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Physiol. Genomics 35: 65-74, 2008. First published July 15, 2008; doi:10.1152/physiolgenomics.00027.2008
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Received 30 January 2008; accepted in final form 8 July 2008.
Physiological Genomics 35:65-74 (2008)
1094-8341/08 $8.00 © 2008 American Physiological Society

Quantitative trait loci for impaired glucose tolerance in nondiabetic SM/J and A/J mice

Natsuko Hada 1, Misato Kobayashi 1, Masato Fujiyoshi 1, Akira Ishikawa 1, Masako Kuga 2, Masahiko Nishimura 2, Shizufumi Ebihara 1, Tamio Ohno 2 and Fumihiko Horio 1

1 Department of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
2 Division of Experimental Animals, Center for Promotion of Medical Research and Education, Graduate School of Medicine, Nagoya University, Nagoya, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The SMXA-5 recombinant inbred strain, which was established from nondiabetic parental SM/J and A/J mice, develops diabetic phenotypes such as impaired glucose tolerance. The combination of diabetogenic genes in the SM/J and A/J genomes impairs glucose tolerance in SMXA-5 mice. Using (SM/J x SMXA-5)F2 mice fed a high-fat diet, we previously detected a diabetogenic locus, T2dm2sa, on chromosome (Chr) 2. The A/J allele at this locus is diabetogenic. The SM.A-T2dm2sa congenic mouse, in which the Chr 2 region of A/J including T2dm2sa was introgressed into SM/J, showed obviously impaired glucose tolerance. These results indicate that SM.A-T2dm2sa mice develop diabetogenic traits due to T2dm2sa with the A/J allele and unknown diabetogenic loci with the SM/J allele. The aim of this study was to dissect these unknown loci, using quantitative trait locus (QTL) analysis in the (A/J x SM.A-T2dm2sa) F2 intercross fed a high-fat diet. The results revealed a highly significant QTL, T2dm4sa, for glucose tolerance on Chr 6 and a significant QTL, T2dm5sa, for glucose tolerance on Chr 11. These loci with the SM/J allele were diabetogenic. The diabetogenic effect of T2dm4sa or T2dm5sa was verified by the impairment of glucose tolerance in the A/J-6SM or A/J-11SM consomic strain, in which Chr 6 or Chr 11 of SM/J is introgressed into A/J, respectively. These results demonstrate that diabetogenic loci exist in the genomes of nondiabetic A/J and SM/J mice and suggest that T2dm2sa with the A/J allele and T2dm4sa and/or T2dm5sa with the SM/J allele elicit impaired glucose tolerance in SM.A-T2dm2sa mice.

recombinant inbred mouse; type 2 diabetes; consomic; mouse chromosome 11; chromosome 6


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE DEVELOPMENT of type 2 diabetes is influenced by multiple genetic and environmental factors (43). Because the interaction of these factors makes the disease heterogeneous, identification of the genes involved in human type 2 diabetes is difficult. Therefore, genetic analysis using experimental animals such as mice or rats is invaluable for the dissection of diabetogenic genes in type 2 diabetes. By using inbred animal models, we can strictly control genetic and environmental components in such analyses. Quantitative trait locus (QTL) mapping analyses have identified the susceptible loci contributing to type 2 diabetes-related traits in diabetic animal models such as GK rat (11, 13), OLETF rat (17, 38, 44), SDT rat (27), NSY mouse (42), NZO mouse (23, 33), TSOD mouse (15), KK mouse (36), KK-Ay mouse (39, 40), and TH mouse (18). In addition, with other animal models, a lot of QTLs for diabetes-related traits have been mapped (7).

The SMXA-5 mouse is one of the 26 SMXA recombinant inbred (RI) strains that have been established from nondiabetic parents, the SM/J and A/J strains (31). Each SMXA RI strain has a mosaic genome composed of chromosomal fragments from SM/J and A/J strains (12). Although the parental strains are nondiabetic, the SMXA-5 mouse develops impaired glucose tolerance and hyperinsulinemia (3, 19). These results indicate that diabetogenic loci within nondiabetic SM/J mice were combined with diabetogenic loci within nondiabetic A/J mice in the genomes of the RI strains, leading to the development of diabetic traits. Moreover, in SMXA-5 mice, a high-fat diet increases the degree of these diabetogenic traits, including glucose intolerance (20). Thus the SMXA-5 mouse is an available model with increased susceptibility to diabetes induced by a high-fat diet.

