From the Department of Internal Medicine, Graduate
School of Medicine, University of Tokyo, Tokyo 113-8655, Japan;
§ Core Research for Evolutional Science and Technology
(CREST), Japan Science and Technology Corporation (JST), Kawaguchi
332-0012, Japan; ¶ Division of Laboratory Animal Science, Animal
Research Center, Tokyo Medical University, Tokyo 160-8402, Japan;
Laboratory for Vertebrate Body Plan, Center for Developmental
Biology, RIKEN, Kobe 650-0047, Japan; and ** Department of
Biochemistry, Dartmouth Medical School, Hanover, New Hampshire
03755
Received for publication, October 29, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Although we and others have generated
IRS-2 knock-out (IRS-2 The pathogenesis of type 2 diabetes involves complex interactions
among multiple physiological defects. Transgenic and knock-out technology to create animal models of type 2 diabetes have made a major
contribution to assessing the function of newly identified molecules
implicated in the regulation of in vivo glucose homeostasis (1, 2). Insulin receptor substrate-1
(IRS-1)1 was originally
identified as the major substrate of the insulin receptor and
insulin-like growth factor-1 receptor tyrosine kinases (3-5)
and represents the prototype for the IRS family of proteins (6-8). To
clarify the physiological roles of IRS-1 in vivo, we (9) and
others (10) have created mice with a targeted disruption of the IRS-1
gene locus. Although homozygous IRS-1-deficient mice (IRS-1 To investigate the role of IRS-2 in vivo, White's group
(15) and we ourselves (16) have generated IRS-2-deficient mice. The
phenotype of these mice was strikingly different from that of
IRS-1 IRS-1 and IRS-2 are required for normal growth and glucose homeostasis
in mice. To determine whether IRS-3 (for review see Ref. 7), one of the
insulin receptor substrates mainly expressed in adipose tissues, is
also involved in the regulation of these processes, Liu et
al. (19) generated mice with a targeted disruption of the
IRS-3 gene. Homozygous IRS-3 knock-out
(IRS-3 To assess the effect of genetic background and also ablation of IRS-3
on IRS-2 Animals and Determination of
Genotype--
IRS-2
Genotype was determined using PCR methods. Genomic DNA was extracted
from the tip of the tail. Primers and PCR conditions for genotyping of
IRS-2 were as follows. The sense primer was 5'-GAAGACAGTGGGTACATGCGAATG-3', and the antisense primer was
5'-CCTCATGGAGGAAGGCACTGCTG-3' from the IRS-2 gene. The sense
primer was 5'-TTCTATCGCCTTCTTGACGAGTTC-3' from a neomycin resistance
gene. These three primers and a genomic DNA template were mixed in a
tube. The thermal cycle reaction consisted of 94 °C for 5 min
followed by 35 cycles of 94 °C (1 min), 60 °C (1 min), 72 °C
(1 min), and then 72 °C for 5 min. The wild-type allele gave 600 base pairs, and the recombinant allele gave 450 base pairs. Primers and
PCR conditions for IRS-3 genotyping were described
previously (19).
Backcrossing of IRS-2 Knock-out Animals with Mice of Strain
129/Sv or C57Bl/6--
We backcrossed
IRS-2 In Vivo Glucose Homeostasis--
Glucose tolerance test is as
follows. Mice were fasted for >16 h before the study. They were then
loaded with 1.5 mg of glucose/gram body weight by intraperitoneal
injection. Blood samples were taken at different time points from the
tail vein, and glucose was measured using an automatic glucometer
(Glutest Pro, Sanwa Chemical Co., Nagoya, Japan). Serum insulin levels
were determined using an insulin radioimmunoassay kit (BIOTRAK,
Amersham Biosciences) with rat insulin as the standard (10, 21).
Insulin tolerance test is as follows. Mice were allowed free access to
food and then were fasted during the study. They were intraperitoneally
challenged with 0.75 milliunits of human insulin (Novolin R, Novo
Nordisk) per gram body weight. Venous blood samples were drawn at
different time points (10, 22).
