1 Section for Molecular Signaling, Department of Cell and Molecular Biology, Lund University, SE-221 00 Lund; 2 Section for Neuroendocrine Cell Biology, Department of Physiological Sciences, Lund University, SE-221 85 Lund; 3 Section for Molecular and Cellular Physiology, Department of Physiological Sciences, Lund University, SE-223 62 Lund; 5 Dept. of Medicine at Malmö University Hospital, Lund University, SE-205 02 Malmo; and 4 Dept. of Medical Biochemistry, Göteborg University, SE-405 30 Goteborg, Sweden
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ABSTRACT |
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To examine
whether islet amyloid polypeptide (IAPP), other than through amyloid
formation, may be of importance in diabetes pathogenesis,
IAPP-deficient mice
(IAPP/
) were
challenged with alloxan (day 0). Diabetes in
IAPP
/
mice was more
severe at day 35, indicated by greater weight loss; glucose
levels were higher in alloxan-treated
IAPP
/
mice, whereas
insulin levels were lower, indicating a greater impairment of islet
function. Accordingly, glucose levels upon intravenous glucose
challenges at days 7 and 35 were consistently higher in
alloxan-treated IAPP
/
mice. At day 35, insulin mRNA expression, but not
-cell
mass, was lower in untreated
IAPP
/
mice. Yet, upon
alloxan administration,
-cell mass and numbers of
-cell-containing islets were significantly more reduced in IAPP
/
mice.
Furthermore, they displayed exaggerated
-cell dysfunction, because
in their remaining
-cells, insulin mRNA expression was significantly
more impaired and the localization of glucose transporter-2 was
perturbed. Thus the lack of IAPP has allowed exaggerated
-cell cytotoxic actions of alloxan, suggesting that there may be beneficial features of IAPP actions in situations of
-cell damage.
gene knockout; -cell mass; insulin messenger ribonucleic acid; glucose transporter-2; glucose tolerance
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INTRODUCTION |
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ISLET AMYLOID POLYPEPTIDE (IAPP, also designated
amylin) is a normal constituent of pancreatic -cells in nearly all
species examined so far (21). Given this conserved expression, as well as the regulation of IAPP expression (19) and secretion (14) by
glucose, a regulatory role for IAPP in glucose homeostasis is likely.
However, despite considerable efforts to clarify this issue, which
include showing that IAPP inhibits insulin release (1) and peripheral
glucose utilization (8), a clear physiological role of the peptide has
yet to be revealed. Nevertheless, recent findings in mice with a
targeted disruption of the IAPP gene (9) favor the idea that IAPP under
normal conditions indeed acts in islets, inhibiting insulin release,
and possibly also in the periphery, restraining insulin-stimulated
glucose utilization. Thus the physiological relevance of the previously
found metabolic actions of IAPP has been reinforced, and it appears
likely that IAPP is an insulin counterregulatory
-cell hormone.
Previously, the debate on the role of IAPP in the pathogenetic events
leading to non-insulin-dependent diabetes mellitus (NIDDM) has focused
on the amyloid-forming capacity of the peptide (13). Whether the
metabolic effects or other actions of IAPP could also play a role in
the pathogenesis of diabetes is not known. Nonetheless, perturbed
glucose-stimulated insulin secretion and insulin resistance are both
hallmarks of NIDDM, and the dual insulin-antagonistic effects of IAPP
(1, 8) could contribute to both these aspects of the disease.
Interestingly, under diabetic conditions in rodents, IAPP is
overexpressed in islets compared with insulin (20), an overexpression
that is matched by an increased ratio of IAPP to insulin for peptide
content in the pancreas (18) and secretion (11). It is therefore
conceivable that the insulin-antagonizing metabolic effects of IAPP
will be pronounced by such an overexpression. This could be harmful in
individuals at risk of developing NIDDM and further contribute to the
development of the disease. Alternatively, the possibility also remains
that overexpression of IAPP could be a beneficial adaptation to islet
perturbations in the development of diabetes. In this scenario, there
are several putative mechanisms by which IAPP could serve to protect
-cells under hyperglycemic and/or diabetic conditions; these include
enhancement of islet microcirculation (28) and limitation of prolonged
-cell depolarization (31). To explore these issues, it would be
desirable to examine the onset and course of experimental diabetes when
the effects of IAPP are eliminated. The feasibility, however, of such
studies is hampered by the lack of the identity of an IAPP receptor(s) and, consequently, reliable IAPP antagonists. However, the recent generation of IAPP-deficient mice
(IAPP
/
) (9) allowed
us to investigate whether a lack of IAPP will affect the development of
diabetes. To this end, we used the
-cell-specific cytotoxic agent
alloxan, a high dose of which induces permanent diabetes in mice.