For the systematic dissection of the diabetogenic loci derived from nondiabetic strains, our first step was to focus on the A/J regions of the SMXA-5 genome by using QTL analysis in (SM/J x SMXA-5)F2 intercross mice fed a high-fat diet (Fig. 1). Such a diet is effective for QTL analysis because it enhances the differences in diabetogenic traits between diabetic and nondiabetic mice. As a result, we mapped highly significant QTLs that control diabetogenic traits and obesity on chromosome (Chr) 2 (21). These QTLs on Chr 2 controlled glucose tolerance, free-fed blood glucose concentration, and body mass index (BMI) (12, 21). This locus was designated T2dm2sa, for type 2 diabetes mellitus 2 in SMXA RI strains, and the A/J allele at this locus was diabetogenic.


Figure 1
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  		Fig. 1. Flow scheme of our studies to identify diabetes-related loci and causative genes existing in SM/J and A/J genomes. RI, recombinant inbred; QTL, quantitative trait locus.

 
Subsequently, to verify the function of T2dm2sa, we produced SM.A-T2dm2sa congenic mice in which the A/J-derived T2dm2sa region was introgressed into SM/J (12) (Fig. 1). Compared with the recipient SM/J mice, SM.A-T2dm2sa mice on a high-fat diet exhibited significantly impaired glucose tolerance and hyperglycemia (21). This result clearly confirmed that diabetogenic loci exist in the genome of the introgressed T2dm2sa region of A/J mice. On the other hand, A/J mice have the diabetogenic allele at T2dm2sa but are nondiabetic. This suggested that the combination of T2dm2sa with the A/J allele and unknown diabetogenic loci with the SM/J allele causes impaired glucose tolerance in SM.A-T2dm2sa mice.

In this study, we performed QTL analysis using (A/J x SM.A-T2dm2sa)F2 intercross mice fed a high-fat diet for the next step in our systematic dissection of the diabetogenic loci derived from nondiabetic strains. All F2 mice in this cross showed that the A/J allele was completely fixed at T2dm2sa (Fig. 1), which was previously mapped as a major diabetogenic locus. Therefore, the T2dm2sa locus did not directly affect the phenotypic variations in F2 mice. The main purpose of this study was to identify the diabetogenic loci with the SM/J allele under the homozygous condition of the A/J allele at the T2dm2sa locus. Moreover, to confirm the functions of the two major QTLs mapped in the present study, we characterized the diabetes-related traits of two consomic strains in which the selected chromosome of SM/J was introgressed into A/J.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals.
SM/J, A/J, and two consomic (A/J-6SM, A/J-11SM) strains were obtained from the Division of Experimental Animals, Center for Promotion of Medical Research and Education, Graduate School of Medicine, Nagoya University. The SM.A-T2dm2sa congenic strain was produced as previously described (21). Male SM/J mice were mated to A/J female mice to produce F1 mice. Male F1 mice were backcrossed to SM/J females to produce the N2 generation. Male heterozygous carriers of Chr 2 intervals between D2Mit295 and D2Mit281 were continuously bred to SM/J mice to produce the next generation. The marker-assisted speed congenic procedure resulted in >99% replacement of the SM/J background genome by N5. N6 congenic animals were intercrossed to produce F1 animals (N6:F1) that were homozygous for the introgressed D2Mit295–D2Mit28 region from A/J. SM.A-T2dm2sa congenic mice possessed the A/J homozygous alleles between D2Mit295 (proximal, 29.3 Mb) and D2Mit28 (distal, 148.4 Mb). At the proximal chromosomal region between D2Mit6 (20.8 Mb) and D2Mit295 (29.3 Mb) and at the distal chromosomal region between D2Mit28 (148.4 Mb) and D2Mit226 (163.0 Mb), the alleles possessed by the SM.A-T2dm2sa congenic mice were unknown.

