bp PCR
product. The primer pair used for
-actin (sense
5'-CATCGTGGGCCGCCCTAGGCAC-3', and antisense
5'-CCGGCCAGCCAGGTCCAGACGC-3') yielded a 451-bp PCR product. The
co-amplified products were analyzed on a 2% agarose gel and visualized
following staining with ethidium bromide.
Protein Analysis--
Western blot analysis was performed by
standard procedures (31) from total cellular lysates of islets. Islets
were disrupted in 5% sodium dodecyl sulfate (SDS), 80 mM
Tris-HCl (pH 6.8), 5 mM EDTA, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride by sonication. Twenty µg
of protein was electrophoresed on 10% SDS-polyacrylamide gels and
transferred to nitrocellulose membranes. To detect IFN- a rabbit
antiserum to human IFN-
(Biogenesis, Bournemouth, UK), diluted at
1:1000, was used.
Histologic Analysis--
For immunohistochemical detection of
IFN- and insulin, sections of pancreas from control and transgenic
mice were fixed for 12 to 24 h in Carnoy's reagent, embedded in
paraffin, and sectioned. Sections were then incubated with a rabbit
anti-human IFN-
antibody (Biogenesis), diluted at 1:100, or with a
guinea pig anti-porcine insulin antibody (Dako, Carpinteria, CA), at
1:100 dilution, overnight at 4 °C. As secondary antibodies goat
anti-rabbit or rabbit anti-guinea pig immunoglobulin G coupled to
peroxidase (Boehringer Mannheim) was used. The 3',3'-diaminobenzidine
was used as the substrate chromogen. Sections were counterstained in
Mayer's hematoxylin. For histologic analysis, sections of pancreas
were stained with hematoxylin/eosin.
Insulin Secretion from Islets--
Batches of six islets were
incubated in a shaking water bath for 90 min at 37 °C in 1 ml of
bicarbonate-buffered salt solution containing bovine serum albumin (5 mg/ml, fraction V, Sigma) supplemented with 2.8 or 16.7 mM
glucose, with 2.8 mM glucose together with either 10 mM leucine plus 10 mM glutamine or 10 mM arginine, or with 16.7 mM glucose plus 5 µM forskolin. At the beginning of the treatments, vials
were gassed with O2:CO2 (95:5%) for 10 min. At
the end of the incubation period supernatants were stored at 20 °C
until the insulin was assayed by RIA (CIS, Biointernational, Gif-Sur-Yvette, France). The method allows the determination of 2.5 milliunits of insulin/ml, with a coefficient of variation within and
between assays of 6 and 8%, respectively.
cAMP Measurements--
For cAMP measurements, islets were
incubated in groups of 30 for 15 min at 37 °C in Hanks' medium (136 mM NaCl, 1.67 mM CaCl2, 0.8 mM MgSO4, 5.4 mM KCl, 0.35 mM Na2HPO4, 0.45 mM
KH2PO4, and 4.2 mM
NaHCO3) containing 2.8 and 16.7 mM glucose or
16.7 mM glucose plus 5 µM forskolin. The
incubation was stopped by adding 6% trichloroacetic acid, and islets
were frozen and stored at 80 °C, pending analysis. The samples
were thawed by sonication on ice and centrifuged at 2000 × g (4 °C) for 15 min. The pellet was re-sonicated in 200 µl of water and used to measure islet cell DNA content. The
supernatant was washed 4 times in 5 volumes of water-saturated diethyl
ether. The aqueous extract was freeze-dried, and the cAMP content was measured by RIA (using 125I-cAMP) as described by the
manufacturer of the assay kit (Amersham Pharmacia Biotech). In order to
increase the sensitivity of the method, samples were acetylated.