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MATERIALS AND METHODS |
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Experimental animals, glucose and insulin determinations, and
tissue processing.
As described elsewhere (9), by use of targeted mutagenesis in embryonic
stem cells, the mouse IAPP gene was disrupted by deletion of a major
part of exon 3, which encodes the mature peptide (7). The deficient
IAPP expression in
IAPP/
mice was
confirmed by the lack of exon 3 in mouse tail DNA subjected to Southern
hybridization and by the lack of IAPP-like immunoreactivity in
pancreatic islets (9) (see Fig. 5, C and D).
In situ hybridization. Insulin mRNA was detected and quantitated in pancreatic sections, using [35S]dATP-labeled 30-mer deoxyribonucleotide probes for in situ hybridization, as previously described in detail (18, 20). Briefly, longitudinal sections of the entire pancreases were prepared to avoid any regional bias; two sections were cut from different depths of the specimens. The sections were deparaffinized, rehydrated, and permeabilized. Before hybridization, the sections were incubated in proteinase K followed by acetic anhydride. Hybridization overnight at 37°C was followed by stringent posthybridization washing. The slides were dipped in autoradiographic photoemulsion and developed after 4 days. Insulin mRNA levels were determined by measuring the mean optical density (OD) of probe labeling in islets with Quantimet Q500MC 1.1 (Leica Cambridge, Cambridge, UK), as previously described in detail (20). Briefly, dark-field images of islets were captured and digitized; the polarity of the images was reversed. Before analysis, the system was calibrated to a standard section, and the grey levels were converted to ODs; all sections were analyzed under identical conditions. The total outline of the probe-labeled cells in each individually analyzed islet was interactively defined, and the mean OD of labeling within that defined area was measured. Data from 10 mice per group were collected; 8.9 ± 0.4 islets per animal were analyzed.
Immunocytochemistry. Single or double indirect immunofluorescence was used (22). Polyclonal antibodies to rat IAPP (486; a kind gift from Dr. D. T. Stein, Southwestern Medical Center, Dallas, TX), to human proinsulin (9003; Euro-Diagnostica, Malmö, Sweden), and to glucose transporter-2 (GLUT-2; AB 1342; Chemicon, Temecula, CA) were employed. Briefly, sections were incubated with primary antibodies at 4°C, either overnight or sequentially for 2 nights (single and double staining, respectively), followed by incubation with FITC- and/or tetramethyl rhodamine isothiocyanate-coupled secondary antibodies. Changing the microscope filters allowed localization of two primary antibodies in an islet.Morphometry.
The total number of islet profiles in sections processed for in situ
hybridization was manually determined; an islet was defined as one or
more cells labeled by the insulin mRNA probes. When this approach is
employed, differences in -cell mass will impact the probability of
identifying insulin mRNA-labeled islets in sections. From each animal
(n = 10 in each group), two sections cut at different
depths were analyzed; an average number for each animal was calculated.
In addition, by use of Quantimet Q500MC 1.1,
-cell mass was assessed
by measuring the area of the total outline of probe-labeled cells in
each islet analyzed for insulin gene expression. Thus an average area
reflecting
-cell mass for each animal (n = 10/group) was
calculated; 8.9 ± 0.4 islets per animal were measured.
Statistical measures.