Parental strains, F1 hybrid, and F2 intercross mice.
A/J female mice were mated to SM.A-T2dm2sa male mice to produce F1 hybrid mice, and F1 mice were intercrossed to produce (A/J x SM.A-T2dm2sa) F2 intercross mice. F1 (17 mice) and F2 (255 mice) generations were produced and maintained in our animal facility. All mice were maintained in a room under conventional conditions at a controlled temperature of 23 ± 3°C and humidity of 55 ± 5% with a 12:2-h light-dark cycle. From 6 wk of age, F2 mice were fed a high-fat diet. The mice were given ad libitum access to drinking water and a high-fat diet. The composition (g/kg diet) of the high-fat diet was as follows: 209 casein, 369 carbohydrate (starch-sucrose, 1:1), 35 AIN93MX mineral mixture (34), 10 AIN93VX vitamin mixture (32), 2 choline chloride, 35 corn oil, 300 lard, and 40 cellulose powder (AVICEL type FD-101, Asahi Chemical Industry, Osaka, Japan). Animal care and experimental procedures were approved by the Animal Research Committee of Nagoya University and were conducted according to Regulations for Animal Experiments in Nagoya University.

Analytic procedures for phenotype determination.
Body weight and anal-nasal length of the mice were measured at the start and after 10 wk of feeding a high-fat diet. BMI was calculated as body weight (g) divided by the square of the anal-nasal length (cm). Blood samples were obtained from the tail vein in nonfasting and fasting mice after 10 wk on the high-fat diet. Serum samples were collected, centrifuged, and stored at –30°C. Serum immunoreactive insulin concentration was measured by radioimmunoassay (ShionoRIA; Shionogi, Osaka, Japan) with rat insulin as a standard. An intraperitoneal glucose tolerance test (IPGTT) was performed after 10 wk on the high-fat diet with the following protocol. After a 14-h fast, blood samples were collected from the tail vein (fasting blood sample, 0 min sample in IPGTT). Then 20% glucose solution was injected intraperitoneally (2 g glucose/kg body wt). Blood samples were collected at 30, 60, and 120 min after injection. Blood glucose concentration was measured with a Glucose-B Test Kit (Wako, Osaka, Japan). The area under the curve (AUC; mg·min/dl) of that concentration was also calculated according to the trapezoid rule from the glucose measurements at fasting (0 min), 30, 60, and 120 min.

Genotypic and linkage analysis.
Genomic DNA was prepared from mouse kidney by salt/ethanol precipitation. A total of 125 microsatellite marker loci, polymorphic between SM/J and A/J, were genotyped in all F2 mice and are shown in Supplemental Table S1.1 We selected available markers for this study from SMXA RI strain distribution patterns (32). Polymerase chain reactions (PCR) were performed according to standard methods (37). PCR products were separated by electrophoresis on a 4% NuSieve (FMC, Rockland, ME) agarose gel and visualized with ethidium bromide staining. Linkage analysis was performed with the MapManager QTXb20 (25, 26) software program. This program is based on interval mapping using the free regression model. The permutation test estimates experiment-wide probability for given likelihood ratio statistics (LRS). Significance was determined by 1-cM steps for 1,000 permutations to provide LRS that were suggestive, significant, or highly significant (6, 10). These categories corresponded to the 37th, 95th, and 99.9th percentiles, respectively. The logarithm of odds (LOD) score was obtained by dividing the LRS by 4.605 (24). Significant linkage was defined in accordance with the guidelines of Lander and Kruglyak (22) as statistical evidence occurring by chance in the genome scan with P < 0.05. The positions for all microsatellite markers were collected from the Ensembl Genome Browser (http://www.ensembl.org/).

Statistical analysis.
All results are expressed as means ± SE. One-way ANOVA and subsequent Tukey-Kramer test were used to compare the means of A/J, SM.A-T2dm2sa, and F1 mice. One-way ANOVA and subsequent Dunnett's test were used to compare the means between A/J and A/J-6SM mice and between A/J and A/J-11SM mice. Differences with P < 0.05 were regarded as significant. All statistical analyses were performed with StatView version 5.0 software (SAS Institute, Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Diabetes-related phenotypes in parental strains, F1, and F2 mice.
Table 1 shows the initial and final body weight, final BMI, free-fed blood glucose concentration, free-fed serum insulin concentration, and blood glucose concentrations during IPGTT in parental (SM.A-T2dm2sa and A/J), (A/J x SM.A-T2dm2sa)F1, and (A/J x SM.A-T2dm2sa)F2 intercross mice after 10 wk on the high-fat diet.