Hormone and Metabolite Assays-- Insulin levels in serum samples were determined by RIA (CIS, Biointernational, Gif-Sur-Yvette, France). Serum glucose concentration was measured enzymatically (GlucoquantR, Boehringer Mannheim). Glucose levels were also determined in blood by using a GlucometerR analyzer (Bayer, Germany). Serum triglycerides were determined enzymatically (GPO-PAP, Boehringer Mannheim). The intraperitoneal glucose tolerance test was performed between 10 and 11 a.m. in fed control and transgenic mice. After anesthetizing the mice with avertin, a blood sample was obtained from the tail vein to measure the basal level of glucose. Mice were subsequently given an intraperitoneal injection of 1 mg of glucose per g of body weight. Blood samples (5 µl) were obtained at different times from the same animals, and the levels of glucose were determined.
Statistical Analysis-- All values are expressed as the mean ± 1 S.E. Statistical analysis was carried out using the Student-Newmann-Keuls test. Differences were considered statistically significant at p < 0.05.
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RESULTS |
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Alteration of Glucose-stimulated Insulin Secretion by Islets
Cultured in the Presence of IFN---
In this study we examined the
role of IFN-
in the diabetic process. Since alteration in
-cell
function might precede the development of type 1 diabetes, we first
analyzed the effect of this cytokine in vitro. Mouse islets
were cultured in the presence of either mouse (103
units/ml) or human (106 units/ml) IFN-
to determine the
effect of these cytokines on insulin secretion. The concentration of
the human IFN-
used was 1000 times that of the mouse to overcome the
species specificity (32). Neither mouse nor human IFN-
caused
morphologic changes in the islets at the concentrations used (data not
shown). To determine the biologic activity of IFN-
in the pancreatic
islets, the expression of
2-microglobulin was analyzed
by RT-PCR in islets treated with either mouse or human IFN-
. The
2-microglobulin and MHC class I heavy chains complex at
the cell surface to form the intact MHC class I antigen (33), and their
expression is often coordinately regulated (34). Both mouse and human
IFN-
induced the expression of
2-microglobulin after
3 days of culture, while islets cultured in the absence of IFNs showed
very low levels of
2-microglobulin mRNA (Fig.
1), suggesting an up-regulation of MHC
class I antigen by IFN-
. The effect of the islet exposure to IFN-
on glucose-induced insulin secretion was next studied. Islets cultured
for 3 days in the presence of either mouse or human cytokine were
incubated for 90 min in the presence of 2.8 or 16.7 mM
glucose. At low glucose concentration, insulin levels in the incubation
medium of islets treated with IFN-
were lower than those of
non-treated islets (Table I). When
control islets were incubated in the presence of 16.7 mM
glucose, a 3-fold increase in insulin secretion was noted. Although
islets treated with either mouse or human IFN-
showed an increase in
insulin secretion at high glucose, a marked reduction (about 50 and
40%, respectively) of the insulin concentration in the incubation
medium of these islets compared with non-treated islets was detected
(Table I). When islets were incubated in the presence of 16.7 mM glucose plus 5 µM forskolin, an adenylate
cyclase activator, non-cytokine-treated islets showed a 4-fold increase
in the insulin release over control islets incubated with glucose alone
(Table I). In contrast, when incubated with glucose plus forskolin the
level of insulin in the incubation medium of IFN-
-treated islets was
60% lower than that of control islets (Table I). Thus, these results
indicated that IFN-
was able to alter
-cell function in
vitro.