Means ± SE for each parameter are given. The statistical measures
used to analyze the data are given in the legend to the respective
figures. Mean OD, area of the total outline of probe-labeled cells, and
number of -cell-containing islets were compared by the
Kruskal-Wallis test followed by Dunn's post hoc test. To statistically evaluate the percent change in these parameters between
IAPP+/+ and
IAPP
/
mice after
alloxan treatment, each data point in the alloxan-treated mice was
expressed as a percentage of the mean value in the respective control
group; these percentages were compared with a one-tailed Mann-Whitney
U-test. The insulin content was compared with a two-tailed Student's t-test. A probability level of P < 0.05 was considered statistically significant.
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RESULTS |
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Body weight and basal plasma glucose and insulin levels.
The alloxan-treated
IAPP/
mice lost
significantly more weight than the age-matched wild type controls (from
34.2 ± 1.0 to 26 ± 1.3 g vs. from 32 ± 1.0 to 30.2 ± 1.2 g;
P < 0.001 for
-values), indicating that they were
metabolically more severely affected by alloxan. As shown in Fig.
1A, this was further corroborated by the basal plasma glucose levels after alloxan treatment, which were
more elevated in the
IAPP
/
mice than in
IAPP+/+ mice. Basal plasma insulin levels determined at the
same time points (Fig. 1B) were consistently lower in the
IAPP
/
mice after
alloxan treatment compared with the alloxan-treated wild type mice,
despite the higher glucose levels in the
IAPP
/
mice, thus
indicating a more extensive impairment of
-cell function. It should
also be noted that there was a trend toward lower basal plasma insulin
levels in IAPP
/
mice
before alloxan treatment (Fig. 1B). A similar finding was made
in additional untreated mice that were analyzed; here, basal plasma
insulin levels were 165 ± 8 pmol/l in
IAPP
/
mice vs. 191 ± 10 pmol/l in IAPP+/+ mice (P = 0.060).
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Intravenous glucose tolerance test.
To further explore the nature of the -cell impairment, IVGTTs at
days 7 and 35 after alloxan injection were performed
(Fig. 2). At both time points, the control
IAPP
/
mice eliminated
glucose faster than the IAPP+/+ mice. This finding is in
agreement with our previous data (9). In contrast, the alloxan-treated
IAPP
/
mice had lost
their enhanced elimination of glucose, compared with the
alloxan-treated IAPP+/+ mice; at both time points after
alloxan treatment, the basal glucose level immediately before glucose
injection was higher in the
IAPP
/
mice and was
still higher at 2 h compared with the IAPP+/+ mice. Thus,
taken together, the basal levels of glucose and insulin and the results
from the glucose challenges demonstrate that alloxan-treated IAPP
/
mice display a
more extensive impairment of
-cell function and, consequently,
exaggerated diabetes compared with their wild type controls.
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Insulin gene expression and storage.
To examine the cellular events underlying the -cell impairment, we
evaluated insulin mRNA expression in islets, using quantitative in situ
hybridization. In control mice, the mean OD of in situ hybridization
labeling was significantly higher in IAPP+/+ mice compared
with IAPP
/
mice
(Figs. 3 and
4). The lower expression of insulin in
IAPP
/
mice suggests
that insulin is synthesized at a lower rate in the absence of IAPP. To
examine whether this reduced insulin mRNA expression correlates with
reduced storage of the hormone, we determined the pancreatic insulin
content in untreated
IAPP
/
and
IAPP+/+ mice, age-matched with the alloxan-treated
IAPP
/
and
IAPP+/+ mice and their controls in the study. Indeed, the
pancreatic insulin content was lower in untreated
IAPP
/
mice than in
their wild type controls (1.0 ± 0.1 vs. 1.4 ± 0.1 nmol/mg;
P = 0.029).
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Immunocytochemistry.
To further confirm the results of the analysis of insulin mRNA
expression, we examined expression of islet-cell constituents at the
protein level with immunocytochemistry. In control IAPP+/+
and IAPP/
mice, the
number of islets and
-cells visibly appeared to be similar (Fig.
5, A, B, and D). As
expected, IAPP-immunoreactive cells were found only in the wild type
mice (Fig. 5, A and C). However, in the alloxan-treated
IAPP
/
mice, islets
containing
-cells appeared to be scarcer and to harbor fewer such
cells than in the alloxan-treated IAPP+/+ mice (Fig.
5, E and F).
|
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Morphometry.