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  		Table 1. Initial and final body weights, BMI, and diabetes-related traits in parental strains, (A/J x SM.A-T2dm2sa)F1, and (A/J x SM.A-T2dm2sa)F2 intercross mice after 10 wk of high fat-diet

 
Final BMI, blood glucose concentrations at 30, 60, and 120 min, AUC during IPGTT, and free-fed serum insulin concentration were significantly higher in SM.A-T2dm2sa mice than in A/J mice, while the initial body weight and final body weight were significantly lower in SM.A-T2dm2sa mice. There was no significant difference in free-fed blood glucose concentration between parental mice (Table 1). SM.A-T2dm2sa mice showed obviously impaired glucose tolerance and hyperinsulinemia compared with A/J mice.

Initial body weight of F1 mice was similar to that of A/J mice and higher than that of SM.A-T2dm2sa mice. Final body weight, final BMI, and free-fed blood glucose concentration of F1 were higher than the respective values of parental mice. Blood glucose concentrations at 30 and 60 min during IPGTT of F1 mice were similar to those of A/J mice and lower than those of SM.A-T2dm2sa mice. Blood glucose concentration at 120 min and AUC during IPGTT of F1 mice were higher than those of A/J mice but lower than those of SM.A-T2dm2sa mice. There was no significant difference in fasting blood glucose concentration (that is, the 0 min value during IPGTT) among SM.A-T2dm2sa, A/J, and F1 mice.

In F2 intercross mice, the means of blood glucose concentrations at 30, 60, and 120 min and AUC during IPGTT lay between those of A/J and SM.A-T2dm2sa mice (Table 1). Figure 2 plots the blood glucose concentration at 120 min during IPGTT for A/J, SM.A-T2dm2sa, F1, and F2 mice. For all parameters measured, the individual values in F2 mice showed wide distributions, exceeding the ranges of values in A/J and SM.A-T2dm2sa mice (Table 1, Fig. 2).


Figure 2
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  		Fig. 2. Blood glucose concentration 120 min after glucose injection during intraperitoneal glucose tolerance text (IPGTT) in SM.A-T2dm2sa (n = 18), A/J (n = 16), (A/J x SM.A-T2dm2sa)F1 (n = 17), and (A/J x SM.A-T2dm2sa) F2 (n = 255) mice after 10 wk of a high-fat diet.

 
QTL mapping in (A/J x SM.A-T2dm2sa) F2 intercross.
We performed QTL analyses for final body weight, final BMI, free-fed blood glucose concentration, blood glucose concentration, and AUC during IPGTT, and free-fed serum insulin concentration using F2 intercross mice. Among the results of QTL analyses performed in this study, the LOD score plot of the AUC during IPGTT for the whole genome scan is shown in Fig. 3. Figure 3 shows that the major QTLs for AUC during IPGTT were detected on Chr 6 and Chr 11.


Figure 3
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  		Fig. 3. Genomewide analysis of (A/J x SM.A-T2dm2sa)F2 mice showing significant linkages on chromosomes 6 and 11 for the area under the curve (AUC) during IPGTT. LOD, logarithm of odds.

 
On Chr 6, we mapped highly significant, significant, and suggestive QTLs that affected blood glucose concentrations at 0, 30, 60, and 120 min and AUC during IPGTT between D6Mit8 and D6Mit373 (Table 2, Fig. 4). A QTL for IPGTT 30 min was found in the region near D6Mit287, with the highest LOD score being 5.4 (highly significance threshold, LOD score ≥ 5.2). This locus was designated T2dm4sa, for type 2 diabetes mellitus 4 in SMXA RI strains. T2dm4sa explained 4–9% of the phenotypic variance in blood glucose concentrations at all time points during IPGTT (Table 2). The mean values of these traits among F2 mice with SM/J homozygous (SM/SM) at D6Mit8–D6Mit373 were higher than the values in those with A/J homozygous (A/A) (Table 2). This indicated that the SM/J allele at QTLs on Chr 6 contributed to the impairment of glucose tolerance. The LOD score curve for each trait showed more than one peak (Fig. 4).