Histological and Immunohistochemical Analysis of
Islets--
Isolated pancreata from 20-week-old mice were
immersion-fixed in Bouin's solution at 4 °C overnight. Tissues were
routinely processed for paraffin embedding, and 2-µm sections were
cut and mounted on silanized slides. Sections were
triple-stained with anti-insulin (1:200), anti-glucagon (1:200), and a
mixture of anti-somatostatin (1:800) and anti-pancreatic polypeptide
(1:800) antibodies (all from DAKO Japan Co., Ltd., Japan). Images of
pancreatic tissues, Islet Isolation and Analysis of Insulin Content and Secretion--
To assess insulin
content, isolated islets were extracted in acid ethanol at Statistical Analysis--
Results were expressed as means ± S.E. (n). Statistical analysis was performed using a
Statview software system (Abacus Concepts Inc., Berkeley, CA).
Statistical differences were analyzed using the Student's t
test for unpaired comparisons. A p < 0.05 value was
considered statistically significant.
Generation of IRS-2 20-30% IRS-2 IRS-2 Markedly Decreased Fat Mass in IRS-2 IRS-3 Insulin-positive Expressions of Pdx1 and GLUT2 in Islets Were Decreased to Nearly
Undetectable Levels in IRS-2 Increased Insulin Secretion in IRS-2 Increased Susceptibility to Diabetes in IRS-2 Although we (16) and others (15) have generated
IRS-2 We found that 20-30% F2 mice carrying a null mutation for the
IRS-2 gene irrespective of the IRS-3 genotype
developed diabetes at 10-20 weeks of age (Fig. 2A).
However, it should be noted that IRS-2 A recent report (28) from another laboratory indicated Pdx1 expression
to be reduced in IRS-2 In summary, IRS-3 does not compensate for the loss of IRS-2 in
maintaining glucose homeostasis and the severity of diabetes in
IRS-2/
) mice, significant
differences were seen between the two lines of
IRS-2
/
mice in the severity of diabetes and
alterations of
-cell mass. It has been reported that although IRS-1
and IRS-3 knock-out mice showed normal blood glucose levels,
IRS-1/IRS-3 double knock-out mice exhibited marked hyperglycemia. Thus,
IRS-1 and IRS-3 compensate each other's functions in maintaining
glucose homeostasis. To assess the effect of genetic background and
also ablation of IRS-3 on IRS-2
/
, we generated
IRS-2/IRS-3 double knock-out
(IRS-2
/
IRS-3
/
) mice by
crossing IRS-3
/
mice (129/Sv and C57Bl/6
background) with our IRS-2
/
mice (CBA
and C57Bl/6 background). Intercrosses of
IRS-2+/
IRS-3+/
mice yielded
nine genotypes, and all of them including
IRS-2
/
IRS-3
/
mice were
apparently healthy and showed normal growth. However, at 10-20 weeks
of age, 20-30% mice carrying a null mutation for the
IRS-2 gene, irrespective of the IRS-3 genotype,
developed diabetes. When mice with diabetes were excluded from the
analysis of glucose and insulin tolerance test,
IRS-2
/
IRS-3
/
showed a
degree of glucose intolerance and insulin resistance similar to those
of IRS-2
/
mice. Both
IRS-2
/
and
IRS-2
/
IRS-3
/
mice had
moderately reduced
-cell mass despite having insulin resistance.
Insulin-positive
-cells were decreased to nearly zero in
IRS-2
/
mice with diabetes. Although Pdx1 and
glucose transporter 2 expressions were essentially unaltered in islets
from IRS-2
/
mice without diabetes, they were
dramatically decreased in IRS-2
/
mice with
diabetes. Taken together, these observations indicate that IRS-3 does
not play a role compensating for the loss of IRS-2 in maintaining
glucose homeostasis and that the severity of diabetes in
IRS-2
/
mice depends upon genetic background,
suggesting the existence of modifier gene(s) for diabetes in mice of
the 129/Sv genetic strain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice) were insulin-resistant, they
maintained normal glucose tolerance via compensatory
-cell
hyperplasia (9, 11). Subsequently, Kahn's group and we ourselves
proposed that IRS-2 (pp190), another insulin receptor substrate, may
play important roles in insulin action, particularly in the liver
(12-14).