To further evaluate the differential impact of alloxan in the
IAPP+/+ and
IAPP/
mice, we
determined their numbers of
-cell-containing islets and
-cell
mass. The lack of IAPP appeared not to have affected the number of such
islets, because in sampled pancreatic sections from both untreated
groups, the number of islets containing insulin mRNA was similar (Fig.
7A). After alloxan treatment, the
number of
-cell-containing islets was reduced in both groups of mice (Fig. 7A); the number of such islets, however, was
significantly lower only in the
IAPP
/
mice; a
reduction to 43 vs. 63% of respective untreated control was seen, and
this reduction was significantly greater in
IAPP
/
mice.
Accordingly, as reflected by area of
-cells labeled for insulin
mRNA,
-cell mass was not significantly different in the control
IAPP+/+ and
IAPP
/
mice (Figs. 3,
A and B, and 7B). Alloxan treatment induced a significant reduction in
-cell mass in both strains of mice (Figs. 3, C and D, and 7B); this reduction to 31 vs.
38% of respective untreated control, however, was again significantly
greater in IAPP
/
mice, as indicated by comparison of percent changes. These findings agree with our observations on insulin-immunoreactive cells (Fig. 5)
and collectively demonstrate that diabetes in the mice was caused by
reduced
-cell mass. Moreover, because both the reduction of
-cell
mass and the signs of
-cell dysfunction are exaggerated in
IAPP-deficient mice, islets lacking IAPP appear to be more vulnerable
to alloxan.
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DISCUSSION |
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There are now two phenomena that may link IAPP to the pathogenesis of
diabetes. First, amyloid deposits are formed from the peptide in islets
from subjects with NIDDM (13). Amyloid formation could be causally
linked to impairment of -cell function, because in monkeys it
precedes the development of diabetes (6) and, in vitro, human IAPP
fibrils induce apoptosis in
-cells (16) and create pores in planar
phospholipid bilayers (17). If amyloid formation, on the other hand, is
a sequel to
-cell perturbation, it may interfere with nutrition of
islet cells and their release of islet hormones and thereby worsen the
condition of the disease. The possibility, however, remains that
amyloid formation is an unharmful event coupled to aging of
-cells;
islet amyloid is also found in elderly nondiabetic individuals (13).
Nevertheless, the findings of islet amyloid and hyperglycemia in
transgenic mice with a targeted overexpression of human IAPP to
-cells again highlight the possibility that IAPP is indeed involved
in the pathogenesis of NIDDM (30).
Second, because IAPP has previously been shown to restrain insulin
release (1) and action (8), metabolic actions of the peptide may be
harmful in diabetes. Hence, the overexpression of IAPP compared with
insulin observed in experimental models of diabetes in rodents could
further contribute to pathogenetic events in the disease (20).
Therefore, it was anticipated that IAPP deficiency in mice would
ameliorate alloxan-induced diabetes. To our surprise, we found that
IAPP-deficient mice developed a more severe form of diabetes when
challenged with alloxan; the basal glucose levels were consistently
higher in alloxan-treated IAPP/
mice, whereas
basal insulin levels were lower. Also, the previously observed
enhancement of glucose elimination under normal conditions in
IAPP
/
mice (9) was
reversed by alloxan treatment; although both groups of alloxan-treated
mice eliminated glucose poorly, after the glucose challenges glucose
levels were consistently higher in
IAPP
/
mice. The
morphological examination revealed that the underlying cause for this
more severe diabetes phenotype is lower
-cell mass and insulin gene
expression in IAPP-deficient mice after alloxan treatment. Moreover,
statistical comparisons of percent changes in
-cell mass, number of
-cell-containing islets, and insulin mRNA levels indicated that
these changes were significantly exaggerated in
IAPP
/
mice,
suggesting that islets lacking IAPP are more vulnerable to alloxan.