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  		Table 2. Suggestive, significant, and highly significant QTLs for diabetes-related traits

 

Figure 4
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  		Fig. 4. LOD score curves for blood glucose concentrations at 30, 60, and 120 min during IPGTT as well as AUC during IPGTT on chromosome 6.

 
On Chr 11, we mapped significant and suggestive QTLs that affected free-fed blood glucose concentration as well as blood glucose concentrations at 0, 30, 60, and 120 min and AUC during IPGTT, between D11Mit8 and D11Mit10 (Table 2, Fig. 5). A QTL for IPGTT 30 min was found in the region near D11Mit122, with the highest LOD score being 4.0 (significance threshold, LOD score ≥ 3.5). This locus was designated T2dm5sa. T2dm5sa explained 4–7% of the phenotypic variance in blood glucose concentrations at all time points during IPGTT (Table 2). The mean values of these traits among F2 mice with SM/J homozygous (SM/SM) at D11Mit122 were higher than the values in those with A/J homozygous (A/A) (Table 2). This indicated that the SM/J allele at QTLs on Chr 11 also contributed to the impairment of glucose tolerance.


Figure 5
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  		Fig. 5. LOD score curves for blood glucose concentration at 30 min and AUC during IPGTT as well as free-fed blood glucose concentration (BG) on chromosome 11.

 
In addition to T2dm4sa and T2dm5sa, we also mapped significant QTLs for diabetes-related traits on Chr 3 and Chr 18. On Chr 18, significant and suggestive QTLs that affected blood glucose concentrations at 60 and 120 min and AUC during IPGTT were mapped (Table 2). The A/J allele at this QTL caused glucose intolerance, and this locus was designated T2dm3sa. On Chr 3, we mapped a significant QTL for free-fed blood glucose concentration (Table 2). The A/J allele at this QTL exhibited increased free-fed blood glucose, and this locus was designated T2dm6sa.

A significant QTL for final body weight was detected in the region near D1Mit36 on Chr 1. In addition, a significant QTL (LOD 3.4) for body weight gain in response to the high-fat diet and a significant QTL (LOD 3.8) for gain in BMI as a result of the high-fat diet were detected at D9Mit296 on Chr 9. These significant QTLs were detected in the same region in which a suggestive QTL for final BMI was detected. On Chr 1, suggestive QTLs for final BMI and free-fed blood glucose were detected; Chr 4 had a QTL for fasting blood glucose; Chr 5 had a QTL for glucose tolerance (IPGTT 30 min); Chr 9 had a QTL for final BMI and fasting blood glucose; Chr 13 had a QTL for final BMI and glucose tolerance (IPGTT 120 min); Chr 16 had a QTL for final body weight and final BMI; and Chr 18 had a QTL for glucose tolerance (IPGTT 60 min, AUC) (Table 2). Each suggestive locus explained 4–6% of the phenotypic variance in diabetes-related traits, as shown in Table 2. The diabetogenic allele that originated in the SM/J allele on Chr 1 and Chr 4, in addition to Chr 6 and Chr 11, was associated with an increased value of each phenotype based on genotype. In contrast, the diabetogenic allele that originated in the A/J allele on Chr 3, Chr 9, Chr 13, and Chr 18 was associated with an increased value of each phenotype based on genotype. On QTLs on Chr 5, the heterozygous allele (A/S) was associated with an increased value of each phenotype based on genotype.

Diabetic phenotype of consomic strains.
To confirm the diabetogenic effects of the SM/J allele at T2dm4sa on Chr 6 and at T2dm5sa on Chr 11, we characterized the diabetes-related traits of A/J-6SM and A/J-11SM consomic strains and compared them with the respective values of A/J mice. These results are shown in Table 3.