/
mice. Thus, homozygous IRS-2-deficient
mice (IRS-2
/
mice) progressively developed
diabetes. IRS-2
/
mice were insulin-resistant as
a result of insulin resistance in the liver but not in skeletal muscle,
and the
-cell mass in IRS-2
/
mice was reduced
to 83% of that in wild-type mice, which was in marked contrast to the
85%
-cell mass increase in IRS-1
/
mice (16).
Thus, liver insulin resistance together with a lack of compensatory
-cell hyperplasia caused diabetes in IRS-2
/
mice. Interestingly, the degree of hyperglycemia was much milder in our
IRS-2
/
mice than in the
IRS-2
/
mice generated by White's group (15,
16). Our IRS-2
/
mice had fasting plasma glucose
(FPG) levels of around 120 mg/dl at 10-16 weeks of age (16), which is
clearly lower than the 350-400 mg/dl at 16 weeks of age in their
IRS-2
/
mice (15). It is also noteworthy that the
-cell mass of our IRS-2
/
mice was reduced to
83% at 6 weeks of age and 51% at 12 weeks of age, respectively, of
that in wild-type mice, which is clearly milder than the 59% reduction
at 4 weeks of age and the 90%~ reduction at 4 months of age,
respectively, in their IRS-2
/
mice (15-17). The
molecular basis for these apparent differences is unclear. However, it
is possible that differences in either genetic background or
environmental factors such as chow may affect phenotypic expression in
IRS-2
/
mice. In this respect, it should be noted
that these quantitative differences in the
IRS-2
/
mice may suggest the existence of a major
modifier gene as was previously reported in
IR+/
IRS-1+/
mice (18).
/
) mice showed normal body weight
throughout development, normal blood glucose and insulin levels, and
normal glucose transport in adipocytes. However, important roles of
IRS-3 in adipocytes and potentially in
-cells may be masked via
compensation by either IRS-1 or IRS-2 in these tissues. In fact, it has
been reported that whereas both IRS-1
/
and
IRS-3
/
mice showed normal FPG, IRS-1/IRS-3
double knock-out mice were marked hyperglycemic (20). Thus, IRS-1 and
IRS-3 compensate for each other's functions in maintaining glucose homeostasis.
/
, we generated IRS-2/IRS-3 double
knock-out (IRS-2
/
IRS-3
/
)
mice by crossing IRS-3
/
mice (129/Sv and C57Bl/6
background) with our IRS-2
/
mice (CBA and
C57Bl/6 background). Intercrosses of
IRS-2+/
IRS-3+/
mice yielded
nine genotypes.
IRS-2
/
IRS-3
/
mice were
viable and showed normal growth. At 10-20 weeks of age, 20-30% mice
carrying a null mutation for the IRS-2 gene developed diabetes. When mice with diabetes were excluded from the analyses of
glucose and insulin tolerance test results,
IRS-2
/
IRS-3
/
mice showed
a degree of glucose intolerance and insulin resistance similar to those
of IRS-2
/
mice, indicating that IRS-3 does not
compensate for the loss of IRS-2 in maintaining glucose homeostasis.
The severity of diabetes in IRS-2
/
mice was
found to be dependent upon genetic background, suggesting the existence
of modifier gene(s) for diabetes in mice of the 129/Sv genetic strain.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice had been
maintained on the original C57BL/6 and CBA hybrid background (16).
IRS-3
/
mice had been maintained on the C57BL/6
and 129/Sv hybrid background (19). IRS-2+/
IRS-3+/
mice were prepared by crosses of
IRS-3
/
male mice and
IRS-2+/
or IRS-2
/
female
mice. IRS-2+/
IRS-3+/
mice
were viable and obtained with the expected Mendelian frequency. IRS-2+/
IRS-3+/
mice were
fertile and were intercrossed to obtain progeny of all combinations of
IRS-2 and IRS-3 deletions. All of the mice were
kept on a 12 h of light period followed by 12 h of dark period cycle. All of the experiments in this study were performed using male mice except in the situation in which female mice were analyzed.
/
animals with 129/SvEvTaconic (Taconic
Farm, Germantown, NY) or C57Bl/6J (CLEA Japan Co., Ltd., Japan) mice.
To date, we have obtained N7 generations with the respective backgrounds.