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Expression of IFN- in Pancreatic
-Cells of Transgenic
Mice--
To study the potential role of IFN-
in the pathogenesis
of type 1 diabetes, we developed a transgenic animal model that
expresses IFN-
specifically in pancreatic
-cells. IFNs are
species-specific. However, to avoid the male sterility observed in mice
expressing high levels of mouse IFN-
(35), the use of the human
IFN-
gene was considered to be more appropriate in transgenic mice. Human IFN-
is active in mouse cells (36-39), although its efficacy was about 1000 times lower than that of mouse IFN-
. Furthermore, here we showed that human IFN-
exerted a clear biologic effect in
mouse islets, suggesting that it might be used instead of mouse cytokine. Thus, transgenic animals that expressed human IFN-
in
-cells, under control of the rat insulin-I promoter (RIP-I/IFN-
), were developed to examine the effect of IFN-
in vivo. The
RIP-I/IFN-
chimeric gene was microinjected into fertilized eggs, and
four transgenic mice were obtained. In this study, we used F1 and F2 heterozygote mice from the transgenic lines RIP-I/IFN-
28
(TgIFN-
-28) and RIP-I/IFN-
56 (TgIFN-
-56). TgIFN-
-28 and
TgIFN-
-56 carried about 5 and 20 intact copies, respectively, of the
RIP-I/IFN-
chimeric gene when analyzed by Southern blot (data not
shown). We used littermates as controls for the transgenic animals. A transcript that hybridized with the IFN-
probe was detected when total RNA obtained from the pancreas of transgenic mice was analyzed by
Northern blot (Fig. 2A).
Although these lines of transgenic mice had integrated different number
of copies of the transgene, similar levels of IFN-
mRNA were
noted in the pancreas of both TgIFN-
-28 and TgIFN-
-56, probably
as a result of the site of integration in the genome. Nevertheless, no
IFN-
mRNA was detected in the pancreas from control animals
(Fig. 2A). No expression of the transgene was detected in
other tissues examined, like liver and kidney (data not shown). Mice
were fed a high carbohydrate diet for 1 week, to induce the expression
of the transgene, and afterward islets were obtained and IFN-
protein was analyzed by Western blot. The expression of IFN-
was
parallel to the presence of IFN-
protein in islets from transgenic
mice, whereas no immunodetectable IFN-
was noted in control islets
(Fig. 2B). Similarly, after immunohistochemical analysis
human IFN-
was also detected in insulin-producing cells of islets
from transgenic mice fed a standard diet (Fig.
3B). However, the expression
of IFN-
protein was variegated, probably resulting from differences
in the level of expression of the transgene in the various
-cells
within the islet. Similar results were obtained in both TgIFN-
-28
and TgIFN-
-56 mice. No IFN-
immunostaining was observed in islets
from control mice (Fig. 3A). Islets from both control and
transgenic mice showed similar insulin immunoreactivity (Fig. 3,
C and D). All these results indicate that high
levels of IFN-
were produced in the
-cells of transgenic mice.
The expression of the transgene did not cause islet lesions, even in
older mice (data not shown).
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Expression of IFN- in Islets Led to an Increase in
2-Microglobulin--
To examine whether human IFN-
was biologically active in the islets of the transgenic mice, the
expression of
2-microglobulin was determined in islets
from control and transgenic mice by RT-PCR. The levels of
2-microglobulin mRNA in islets from control mice were very low. However, islets from both TgIFN-
-28 and TgIFN-
-56 showed a high induction (about 5-fold) in the expression of
2-microglobulin mRNA (Fig. 1). These results
suggested that human IFN-
produced by the
-cells might induce MHC
class I antigen in islets of transgenic mice. The results described
below were obtained from the TgIFN-
-28 line. However, a similar
phenotype was observed in the TgIFN-
-56 animals.
IFN- Expression in Islets Altered
-Cell Function--
To
discern whether the glucose-stimulated insulin secretion in
-cells
from the transgenic mice was altered, islets from control and
transgenic mice were isolated, and the insulin release in response to
2.8 and 16.7 mM glucose was measured (Fig.
4A). Although at both glucose
concentrations islets from transgenic mice secreted less insulin than
islets from non-transgenic animals, the reduction was higher (about
60%) at 16.7 mM glucose. The concentration of insulin in
the medium of islets from transgenic animals incubated with 16.7 mM glucose was similar to that released by islets from control animals incubated with 2.8 mM glucose (Fig.