Because it is the lack of IAPP that separates
IAPP/
from
IAPP+/+ mice at the genetic level, it is reasonable to
assume that this lack is specifically responsible for the diabetes
phenotype in IAPP-deficient mice that we describe here. Along these
lines, it was recently shown that the targeted expression of calcitonin gene-related peptide (CGRP) to
-cells in nonobese diabetic mice prevents diabetes or decreases its incidence in such male and female
mice, respectively (15). Because IAPP and CGRP exert similar effects in
islets, possibly due to activation of the same receptors (27), it is
not surprising that
-cell-targeted overexpression of CGRP results in
a phenotype which, in effect, is an opposite of the exaggerated
diabetes phenotype associated with IAPP deficiency in our genetic
model. Moreover, in a transgenic mouse overexpressing human IAPP in
-cells, insulin mRNA expression and storage are increased (5, 29);
again, in this regard, this phenotype is the opposite of that which we
describe here, in which insulin mRNA expression and storage already
were lower in untreated IAPP-deficient mice compared with wild type
control mice. Taken together with our data, the experiments with
targeted expression of IAPP/CGRP to
-cells (5, 15, 29) argue that
the phenotypes of these mice, as well as that of the
IAPP
/
mice, are
specific for the overexpression or lack of these peptides. Moreover,
the previously described metabolic phenotype in male IAPP
/
mice, i.e., an
exaggerated insulin response to glucose, was corrected by a randomly
integrated
-cell-specific human IAPP transgene (9), again confirming
that it is the lack of IAPP that is responsible for the phenotype observed.
Several possible mechanisms may underlie the aggravated diabetes in the
IAPP-deficient mice. Khachatryan et al. (15) reason that a local
immunomodulatory action of CGRP prevents diabetes in their model. Local
immunomodulation may involve control of islet circulation. Indeed, IAPP
is known to act as a vasodilator (4) and has previously also been shown
to increase the fractional blood flow through islets (28). Lack of such
a blood flow increase in IAPP-deficient mice may impair islet
regeneration after the alloxan insult. IAPP has also been shown to
potently promote growth in cultured renal cells (10). Perhaps IAPP may
act as a growth factor in islets, an effect that is lacking in
IAPP-deficient mice and that could explain the persistence of impaired
islet function. Also, it has previously been suggested that IAPP may limit prolonged depolarization of -cells with ensuing elevation of
intracellular Ca2+ levels, because IAPP hyperpolarizes the
plasma membrane of patch-clamped
-cells (31). In IAPP-deficient
mice, lack of such action, which we assume serves to protect
-cells
from toxic effects of hyperglycemia, may aggravate
-cell damage in
diabetes. In addition, the
-cell cytotoxic actions of alloxan may be
associated with excessive cycling of Ca2+ through the
mitochondrial membranes, which eventually are damaged (26). This leads
to a decreased ability of mitochondria to retain Ca2+,
their subsequent uncoupling, and impairment of ATP production, depriving the
-cell of energy. Therefore, if one role for IAPP is to
reduce intracellular Ca2+ levels through membrane
hyperpolarization (31), lack of this mechanism in the IAPP-deficient
mice may potentiate the cytotoxic action of alloxan, presumably caused
by a combination of cellular energy deprivation and constant elevation
of intracellular Ca2+.
Because GLUT-2 is responsible for cellular uptake of both glucose and
alloxan (23), increased GLUT-2 expression could form the basis for both
the increased insulin response to glucose (9) and -cell
susceptibility to alloxan in
IAPP
/
mice. However,
immunocytochemistry revealed no differential expression of GLUT-2 at
the protein level in the untreated mice. Interestingly, a perturbed
cellular expression of GLUT-2 was found in the alloxan-treated IAPP
/
mice: although
GLUT-2 normally (and in alloxan-treated IAPP+/+ mice) is
localized to the plasma membrane (25), immunofluorescence in
alloxan-treated IAPP
/
mice indicated a cytoplasmic localization for GLUT-2. It has previously
been reported that GLUT-2 is localized to the cytoplasm of
-cells in
islets grafted to streptozotocin-diabetic rats (12). Moreover, it has
previously been demonstrated that perturbed islet expression of GLUT-2
in experimental models of diabetes, e.g., Zucker diabetic fatty rats
(24), accompanies progression of prediabetes to overt diabetes. Thus,
in IAPP-deficient mice, the perturbed GLUT-2 expression may indicate
-cell failure, because it is likely that GLUT-2 in the cytoplasm
will not function properly, hence impairing glucose responsiveness.