-cells,
-cells, and
plus PP cells were
captured on the monitor screen of a computer through a microscope
connected to a CCD camera (Olympus Co., Ltd., Tokyo, Japan) as
described previously (10, 23). The areas of pancreata,
-cells,
-cells, and
plus PP cells were traced manually and analyzed with
Win ROOF software (Mitani Co., Ltd., Tokyo, Japan). The masses of
-cells,
-cells, and
plus PP cells were calculated as the
proportion of the respective area to the area of the whole pancreas as
described previously (10, 16). >50 islets were analyzed per mouse
in respective groups. Immunostaining with anti-duodenal homeobox factor-1 (Pdx1) antibody (24) was performed as described elsewhere (25). Rabbit anti-glucose transporter 2 (GLUT2) antibody was purchased
from Chemicon International, Inc. (Temecula, CA).
-Cell Preparation--
Islets were
isolated as described previously (26, 27). After clamping the common
bile duct at a point close to the duodenal outlet, 2.5 ml of
Krebs-Ringer bicarbonate buffer (129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM
CaCl2, 5 mM NaHCO3, and 10 mM HEPES at pH 7.4) containing collagenase (Sigma) was injected into the duct. The swollen pancreas was taken out and incubated at 37 °C for 3 min. The pancreas was dispersed by
pipetting and washed twice with Krebs-Ringer bicarbonate buffer. Islets were collected manually.
20 °C
for measurement of insulin content by RIA. Insulin secretion from
islets was measured using Krebs-Ringer bicarbonate buffer with a basal
glucose concentration of 2.8 mM unless otherwise stated.
Static incubation was performed with 10 islets/tube at 37 °C for
1 h after preincubation with the basal glucose concentration for
20 min (26, 27). Insulin levels were determined using an insulin
radioimmunoassay kit with rat insulin as the standard.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
IRS-3
/
Mice--
Intercrosses of
IRS-2+/
IRS-3+/
mice yielded
nine genotypes. Of 365 male offspring, 18 IRS-2
/
IRS-3
/
males were
viable (Fig. 1A). The
proportion of mice carrying a null mutation for the IRS-2
gene was similar to that expected (IRS-2
/
IRS-3+/+, 6.6 actual
versus 6.25% expected;
IRS-2
/
IRS-3+/
, 8.0 actual
versus 12.5% expected;
IRS-2
/
IRS-3
/
, 4.9 actual
versus 6.25% expected). All of the murine genotypes including IRS-2
/
IRS-3
/
were apparently healthy and showed normal growth. At 6 weeks of age,
there were no differences in body weight (Fig. 1B) or FPG
levels (Fig. 1C) among the nine genotypes, although mice
carrying a null mutation for the IRS-2 gene showed insulin
elevation (Fig. 1D).
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Fig. 1.
Generation of
IRS-2 /
IRS-3
/
mice. A, number of offspring derived from intercrosses
of IRS-2+/
IRS-3+/
. Body
weight (B), fasting blood glucose levels (C), and
serum insulin levels (D) in mice with the nine genotypes
derived from IRS-2+/
IRS-3+/
intercrosses at 6 weeks.
/
Male Mice Developed
Diabetes--
At 10-20 weeks of age, 20-30% male mice carrying a
null mutation for the IRS-2 gene, irrespective of the
IRS-3 genotype, developed "diabetes" (Fig.
2A). In this
paper, when FPG was elevated to >200 mg/dl, it was defined as
diabetes. Some of the mice showed FPG >300 mg/dl and body weight loss
and died because of dehydration. By contrast, female mice carrying a
null mutation for the IRS-2 gene neither developed diabetes
nor died by 20 weeks of age. Both male and female
IRS-2
/
mice of the CBA and C57Bl/6 strain never
developed such diabetes (15).
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Fig. 2.
IRS-2 /
IRS-3
/
mice showed glucose intolerance and insulin resistance similar to those
of IRS-2
/
mice. A,
fasting blood glucose levels at 10-20 weeks of age in the nine
genotypes derived from
IRS-2+/
IRS-3+/
intercrosses.