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  		Table 3. Initial body weight, final body weight, BMI, and diabetes-related traits in A/J, A/J-6SM, and A/J-11SM consomic mice fed high-fat diet

 
Body weight at the start and after 10 wk on the high-fat diet as well as final BMI were significantly lower in A/J-6SM mice than in A/J mice. The blood glucose concentration at 120 min and AUC during IPGTT were significantly higher in A/J-6SM mice than in A/J mice. However, there were no significant differences in free-fed blood glucose concentration and free-fed serum insulin concentration between A/J-6SM mice and A/J mice.

Initial body weight, final body weight, and final BMI after 10 wk of the high-fat diet were not different between A/J-11SM and A/J mice. The free-fed blood glucose concentration was higher in A/J-11SM mice than in A/J mice after 10 wk on the high-fat diet (Table 3). However, there was no significant difference in the free-fed serum insulin concentration between the two strains. Blood glucose concentrations at 30 and 120 min and AUC during IPGTT were significantly higher in A/J-11SM mice than in A/J mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Recently we mapped a diabetogenic locus, T2dm2sa, whose A/J allele contributed to the impairment of glucose tolerance, by using the (SM/J x SMXA-5) F2 intercross. SM.A-T2dm2sa congenic mice, in which the T2dm2sa region of A/J mice was introgressed into nondiabetic SM/J mice, showed obviously impaired glucose tolerance and hyperglycemia (21). Because A/J mice are nondiabetic, this finding suggested that the combination of T2dm2sa with the A/J allele and unknown diabetogenic loci with the SM/J allele leads to impaired glucose tolerance in SM.A-T2dm2sa. Figure 1 shows the flow scheme of our systematic dissection of diabetogenic loci from the previous studies to the present study. In the present study, we systematically performed genetic dissection of unknown diabetogenic loci other than T2dm2sa that contribute to the development of diabetes-related phenotypes in SM.A-T2dm2sa, by using (A/J x SM.A-T2dm2sa)F2 intercross mice. The A/J homozygous region including T2dm2sa on Chr 2 was fixed in genomes of all F2 intercross mice (Fig. 1). On the chromosomal region except for this A/J homozygous region, each intercross mouse had a mosaic genome composed of chromosomal fragments from A/J and SM/J strains. The present QTL analysis revealed the existence of a highly significant QTL, T2dm4sa, for glucose tolerance on Chr 6 (D6Mit8–D6Mit373) and a significant QTL, T2dm5sa, for glucose tolerance on Chr 11 (D11Mit8–D11Mit10). The SM/J allele at these loci increased the fasting blood glucose concentration and caused glucose intolerance. Moreover, a significant QTL, T2dm3sa, for glucose tolerance on Chr 18 (D18Mit103–D18Mit7) and a significant QTL, T2dm6sa, for free-fed blood glucose on Chr 3 (D3Mit28–D3Mit194) were detected, and the A/J allele at these loci caused glucose intolerance and hyperglycemia (Fig. 1).

On Chr 6, the chromosomal region (T2dm4sa) having a significant linkage with blood glucose concentration at 30 min during IPGTT was D6Mit287–D6Mit373, and this is homologous to human chromosomes 2p13–p11, 3p26–p25, 3p14–12, 3q21, 10q11, 12p13–11, and 22q11. This region includes the gene Ppar{gamma} encoding peroxisome proliferator-activated receptor {gamma}, Iapp encoding islet amyloid polypeptide, and Adipor2 encoding adiponectin receptor 2. It was reported that the single nucleotide polymorphisms (SNPs) of these genes are associated with type 2 diabetes in humans (2, 9, 30, 35). At present, we consider these genes as T2dm4sa candidates. In QTL analysis using (NSY x C3H/He)F2 intercross mice, QTLs for glucose tolerance, serum insulin level, and liver fat were mapped near the T2dm4sa region on Chr 6 (16), so there is a possibility that T2dm4sa possesses common diabetogenic genes acting in different strains.