It has previously been shown that glucose hinders -cell cytotoxicity
conferred by alloxan (2), an effect conceivably due to a competition of
glucose with alloxan for GLUT-2-mediated transport into
-cells (23).
In addition, alloxan by itself is a
-cell secretagogue. This raises
the possibility that the IAPP-deficient mice may also exhibit an
exaggerated insulin response to alloxan, which would enhance glucose
elimination. Under such circumstances, lower ambient glucose may result
in reduced protection of
-cells to alloxan. Whether this holds true
in the present case is not known, but a way to circumvent this would be
to clamp plasma glucose during alloxan administration. Also, plasma
glucose and insulin levels could be determined upon administration of alloxan.
The reason for lower insulin mRNA expression and storage in the
IAPP-deficient mice, and whether they contribute to the exaggerated diabetes phenotype in
IAPP/
mice, are
unclear. It has been suggested that the increased insulin expression,
storage, and release in transgenic mice overexpressing human IAPP may
represent an adaptation to impaired insulin sensitivity (5, 29),
because IAPP has previously been implicated in such impairment (8).
Interestingly, female
IAPP
/
mice display a
similar enhancement of glucose disposal to that in male
IAPP
/
mice (9) but
lack the exaggerated glucose-stimulated insulin response; this suggests
that the lack of IAPP in these mice confers an enhanced sensitivity to
insulin. If this holds true, lower insulin mRNA expression and storage
in the IAPP-deficient mice could have evolved in response to enhanced
sensitivity to insulin. It is possible that such enhanced sensitivity
may be of little importance in mice after alloxan treatment and that
the lower insulin mRNA expression and storage in the IAPP-deficient
mice will make them more susceptible to the
-cell cytotoxic actions of alloxan.
The increased susceptibility to alloxan in
IAPP/
mice is also of
interest in light of the emerging concept of
-cell rest as a means
of hindering future diabetes development (3). This would infer that the
increased susceptibility to alloxan in
IAPP
/
mice to some
extent may be due to a lack of
-cell inhibition (1, 9) caused by the
IAPP deficiency. This, however, needs to be examined in more detail.
In conclusion, we have found that IAPP-deficient mice display a more
severe diabetes phenotype than wild type mice upon a challenge with
alloxan, due to a greater impairment of -cell function. This
impairment is explained by lower
-cell mass and insulin expression
in the IAPP-deficient mice after alloxan treatment compared with the
alloxan-treated wild type mice. Because these perturbations were
significantly greater in the IAPP-deficient mice, lack of IAPP from
islets may confer an increased susceptibility to the diabetogenic
actions of alloxan. In addition, lower insulin mRNA expression and
storage in IAPP-deficient mice may be a contributing factor in the
development of diabetes in such mice. The mechanism(s) underlying the
observed phenomenon conceivably involves local actions of IAPP.
Unraveling of these is likely to shed new light on the pathogenesis of diabetes.
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ACKNOWLEDGEMENTS |
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We thank Lena Kvist, Ulrika Gustavsson, Lilian Bengtsson, Doris Persson, and Eva Hansson for technical assistance.
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FOOTNOTES |
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This study was supported by the Swedish Medical Research Council (Project nos. 12X-4499 and 14X-6834), the Swedish Cancer Research Fund, the Swedish Diabetes Association, the Swedish Society for Medical Research, the Swedish Society of Medicine, Göteborg Medical Society, Novo Nordisk, the Inga-Britt and Arne Lundberg, Albert Påhlsson, Trygg Hansa Research, Åke Wiberg, and Crafoord Foundations, and by the Faculty of Medicine at the Universities of Lund and Goteborg.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Mulder, Gifford Labs. for Diabetes Research, Dept. of Biochemistry and Internal Medicine, Southwestern Medical School, University of Texas at Dallas, Room Y8.214, 5323 Harry Hines Blvd., Dallas, TX 75235-8854 (E-mail: mulder{at}utsw.swmed.edu).
Received 24 May 1999; accepted in final form 25 October 1999.
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