B, glucose tolerance test in 20-week-old wild-type mice,
IRS-3
/
mice, IRS-2
/
mice
without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Plasma glucose (upper panel) and serum insulin
levels (lower panel) were measured at the indicated time
points. Values are expressed as the means ± S.E. of the values
obtained from wild-type mice (circles, n = 6), IRS-3
/
mice (squares,
n = 10), IRS-2
/
mice
(diamonds, n = 11), and
IRS-2
/
IRS-3
/
mice
(triangles, n = 5). C, insulin
tolerance test in 30-week-old wild-type mice,
IRS-3
/
mice, IRS-2
/
mice
without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Mice were allowed food ad libitum and then given
0.75 milliunits of human insulin/gram of body weight. Values are
expressed as means ± S.E. of the values from wild-type mice
(circles, n = 7),
IRS-3
/
mice (squares,
n = 9), IRS-2
/
mice (diamonds,
n = 12), and
IRS-2
/
IRS-3
/
mice
(triangles, n = 6). D, serum-free
fatty acid levels in 30-week-old wild-type mice,
IRS-3
/
mice, IRS-2
/
mice
without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Values are expressed as means ± S.E. of the values
obtained from each group (n = 4). *, p < 0.05; **, p < 0.01 compared with wild-type mice; #,
p < 0.05; ##, p < 0.01 compared with
IRS-2
/
mice.
/
IRS-3
/
Mice Showed a Degree
of Glucose Intolerance and Insulin Resistance Similar to Those of
IRS-2
/
Mice--
We carried out glucose tolerance test
in wild-type mice, IRS-3
/
mice,
IRS-2
/
mice without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes (Fig. 2B). IRS-3
/
mice
showed glucose and insulin levels similar to those of wild-type mice.
IRS-2
/
IRS-3
/
mice showed
glucose intolerance similar to that of IRS-2
/
mice (Fig. 2B, upper panel).
IRS-2
/
mice showed ~1.5-fold higher insulin
levels than wild-type mice and IRS-3
/
mice.
IRS-2
/
IRS-3
/
mice showed
a 1.7- and a 1.9-fold higher insulin level than wild-type mice and
IRS-3
/
mice, respectively, but there were no
statistically significant differences between
IRS-2
/
and
IRS-2
/
IRS-3
/
mice. At 30 weeks of age, IRS-3
/
mice became slightly
insulin-resistant compared with wild-type mice as assessed by the
insulin tolerance test (Fig. 2C). When mice with diabetes
were excluded from the analysis,
IRS-2
/
IRS-3
/
mice showed
a degree of insulin resistance similar to that of IRS-2
/
mice. When we determined serum-free fatty
acid levels for the four genotypes at 20-30 weeks of age,
IRS-2
/
and
IRS-2
/
IRS-3
/
mice without
diabetes had lower free fatty acid levels of than wild-type and
IRS-3
/
mice (Fig. 2D).
/
Mice with
Diabetes--
We measured body weight (Fig.
3A), epididymal fat mass (Fig.
3B), and fed blood glucose levels (Fig.
3C) in wild-type mice, IRS-3
/
mice,
IRS-2
/
mice without diabetes,
IRS-2
/
IRS-3
/
mice without
diabetes, and IRS-2
/
mice with diabetes.
Although there were no differences in body weight among the nine
genotypes at 6 weeks of age (Fig. 1C),
IRS-2
/
and
IRS-2
/
IRS-3
/
mice were
significantly heavier than wild-type mice at 20 weeks of age (Fig.
3A). Moreover,
IRS-2
/
IRS-3
/
mice were
heavier than IRS-2
/
mice albeit not
significantly, presumably because of slightly higher insulin levels
than in IRS-2
/
mice (Fig. 3A).
IRS-2
/
mice with diabetes had body weights
similar to those of IRS-2
/
without diabetes
although they were lighter than
IRS-2
/
IRS-3
/
mice without
diabetes (Fig. 3A), but IRS-2
/
mice
with diabetes had a significantly smaller fat mass than IRS-2
/
or
IRS-2
/
IRS-3
/
mice without
diabetes (Fig. 3B).
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Fig. 3.