A/J-6SM consomic mice on the high-fat diet showed significantly lower glucose tolerance than A/J mice, whereas this strain had significantly lower body weight and BMI. These results verified that T2dm4a with the SM/J allele on Chr 6 exerts effects on the development of diabetes-related phenotypes, and also that loci controlling body weight and obesity exist on Chr 6. Previously, QTLs for diabetes-related traits were detected by analyses using (SM/J x A/J)F2 intercross mice (5), SMXA RI strains (4, 19), and (SM/J x SMXA-5)F2 intercross mice (21). QTLs detected on the same chromosome between the present study and the previous studies using SMXA-related strains are shown in Table 4. Suggestive QTLs controlling final body weight and final BMI were detected on Chr 6 in the previous studies (4, 5, 19). Because the A/J allele at these loci caused increased body weight and BMI, the present results from A/J-6SM consomic mice verify the effectiveness of these loci. The suggestive QTL for free-fed blood glucose concentration was also detected at D6Mit287 in a previous study (19) using SMXA-RI strains fed a high-carbohydrate diet (Table 4). The A/J allele at this locus increased blood glucose concentration. At D6Mit287 in the present study, we also detected T2dm4sa, at which the SM/J allele caused glucose intolerance. The contradiction in the allele effects of these two QTLs detected at D6Mit287 might be due to the difference in the experimental diets between the studies (high-carbohydrate diet vs. high-fat diet). It is unknown whether the separate diabetogenic locus exists near D6Mit287 in either SM/J or A/J mice. At present, we do not have any other explanation for this contradiction in the allele effects of these two QTLs at D6Mit287.


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  		Table 4. QTLs detected on same chromosome between present study and previous study using SMXA RI-related strains

 
On Chr 11, the chromosomal region (T2dm5sa) with significant linkage with blood glucose concentration at 30 min during IPGTT was D11Mit8–D11Mit10, and this is homologous to human chromosomes 17q11–21 and 17p23–p25. The region from 62 to 71 Mb of mouse Chr 11 is a linkage hot spot for diabetes-related traits (7), and the following five diabetogenic loci were identified. We previously identified (21) a suggestive QTL for glucose tolerance in this region, using (SM/J x SMXA-5)F2 mice (Table 4). Loci for plasma insulin level have been identified in the NZO mouse (23) and the M16 mouse (1). A cross between C57BLKS/J and HRS mice homozygous for the fat mutation revealed a locus for blood glucose (8). Finally, Ueda et al. (42) mapped a QTL for glucose tolerance near the T2dm5sa region on Chr 11 using (NSY x C3H/He)F2. They considered Tcf2 encoding hepatocyte nuclear factor-1β as one of the candidate genes for their QTL on Chr 11 (41). In addition to Tcf2, this region includes the gene GIP encoding gastric inhibitory polypeptide, which, as incretin, induces insulin secretion. Moreover, it was recently suggested that GIP is associated with obesity-induced insulin intolerance (14, 28). At present, we consider these genes as T2dm5sa candidates. T2dm5sa might also possess common diabetogenic genes acting in a diverse set of strains. T2dm5sa with the SM/J allele causes impaired glucose tolerance. As shown in Table 4, the SM/J allele at the suggestive locus previously mapped near D11Mit15 in (SM/J x SMXA-5)F2 mice also caused impaired glucose tolerance. The allele effects of these two loci are consistent.

Blood glucose concentration at 30 min during IPGTT was significantly higher in A/J-11SM consomic mice than in A/J mice. However, serum insulin concentration and final BMI in A/J-11SM consomic mice were not different from those in A/J mice. The glucose intolerance in A/J-11SM mice might be due to impairment of glucose-induced insulin secretion, but not to insulin resistance. These results verified that diabetogenic loci exist on Chr 11 of SM/J.

On Chr 18 T2dm3sa, which was significantly linked to blood glucose concentration at 120 min during IPGTT, was mapped near D18Mit8 (74.6 Mb), and the A/J allele at this locus caused glucose intolerance. Previously, by analysis using 19 SMXA-RI strains on a high-carbohydrate diet, we mapped a suggestive QTL for free-fed blood glucose concentration and glucose tolerance on Chr 18 (19) (Table 4). This suggestive locus with the A/J allele also leads to hyperglycemia and glucose intolerance. On Chr 18, a diabetogenic locus for free-fed blood glucose concentration in crosses between NON and NZO mice has also been mapped at D18Mit17 (43.3 Mb) (23).