4A). When amino acid-stimulated insulin secretion was
studied no decrease was detected in islets from transgenic mice
compared with control mice. Isolated islets cultured in the presence of
2.8 mM glucose together with 10 mM leucine plus
10 mM glutamine or with 10 mM arginine,
released more insulin (about 50 and 25% increase, respectively) than
islets from control animals (Fig. 4B). Thus, stimulation with leucine plus glutamine and with arginine counteracted the IFN-
-induced reduction of glucose-stimulated insulin secretion. When
insulin secretion was studied in islets incubated in the presence of
16.7 mM glucose plus 5 µM forskolin, islets
from control mice showed a 3-fold increase in the insulin release
compared with control islets incubated only in the presence of glucose (Fig. 4A). A similar induction in insulin secretion (about
3-fold) was also observed when transgenic islets were incubated with
forskolin and 16.7 mM glucose. However, the level of
insulin in the incubation medium of islets from transgenic mice
expressing IFN-
was 60% lower than that of islets from control mice
(Fig. 4A). Furthermore, islets from transgenic mice
expressing IFN-
showed a decrease in the cAMP content when incubated
with low or high glucose concentration (Table
II). This suggests that IFN-
might
either exert a direct negative effect on cAMP production or interfere
with the effects of glucose on cAMP production in the
-cells.
Incubation of islets from control mice with glucose plus forskolin led
to an increase (about 7-fold) in cAMP content. Although a similar
increase was noted in islets from transgenic mice, the levels of cAMP
reached were 40% lower than those detected in control islets (Table
II).
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Transgenic Mice Expressing IFN- Developed a Prediabetic
State--
Transgenic mice expressing the RIP/IFN-
chimeric gene
were mildly hyperglycemic and showed a lower serum concentration of insulin than control mice (about 40% reduction) and a 2-fold increase in serum triglycerides (Table III). In
addition, high levels of blood glucose were detected in transgenic mice
when intraperitoneal glucose tolerance tests were performed. In
contrast to control mice, the glucose concentration reached in
transgenic animals had not returned to basal values 180 min after
glucose administration (Fig. 5). The
alterations in the serum parameters and the impaired response to an
intraperitoneal glucose tolerance test suggested that transgenic mice
expressing the RIP/IFN-
chimeric gene had developed a
prediabetic state.
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Treatment of Transgenic Mice with Multiple Low Doses of
Streptozotocin Led to Type 1 Diabetes--
Streptozotocin is a toxin
that has direct cytotoxic effects on -cells when injected at high
doses. In contrast, multiple low doses of streptozotocin (MLDS) lead to
inflammatory autoimmune-mediated destruction of
-cells (40). Certain
strains of mice are highly susceptible to MLDS, such as outbred CD-1
mice, whereas others, such as C57Bl6/SJL mice, are resistant and only
exhibit a mild increase in blood glucose levels without insulitis.
Transgenic mice were treated with MLDS, to maximize the autoimmune
response and minimize the direct toxicity of this drug on the
-cells. Daily injections (intraperitoneal) of 40 mg/kg
streptozotocin for 5 consecutive days were performed, and blood samples
were taken from the tail vein at 7, 14, 21, and 28 days after MLDS treatment. Transgenic mice expressing the RIP-I/IFN-
chimeric gene
reached blood glucose levels characteristic of diabetes 14 days after
treatment, whereas control mice presented normal levels of serum
glucose (Fig. 6A).
Immunohistochemical analysis of the pancreas of control mice, following
28 days of treatment, revealed that islets had a slightly lower number
of insulin-containing cells, but they did not show any inflammatory
infiltration (Fig. 7A). In
contrast, not only did islets from transgenic mice show clearly reduced
numbers of insulin-containing cells (Fig. 7B) but also 50%
of the animals presented insulitis leading to the destruction of the
islets (Fig. 7, C and D).