Markedly decreased fat mass in
IRS-2 /
mice with diabetes. Body
weight (A) and epididymal fat mass (B) were
determined in wild-type mice, IRS-3
/
mice,
IRS-2
/
mice without diabetes,
IRS-2
/
IRS-3
/
mice without
diabetes, and IRS-2
/
mice with diabetes. Values
are expressed as means ± S.E. of the values obtained from each
group (n = 4). C, fed blood glucose levels
in the respective groups. Values are expressed as the means ± S.E. of the values obtained from wild-type mice (n = 13), IRS-3
/
mice (n = 18),
IRS-2
/
mice without diabetes (n = 18), IRS-2
/
IRS-3
/
mice
without diabetes (n = 11), and
IRS-2
/
mice with diabetes (n = 5). *, p < 0.05; **, p < 0.01 compared with wild-type mice; #, p < 0.05; and ##,
p < 0.01 compared with IRS-2
/
mice without diabetes.
/
Knock-out Mice Had a Normal
-Cell
Mass--
Fig. 4 shows the results of
immunostaining of pancreatic islets from 20-week-old wild-type mice,
IRS-3
/
mice, IRS-2
/
mice
without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Both IRS-2
/
mice without diabetes and
IRS-2
/
IRS-3
/
mice without
diabetes had smaller islets than wild-type and
IRS-3
/
mice. Upon quantitation, the
-cell
mass in IRS-3
/
mice was similar to that of
wild-type mice. As we reported previously (16), the
-cell mass in
IRS-2
/
mice was reduced to ~50% of that in
wild-type mice. The
-cell amount in
IRS-2
/
IRS-3
/
mice was
similar to that in IRS-2
/
mice.
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Fig. 4.
Normal -cell mass in
IRS-3
/
mice and reduced
-cell mass in
IRS-2
/
mice without diabetes.
Histological analysis of pancreatic islets in 20-week-old wild-type
mice, IRS-3
/
mice, IRS-2
/
mice without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Pancreatic sections were triple-stained with anti-insulin,
anti-glucagon, and cocktails of anti-somatostatin and anti-pancreatic
polypeptide antibodies. Representative islet images captured on a
computer are shown. Bars indicate 100 µm.
-Cells Were Markedly Decreased to Nearly Zero
in IRS-2
/
Mice with Diabetes--
We next performed
histological analyses of pancreata from IRS-2
/
mice without diabetes and IRS-2
/
mice with
diabetes. Insulin-positive
-cells were dramatically decreased to
nearly zero in IRS-2
/
mice with diabetes (Fig.
5, compare C with A
and B).
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Fig. 5.
Pdx1 and GLUT2 expressions in islets were
normal in IRS-2 /
mice without diabetes
but markedly decreased to nearly undetectable levels in those with
diabetes. Histological analysis of pancreatic islets in wild-type
mice, IRS-2
/
mice without diabetes, and those
with diabetes at 20-30 weeks. A-C, pancreatic sections
were triple-stained with anti-insulin (brown), anti-glucagon
(red), and cocktails of anti-somatostatin and
anti-pancreatic polypeptide antibodies (blue).
D-F, pancreatic sections were double-stained with anti-Pdx1
(brown) and cocktails of anti-glucagon, anti-somatostatin,
and anti-pancreatic polypeptide antibodies (red).
G-I, pancreatic sections were stained with anti-GLUT2
antibody (red). Representative islet images captured on a
computer are shown. Bars indicate 100 µm.
/
Mice with
Diabetes--
Transcription factor duodenal homeobox factor-1 (Pdx1)
is known to regulate the expression of multiple genes such as insulin, glucokinase, and GLUT2 in
-cells, thereby maintaining normal
-cell function. Therefore, we further examined Pdx1 and GLUT2 expression in islets. Although the Pdx1 mRNA level was reported to
be reduced in IRS-2
/
islets as compared with
wild-type islets (28), its expression level was essentially unaltered
in islets from IRS-2
/
mice without diabetes
(Fig. 5E). Moreover, GLUT2 expression was preserved (Fig.
5H). Interestingly, however, IRS-2
/
mice with diabetes had markedly decreased Pdx1 and GLUT2 expressions in
-cells (Fig. 5, F and I). Their expressions
were severely decreased even in IRS-2
/
mice with
a blood glucose level of 200-300 mg/dl under fed conditions (data not shown).
/
and
IRS-2
/
IRS-3
/
Mice without
Diabetes--
We next studied islet function in 20-week-old wild-type
mice, IRS-3
/
mice, IRS-2
/
mice without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Islets from IRS-3
/
mice showed normal
insulin content and secretion (Fig. 6,
A and B). By contrast, there was a significant
reduction in insulin content per islet from
IRS-2
/
mice and
IRS-2
/
IRS-3
/
mice without
diabetes compared with wild-type mice (Fig. 6A). However,
when insulin content was normalized by cell number per islet, the
insulin content was essentially unaltered in
IRS-2
/
and
IRS-2
/
IRS-3
/
islets (data
not shown). These results were consistent with our previously reported
results (16). When glucose-induced insulin secretion was normalized by
islet insulin content, it was significantly higher at 11.1 mM glucose from IRS-2
/
and
IRS-2
/
IRS-3
/
islets than
that from wild-type islets (Fig. 6B).
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Fig. 6.
Increased insulin secretion in
IRS-2 /
without diabetes and
IRS-2
/
IRS-3
/
without diabetes. A, insulin contents per islet in
wild-type mice, IRS-3
/
,
IRS-2
/
without diabetes, and
IRS-2
/
IRS-3
/
mice without
diabetes. Values are expressed as the means ± S.E.
(n = 9). B, insulin secretion normalized by
insulin content per islet at 2.8 and 11.1 mM glucose.
Values are expressed as means ± S.E. (n = 4).
Similar results were obtained from two independent experiments. *,
p < 0.05; **, p < 0.01 compared with
wild-type mice.
/
Mice
of the 129/Sv Genetic Strain--
We noted that 20-30% of
F2 mice carrying a null mutation for the IRS-2 gene,
irrespective of the IRS-3 genotype, developed diabetes at
10-20 weeks of age (Fig. 2A). Fig.
7 shows the frequency of animals with
diabetes in each situation. IRS-2
/
mice of the
CBA and C57Bl/6 strain, which is our original genetic background, never
developed such diabetes (16). We therefore assumed that the difference
in blood glucose levels between our original
IRS-2
/
mice and other
IRS-2
/
animals was at least in part because of
the difference in genetic background. The results suggest the existence
of a modifier gene(s) for diabetes in mice of the 129/Sv genetic
strain. However, it is also possible that there are suppressor gene(s)
for the development of diabetes in the CBA murine genetic strain and
that IRS-3
/
animals with the 129/Sv and C57Bl/6
genetic background carry the responsible gene(s). To exclude these
possibilities and also to confirm our hypothesis that the
susceptibility to diabetes of IRS-2
/
animals
increases as the contribution of the 129/Sv genetic background increases, we backcrossed our original IRS-2
/
animals directly with 129/SvEvTaconic or C57Bl/6J mice. To date, we
have obtained N7 generations with the respective backgrounds. We
analyzed the phenotypes of offspring from N2 intercrosses of either the
129/Sv or the C57Bl/6J genetic background. Although three of six
IRS-2
/
animals with the 129/Sv background
developed diabetes by 20 weeks of age, none of >50
IRS-2
/
animals with the C57BL/6J background
developed diabetes (Fig. 7). These results are consistent with the idea
that the increased susceptibility to diabetes in
IRS-2
/
mice is because of a genetic contribution
of 129/Sv.
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Fig. 7.
Proportion of animals with diabetes among
offspring from N2 intercrosses with either the 129/Sv or the C57Bl/6J
genetic background. **, p < 0.01 compared with
IRS-2 /
mice derived from N2 intercrosses with
the 129/Sv background; ##, p < 0.01 compared with
those having the C57Bl/6 background.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice, significant differences in the
phenotypes of IRS-2
/
mice between the two groups
were seen in the severity of diabetes and alterations of
-cell mass.
We had assumed that differences in either genetic background or
environmental factors such as chow might affect the phenotypic
expression of IRS-2
/
mice. On the other hand, it
has been reported that whereas IRS-1
/
and
IRS-3
/
mice showed normal FPG, IRS-1/IRS-3
double knock-out mice were markedly hyperglycemic (20). Thus, IRS-1 and
IRS-3 compensate each other's functions in maintaining glucose
homeostasis. In this study, to assess the effect of genetic background
and also ablation of IRS-3 on IRS-2
/
mice, we generated
IRS-2
/
IRS-3
/
mice by
crossing IRS-3
/
mice (129/Sv and C57Bl/6
background) with our IRS-2
/
mice (CBA and
C57Bl/6 background).
IRS-2
/
IRS-3
/
mice showed
glucose tolerance (Fig. 2),
-cell mass (Fig. 4), and
-cell
function (Fig. 6) similar to those of IRS-2
/
mice. Thus, no major differences were found between
IRS-2
/
IRS-3
/
and
IRS-2
/
mice in contrast to the marked
differences between IRS-1/IRS-3 double knock-out mice and
IRS-1
/
or IRS-3
/
single
knock-out mice (20). We attributed this to the difference in tissue
distribution of IRS proteins. Thus, IRS-1 is abundantly expressed in
skeletal muscle and adipose tissues, and IRS-2 is abundantly expressed
in the liver, whereas IRS-3 is predominantly expressed in adipose
tissues. Therefore, IRS-1/IRS-3 double knock-out leads to nearly a
total loss of function of IRS proteins in adipose tissue. By contrast,
because major tissues in which IRS-2 and IRS-3 are expressed are
different and IRS-1 can compensate for the loss of functions,
IRS-2/IRS-3 double knock-out mice showed phenotypes similar to those of
IRS-2 single knock-out mice.
/
mice of
the CBA and C57Bl/6 strain never developed such diabetes (16). In
addition, although 50% N2 IRS-2
/
mice with the
129/Sv background developed diabetes by 20 weeks of age, none of >50
N2 IRS-2
/
animals with the C57BL/6J background
developed such diabetes (Fig. 7). These results support the idea that
increased susceptibility to diabetes in IRS-2
/
mice is related to the 129/Sv genetic strain. In this respect, the
backcrosses of the insulin receptor mutant animals with mice of strain
129/Sv or C57Bl/6 have already revealed that the 129/Sv strain contains
diabetic genes (29). The mapping and cloning of the modifier gene(s) in
the 129/Sv murine strain that appear to interact with IRS-2 in the
regulation of
-cell mass should facilitate the understanding of the
role of IRS-2 in the regulation of
-cell mass and reveal novel
molecular targets for drug development aimed at ameliorating
-cell
mass reduction in diabetic patients.
/
islets compared with
wild-type islets, suggesting the existence of a pathway from IRS-2 to
Pdx1. By contrast, our results demonstrate Pdx1 expression to be
essentially unaltered in islets from IRS-2
/
mice
without diabetes while being dramatically decreased in islets from
those with diabetes (Fig. 5, E and F). Moreover,
DNA microarray analysis as well as TaqMan PCR analysis revealed Pdx1
expression to be unaltered in islets from our original
IRS-2
/
mice without
diabetes.2 These results
indicate that IRS-2 deficiency by itself is not sufficient to decrease
Pdx1 expression in
-cells and that either genetic background or
hyperglycemia itself plays a role in reducing Pdx1 expression.
Moreover, it seems likely that markedly decreased insulin content and
GLUT2 expression are linked to the development of diabetes in
IRS-2
/
mice (Fig. 5).
/
mice depends upon the genetic background,
suggesting the existence of a modifier gene(s) for diabetes in mice of
the 129/Sv genetic strain.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Eri Yoshida, Miharu Nakashima, Ayumi Nagano, and Hiroshi Chiyonobu for their excellent technical assistance and mouse husbandry.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant for Life & Socio-Medical Science from the Kanae Foundation; a grant by Tanabe Medical Frontier Conference (to Y. T.); Grant DK41816 from the National Institutes of Health (to G. E. L.); Grant 192125 from the Juvenile Diabetes Foundation International, Grant-in-aid 10NP0201 for Creative Scientific Research from the Japan Society for the Promotion of Science; a grant-in-aid for Scientific Research on Priority Areas (C); a grant-in-aid for the Development of Innovative Technology from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and Health Science Research Grants (Research on the Human Genome and Gene Therapy) from the Ministry of Health and Welfare (to T. K.).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.
To whom correspondence should be addressed: Dept. of Metabolic
Diseases, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: 81-3-5800-8818; Fax:
81-3-5689-7209; E-mail: kadowaki-3im@h.u-tokyo.ac.jp.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M211045200
2 R. Suzuki, K. Tobe, Y. Terauchi, and T. Kadowaki, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IRS, insulin receptor substrate; FPG, fasting plasma glucose; Pdx1, duodenal homeobox factor-1; GLUT, glucose transporter.
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