On Chr 3 T2dm6sa, significantly linked to free-fed blood glucose concentration, was mapped near D3Mit12, and the A/J allele at this locus caused hyperglycemia. Our previous studies (4, 5, 19, 21) found no evidence of any diabetogenic QTL in this region. In the region near T2dm6sa, a suggestive locus for fasting blood glucose was mapped by QTL analysis in crosses between C57BL/6J and KK-Ay mice (40).

On Chr 1, we detected a significant QTL for final body weight near D1Mit36, and the A/J allele at this locus increased body weight and BMI. As shown in Table 4, the A/J allele at the suggestive QTL for body weight detected in the previous study (4) also increased body weight.

In the analysis of diabetic phenotypes, glucose tolerance is recognized as a reliable and stable index for evaluating the diabetogenic state. The value of AUC during IPGTT comprehensively indicates the glucose tolerance of mice. In the present study, the loci with significant linkages to AUC during IPGTT were T2dm4sa and T2dm5sa. We previously demonstrated that T2dm2sa had a highly significant linkage with AUC during IPGTT in (SM/J x SMXA-5)F2 intercross mice fed a high-fat diet (21). Table 5 presents the severity of impaired glucose tolerance and the allele distribution patterns of these three loci (T2dm2sa, T2dm4sa, and T2dm5sa) in SM/J, A/J, SMXA-5, SM.A-T2dm2sa, A/J-6SM, and A/J-11SM mice. The value of AUC of SMXA-5 mice measured in our previous study (21) under the same conditions as in this study was similar to that in SM.A-T2dm2sa mice. In addition, the values of AUC of SM.A-T2dm2sa and SMXA-5 mice were higher than those of A/J-6SM and A/J-11SM mice. Table 5 shows that four glucose-intolerant strains (SMXA-5, SM.A-T2dm2sa, A/J-6SM, and A/J-11SM), established from SM/J and A/J, each possessed both the A/J allele at T2dm2sa and the SM/J allele at T2dm4sa and/or T2dm5sa. On the other hand, parental SM/J and A/J strains, which do not exhibit glucose intolerance, possessed either the A/J allele at T2dm2sa or the SM/J allele at T2dm4sa and T2dm5sa. These findings suggest that the combination of the A/J allele at T2dm2sa and the SM/J allele at T2dm4sa and/or T2dm5sa is essential for impaired glucose tolerance. In particular, we speculate that the severely impaired glucose tolerance of SM.A-T2dm2sa mice is caused by the combination of all diabetogenic alleles at these three loci.


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  		Table 5. Allele distribution patterns of diabetic QTLs and severity of glucose intolerance in parental strains of SMXA-RI, SMXA-5, congenic, and consomic strains

 
In conclusion, we systematically dissected diabetogenic QTLs for impaired glucose tolerance, using (A/JxSM.A-T2dm2sa)F2 intercross mice in addition to T2dm2sa. This study demonstrates that nondiabetic SM/J and A/J have diabetogenic loci, such as T2dm4sa and T2dm5sa in addition to T2dm2sa. The combination of the SM/J allele of T2dm4sa and T2dm5sa and the A/J allele of T2dm2sa elicits severe glucose intolerance in SM.A-T2dm2sa mice. We consider that such diabetogenic loci, the combination of which leads to diabetes, play important roles in the pathogenesis of human type 2 diabetes. Dissection of this kind of diabetogenic loci will contribute to an understanding of the complex mechanisms underlying human type 2 diabetes.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by a Grant-in-Aid for Scientific Research (B) (no. 17380084) from the Japan Society for the Promotion of Sciences; a grant from the Kao Research Council for the Study of Healthcare Sciences, Japan; a grant from the Kieikai Research Foundation, Japan; and a grant from the Elizabeth Arnold Fuji Foundation, Japan.


   FOOTNOTES
 
Address for reprint requests and other correspondence: F. Horio, Dept. of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya Univ., Nagoya 464-8601, Japan (e-mail: horiof{at}agr.nagoya-u.ac.jp).

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.

1 The online version of this article contains supplemental material. Back


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 INTRODUCTION
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 RESULTS
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 REFERENCES
 

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