The pathogenesis of inflammation and fibrosis in the pancreatic islets in diabetes is largely unknown. Spontaneously diabetic Torii (SDT) rats exhibit inflammation and fibrosis in and around the islets during the development of the disease. We investigated genetic factors for diabetes, islet inflammation, and fibrosis in the SDT rat. We produced F1 and F2 rats by intercross between SDT and F344 rats, examined the onset of diabetes, glucose tolerance, and histology of the pancreas, and performed genetic analysis of these traits. We then established a congenic strain carrying the SDT allele at the strongest diabetogenic locus on the F344 genetic background and characterized glucose tolerance and histology of the pancreas. F1 rats showed glucose intolerance and inflammatory changes mainly in the islets. Genetic analysis of diabetes identified a major locus on chromosome 3, designated Dmsdt1, at which a dominantly acting SDT allele was involved. Quantitative trait locus (QTL) analysis of glucose tolerance revealed, in addition to Dmsdt1 [logarithm of odds (LOD) 5.3 near D3Mit12], three other loci, designated Dmsdt2 (LOD 4.2 at D8Rat46), Dmsdt3 (LOD 3.8 near D13Arb5), and Dmsdt4 (LOD 5.8 at D14Arb18). Analysis of a congenic strain for Dmsdt1 indicates that the dominantly acting SDT allele induces islet inflammation and fibrosis. Thus we have found a major locus on chromosome 3 for islet inflammation and fibrosis in the SDT rat. Identification of the genes responsible should provide insight into the pathogenesis of diabetes.
- congenic analysis
- genetic factor
- glucose tolerance
- quantitative trait locus
diabetes mellitus is a multifactorial disease involving interaction of genetic and environmental factors. Spontaneous animal models of diabetes are widely used in research and have provided valuable information on the nature of the disease. Inflammation and fibrosis in pancreatic islets have been described in human type 2 diabetes (4) and in spontaneous animal models of type 2 diabetes, including the Goto-Kakizaki (GK) rat (12), the Zucker diabetic fatty rat (33), and the Otsuka Long-Evans Tokushima fatty (OLETF) rat (16). A role of inflammation and fibrosis in the pathogenesis of type 2 diabetes has been suggested (17), but the mechanism and genetic factors remain to be elucidated. Genetic, pathophysiological, and histological analyses using animal models should provide useful information for understanding the molecular mechanism of the inflammatory process in the development of the disease.
The spontaneously diabetic Torii (SDT) rat was established from an outbred colony of Sprague-Dawley rats in 1997 as a nonobese diabetic animal model (37, 38). Male SDT rats develop diabetes at 15 wk of age or later and show 100% incidence of diabetes by 40 wk of age. Pathological changes including microvascular congestion and hemorrhage in pancreatic islets appear from ∼8 wk of age and are followed by inflammation and fibrosis in and around the islets. These pathological changes in the pancreas of SDT rats differ from those of typical autoimmune type 1 diabetes, in which lymphocyte infiltration into pancreatic islets is observed. These inflammatory conditions continue for several months and are accompanied by a decrease in the number of pancreatic β-cells (27). Thus hyperglycemia in SDT rats is thought to be caused by destruction of pancreatic β-cells. After several weeks from the onset of diabetes, nearly all pancreatic β-cells disappear in SDT rats. The main diabetic complication of SDT rats is ocular, including cataracts, proliferative retinopathy, and neovascular glaucoma (15, 37, 38).
To identify genes responsible for diabetes in SDT rats, we previously performed a quantitative trait locus (QTL) analysis for glucose tolerance at 20 wk of age using [Brown Norway (BN)×SDT]F1×SDT backcrossed rats (26). As a result, Gisdt1, 2, and 3, QTLs associated with glucose intolerance, were mapped on rat chromosomes 1, 2, and X, respectively. In addition, it was found that none of the backcrossed rats developed diabetes by 20 wk of age. While the backcross analysis used in that study was suitable for identification of recessively acting genetic factors, unidentified dominantly acting genetic factors may also be involved in the development of the disease. Inflammation and fibrosis were observed in and around the islets in F1 animals (Fuse M, Masuyama T, unpublished observation), suggesting the involvement of dominantly acting genetic factors in the inflammatory process.
In this study, using genetic analysis of diabetes on (F344×SDT)F2 rats and further congenic analysis, we identified a major locus for islet inflammation and fibrosis, at which a dominantly acting SDT allele is involved.
MATERIALS AND METHODS
SDT rats were provided by the Association of the Spontaneously Diabetic Torii rat study (2001–2007) and CLEA Japan (2007–present). F344 rats were purchased from Japan SLC (Shizuoka, Japan). For genetic analysis, male SDT rats were crossed with female F344 rats to obtain F1 progeny. Female and male F1 rats were further crossed to obtain 167 male F2 rats. All animals were maintained under specific pathogen-free (SPF) conditions at 23 ± 2°C and 55 ± 10% relative humidity with a 12:12-h light-dark cycle and were provided with water and commercial diet CE-2 (CLEA Japan) at the Institute for Experimental Animals, Kobe University School of Medicine. All animal experiments were approved by the Committee on Animal Experimentation, Kobe University School of Medicine (Permission No. P-031207) and carried out in accordance with the “Guidelines for Animal Experimentation at Kobe University.”
All animals were checked for nonfasting urinary glucose levels with TES-TAPE (Shionogi, Osaka, Japan) once a week until 60 wk of age. TES-TAPE is a test paper used to measure urinary glucose levels. