Impact of Genetic Background and Ablation of Insulin Receptor Substrate (IRS)-3 on IRS-2 Knock-out Mice*

Yasuo TerauchiDagger §, Junji MatsuiDagger , Ryo SuzukiDagger §, Naoto KubotaDagger §, Kajuro Komeda, Shinichi Aizawa||, Kazuhiro EtoDagger §, Satoshi KimuraDagger , Ryozo NagaiDagger , Kazuyuki TobeDagger §, Gustav E. Lienhard**, and Takashi KadowakiDagger §DaggerDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although we and others have generated IRS-2 knock-out (IRS-2-/-) mice, significant differences were seen between the two lines of IRS-2-/- mice in the severity of diabetes and alterations of beta -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 beta -cell mass despite having insulin resistance. Insulin-positive beta -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-/- mice) were insulin-resistant, they maintained normal glucose tolerance via compensatory beta -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).

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-/- 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 beta -cell mass in IRS-2-/- mice was reduced to 83% of that in wild-type mice, which was in marked contrast to the 85% beta -cell mass increase in IRS-1-/- mice (16). Thus, liver insulin resistance together with a lack of compensatory beta -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 beta -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).

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-/-) 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 beta -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.

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. 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
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Animals and Determination of Genotype-- IRS-2-/- 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.

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-/- 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.

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, beta -cells, alpha -cells, and delta  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, beta -cells, alpha -cells, and delta  plus PP cells were traced manually and analyzed with Win ROOF software (Mitani Co., Ltd., Tokyo, Japan). The masses of beta -cells, alpha -cells, and delta  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).

Islet Isolation and beta -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.

Analysis of Insulin Content and Secretion-- To assess insulin content, isolated islets were extracted in acid ethanol at -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.

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.

    RESULTS
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INTRODUCTION
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RESULTS
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Generation of IRS-2-/-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.

20-30% IRS-2-/- 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-2-/-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).

Markedly Decreased Fat Mass in IRS-2-/- 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.

IRS-3-/- Knock-out Mice Had a Normal beta -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 beta -cell mass in IRS-3-/- mice was similar to that of wild-type mice. As we reported previously (16), the beta -cell mass in IRS-2-/- mice was reduced to ~50% of that in wild-type mice. The beta -cell amount in IRS-2-/-IRS-3-/- mice was similar to that in IRS-2-/- mice.


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Fig. 4.   Normal beta -cell mass in IRS-3-/- mice and reduced beta -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.

Insulin-positive beta -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 beta -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.

Expressions of Pdx1 and GLUT2 in Islets Were Decreased to Nearly Undetectable Levels in IRS-2-/- 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 beta -cells, thereby maintaining normal beta -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 beta -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).

Increased Insulin Secretion in IRS-2-/- 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.

Increased Susceptibility to Diabetes in IRS-2-/- 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although we (16) and others (15) have generated IRS-2-/- mice, significant differences in the phenotypes of IRS-2-/- mice between the two groups were seen in the severity of diabetes and alterations of beta -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), beta -cell mass (Fig. 4), and beta -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.

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-/- 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 beta -cell mass should facilitate the understanding of the role of IRS-2 in the regulation of beta -cell mass and reveal novel molecular targets for drug development aimed at ameliorating beta -cell mass reduction in diabetic patients.

A recent report (28) from another laboratory indicated Pdx1 expression to be reduced in IRS-2-/- 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 beta -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).

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 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.

Dagger Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Moller, D. E. (1994) Diabetes 43, 1394-1401[Abstract]
2. Terauchi, Y., and Kadowaki, T. (2002) Endocr. J. 49, 247-264[Medline] [Order article via Infotrieve]
3. White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186[Medline] [Order article via Infotrieve]
4. Kadowaki, T., Koyasu, S., Nishida, E., Tobe, K., Izumi, T., Takaku, F., Sakai, H., Yahara, I., and Kasuga, M. (1987) J. Biol. Chem. 262, 7342-7350[Abstract/Free Full Text]
5. Shemer, J., Ademo, M., Wilson, G. L., Heffez, D., Zick, Y., and LeRoith, D. (1987) J. Biol. Chem. 262, 15476-15482[Abstract/Free Full Text]
6. Sun, X. J., Wang, L. M., Zhang, Y., Yenush, L., Myers, M. G., Jr., Glasheen, E., Lane, W. S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177[CrossRef][Medline] [Order article via Infotrieve]
7. Lavan, B. E., Lane, W. S., and Lienhard, G. E. (1997) J. Biol. Chem. 272, 11439-11443[Abstract/Free Full Text]
8. Lavan, B. E., Fantin, V. R., Chang, E. T., Lane, W. S., Keller, S. R., and Lienhard, G. E. (1997) J. Biol. Chem. 272, 21403-21407[Abstract/Free Full Text]
9. Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S. (1994) Nature 372, 182-186[CrossRef][Medline] [Order article via Infotrieve]
10. Araki, E., Lipes, M. A., Patti, M. E., Brüning, J. C., Haag, I. B., Johnson, R. S., and Kahn, C. R. (1994) Nature 372, 186-190[CrossRef][Medline] [Order article via Infotrieve]
11. Terauchi, Y., Iwamoto, K., Tamemoto, H., Komeda, K., Ishii, C., Kanazawa, Y., Asanuma, N., Aizawa, T., Akanuma, Y., Yasuda, K., Kodama, T., Tobe, Y., Yazaki, Y., and Kadowaki, T. (1997) J. Clin. Invest. 99, 861-866[Abstract/Free Full Text]
12. Patti, M. E., Sun, X. J., Brüning, J. C., Araki, E., Lipes, M. A., White, M. F., and Kahn, C. R. (1995) J. Biol. Chem. 270, 24670-24673[Abstract/Free Full Text]
13. Tobe, K., Tamemoto, H., Yamauchi, T., Aizawa, S., Yazaki, Y., and Kadowaki, T. (1995) J. Biol. Chem. 270, 5698-5701[Abstract/Free Full Text]
14. Yamauchi, T., Tobe, K., Tamemoto, H., Ueki, K., Kaburagi, Y., Yamamoto-Honda, R., Takahashi, Y., Yoshizawa, F., Aizawa, S., Akanuma, Y., Sonenberg, N., Yazaki, Y., and Kadowaki, T. (1996) Mol. Cell. Biol. 16, 3074-3084[Abstract]
15. Withers, D. J., Gutierrez, J. S., Towery, H., Burks, D. J., Ren, J. M., Previs, S., Zhang, Y., Bernal, D., Pons, S., Shulman, G. I., Bonner-Weir, S., and White, M. F. (1998) Nature 391, 900-904[CrossRef][Medline] [Order article via Infotrieve]
16. Kubota, N., Tobe, K., Terauchi, Y., Eto, K., Yamauchi, T., Tsubamoto, Y., Komeda, K., Nakano, R., Miki, H., Suzuki, R., Satoh, S., Sekihara, H., Sciacchitano, S., Akanuma, Y., Aizawa, S., Nagai, R., Kimura, S., Taylor, S. I., and Kadowaki, T. (2000) Diabetes 49, 1880-1889[Abstract]
17. Withers, D. J., Burks, D. J., Towery, H. H., Altamuro, S. L., Flint, C. L., and White, M. F. (1999) Nat. Genet. 23, 32-40[CrossRef][Medline] [Order article via Infotrieve]
18. Brüning, J. C., Winnay, J., Bonner-Weir, S., Taylor, S. I., Accili, D., and Kahn, C. R. (1997) Cell 88, 561-572[Medline] [Order article via Infotrieve]
19. Liu, S. C., Wang, Q., Lienhard, G. E., and Keller, S. R. (1999) J. Biol. Chem. 274, 18093-18099[Abstract/Free Full Text]
20. Laustsen, P. G., Michael, M. D., Crute, B. E., Cohen, S. E., Ueki, K., Kulkarni, R. N., Keller, S. R., Lienhard, G. E., and Kahn, C. R. (2003) Genes Dev., in press
21. Terauchi, Y., Tsuji, Y., Satoh, S., Minoura, H., Murakami, K., Okuno, A., Inukai, K., Asano, T., Kaburagi, Y., Ueki, K., Nakajima, H., Hanafusa, T., Matsuzawa, Y., Sekihara, H., Yin, Y., Barrett, J. C., Oda, H., Ishikawa, T., Akanuma, Y., Komuro, I., Suzuki, M., Yamamura, K., Kodama, T., Suzuki, H., Koyasu, S., Aizawa, S., Tobe, K., Fukui, Y., Yazaki, Y., and Kadowaki, T. (1999) Nat. Genet. 21, 230-235[CrossRef][Medline] [Order article via Infotrieve]
22. Kubota, N., Terauchi, Y., Miki, H., Tamemoto, H., Yamauchi, T., Komeda, K., Nakano, R., Ishii, C., Sugiyama, T., Eto, K., Tsubamoto, T., Okuno, A., Murakami, K., Ezaki, O., Hasegawa, G., Naito, M., Toyoshima, Y., Tanaka, S., Shiota, K., Aizawa, S., Nagai, R., Tobe, K., Kimura, S., and Kadowaki, T. (1999) Mol. Cell 4, 597-609[Medline] [Order article via Infotrieve]
23. Ishii, C., Kawazu, S., Utsugi, T., Ito, Y., Ohno, T., Kato, N., Shimizu, M., Tomono, S., Nagai, R., and Komeda, K. (1996) Diabetes Res. 31, 1-18
24. Watada, H., Kajimoto, Y., Umayahara, Y., Matsuoka, T., Kaneto, H., Fujitani, Y., Kamada, T., Kawamori, R., and Yamasaki, Y. (1996) Diabetes 45, 1478-1488[Abstract]
25. Kaneto, H., Kajimoto, Y., Miyagawa, J., Matsuoka, T., Fujitani, Y., Umayahara, Y., Hanafusa, T., Matsuzawa, Y., Yamasaki, Y., and Hori, M. (1999) Diabetes 48, 2398-2406[Abstract]
26. Eto, K., Tsubamoto, Y., Terauchi, Y., Sugiyama, T., Kishimoto, T., Takahashi, N., Yamauchi, N., Kubota, N., Murayama, S., Aizawa, T., Akanuma, Y., Aizawa, S., Kasai, H., Yazaki, Y., and Kadowaki, T. (1999) Science 283, 981-985[Abstract/Free Full Text]
27. Eto, K., Suga, S., Wakui, M., Tsubamoto, Y., Terauchi, Y., Taka, J., Aizawa, S., Noda, M., Kimura, S., Kasai, H., and Kadowaki, T. (1999) J. Biol. Chem. 274, 25386-25392[Abstract/Free Full Text]
28. Kushner, J. A., Ye, J., Schubert, M., Burks, D. J., Dow, M. A., Flint, C. L., Dutta, S., Wright, C. V., Montminy, M. R., and White, M. F. (2002) J. Clin. Invest. 109, 1193-1201[Abstract/Free Full Text]
29. Kido, Y., Philippe, N., Schaffer, A. A., and Accili, D. (2000) Diabetes 49, 589-596[Abstract]


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