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Influence of the Genetic Background in IFN--mediated Diabetic
Process--
To assess the extent to which genetic factors could be
involved in the development of the disease, C57Bl6/SJL transgenic mice expressing the RIP/IFN-
chimeric gene were back-crossed to CD-1 mice. The aim of this back-cross was to obtain transgenic mice with the
genetic background of CD-1 mice, which are more susceptible to develop
insulitis. N1, N2, and N3 generations were obtained. N1 transgenic mice
showed a similar phenotype to C57Bl6/SJL mice, i.e.
hypoinsulinemia, mild hyperglycemia, and an altered glucose tolerance
test (data not shown), without progression to overt diabetes. In
contrast, when treated with MLDS, N1 transgenic mice presented higher
glycemia than MLDS-treated C57Bl6/SJL transgenic mice (Fig.
6B), reaching values characteristic of diabetes 7 days after
MLDS injection (Fig. 6B). In contrast, control N1 mice
maintained normal values of glycemia at that time, while a slight
increase was noted around day 14 after treatment, although glucose
levels were always significantly lower than those observed in N1
transgenic mice (Fig. 6B). Histologic analysis of the
pancreas showed that 100% of the MLDS-treated N1 transgenic mice
developed insulitis, whereas none of the control mice had histologic
alterations.
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DISCUSSION |
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The early stages of type 1 diabetes are characterized by a
selective inability to secrete insulin in response to glucose, coupled
to a better response to other secretagogues (10). The deficient glucose
response may be a result of the autoimmune process directed toward the
-cells. Several cytokines may be mediators of immunologic damage of
the
-cells, and culture of pancreatic islets in the presence of
these cytokines results in alterations in their function (11, 12, 41).
In this study we showed that IFN-
may alter pancreatic
-cell
function both in vitro and in vivo. Results
obtained in islets cultured in the presence of IFN-
or islets from
transgenic mice expressing the RIP/IFN-
chimeric gene suggest that
IFN-
might alter the signals that lead to an increase in insulin
secretion in response to glucose. Similarly, a decrease in the insulin
release was noted after incubation of both rat insulinoma cells and rat
pancreatic islets with rat IFN-
(42). This decrease was correlated
with the concentration and the time of incubation with the cytokine
(42). However, while isolated mouse pancreatic islets incubated with
either IFN-
or IFN-
show 50% inhibition of glucose-stimulated
(pro)insulin biosynthesis (43), no change in insulin secretion was
detected, probably as a result of the time of incubation and the
concentration of the cytokines used. Similarly, IL-1
markedly
suppresses glucose-stimulated insulin secretion (10, 41). In contrast,
we found that incubation of islets from transgenic mice with leucine
plus glutamine or with arginine counteracted the IFN-
reduction of
glucose-stimulated insulin secretion. It is conceivable that a shift in
islet metabolism, favoring amino acid oxidation and decreasing glucose
metabolism, might be part of the metabolic responses of the
-cells
to an increase in IFN-
expression. Similar results have been
reported in islets cultured in the presence of IL-1
(10). Exposure
to IL-1
led to a marked reduction of glucose-stimulated insulin secretion, but treatment with arginine plus different glucose concentrations and leucine plus glutamine counteracted the
IL-1
-induced reduction of insulin release (10).
Glucose-stimulated insulin secretion is also subject to stimulatory and
inhibitory modulation by hormones and neurotransmitters (44). Glucagon
or glucagon-like peptide-1 (GLP-1) activates adenylate cyclase, leading
to an increase in the cellular cAMP concentration. However, the role of
cAMP in insulin secretion is not a primary one. Protein kinase A
activation potentiates, but does not initiate, insulin secretion (45).
After glucose plus forskolin incubation, a marked reduction in insulin
release and cAMP concentration was detected in islets cultured in the presence of IFN- and islets from transgenic mice expressing IFN-
compared with islets from control mice not treated with the cytokine. Similar inhibitory effects have been described in islets incubated with
IL-1
(41, 46). These findings suggest that both glucose- and
cAMP-mediated steps (or glucose potentiation of cAMP-mediated steps) in
insulin secretion are targets for the action of IFN-
or IL-1
.
The alterations in pancreatic -cell function resulting from the
local expression of IFN-
led to changes in serum parameters in the
transgenic mice. They showed mild hyperglycemia and hypoinsulinemia, and also altered glucose tolerance test. Moreover, IFN-
injected into healthy subjects led to an impairment of glucose tolerance (47).
These findings suggest that transgenic mice expressing IFN-
developed a prediabetic state, without progression to type 1 diabetes.
Similarly, no incidence of diabetes was detected in transgenic mice
expressing IFN-
in
-cells with a C57Bl/6 background (22).
However, IFN-
transgenic mice back-crossed to CD1 mice became
diabetic with a cumulative incidence of more than 50% (22). An
additional factor or a more susceptible genetic background might be
required to induce type 1 diabetes in transgenic mice expressing
IFN-
. In this regard, although in a small percentage (9%), IFN-
transgenic mice with a CD-1 genetic background spontaneously developed
overt diabetes. This low percentage might result from the fact that the
-cells from these transgenic mice produce human instead of mouse
IFN-
, and the human cytokine might be less effective in inducing the
immune response. However, results obtained in MLDS-treated transgenic
mice indicated that IFN-
expression exacerbated the toxic effect of
streptozotocin and led to type 1 diabetes. Expression of IFN-
in
islets of transgenic mice might have a synergistic action with other
cytokines, since islet expression of IFN-
, IL-6, and tumor necrosis
factor-
has been observed after MLDS treatment (48). Similarly, IL-1
administration promotes MLDS-induced insulitis in strains of mice
resistant to the effects of this treatment (49). Furthermore, it has
been reported that one line of transgenic mice expressing IFN-
in
the pancreas at levels sufficient to induce mild insulitis, but
insufficient to induce type 1 diabetes, show hyperglycemia when treated
with MLDS (48), indicating that IFN-
potentiates the effects of
streptozotocin in islets. Moreover, simultaneous treatment with MLDS
and the type I IFN inducer poly(I/C) exacerbates MLDS-induced insulitis in mice (48). In addition, poly(I/C) treatment has a diabetogenic effect in BB rats, either by inducing the development of diabetes in
the diabetes-resistant BB strain or by accelerating the onset of
diabetes in the diabetes-prone strain (48).
All the findings reported here indicate that IFN- may be directly
involved in the pathogenesis of type 1 diabetes. Thus, undesirable
effects of long term treatment with IFN-
to counteract other
diseases (like chronic hepatitis or multiple sclerosis) cannot be ruled
out. In this regard, it has been reported that long term therapeutic
use of type I IFNs induces autoimmunity (50-52) as well as the
appearance of type 1 diabetes after treatment with type I IFNs
(53-55), supporting the hypothesis that both IFN-
and IFN-
might
lead to diabetes mellitus.
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ACKNOWLEDGEMENTS |
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We thank T. E. Wagner for the
IFN--containing plasmid; R. Casamitjana for RIA insulin
determination assistance; R. Rycroft and I. Robbins for critical review
of the manuscript; and C. H. Ros, M. Moya, and A. Vilalta for
excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by Fondo Investigación Sanitaria Grant 95/1758, Direcciò General de Recerca, Generalitat de Catalunya Grant GRQ94-2013, and Fundación Ramón Areces, Spain.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.
Recipients of predoctoral fellowships.
§ Recipient of a postdoctoral fellowship from INSERM, France.
¶ Recipient of Postdoctoral Fellowship 1995SGR00518 from Direcciò General de Recerca, Generalitat de Catalunya.
To whom correspondence should be addressed. Tel.:
34-3-5811043; Fax: 34-3-5812006; E-mail:
fatima.bosch{at}blues.uab.es.
1 The abbreviations used are: IL, interleukin; IFN, interferon; MHC, major histocompatibility complex; RIP-I, rat insulin I promoter; kb, kilobase pair(s); bp, base pair(s); RT-PCR; reverse transcriptase-polymerase chain reaction; RIA, radioimmunoassay; MLDS, multiple low doses of streptozotocin.
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REFERENCES |
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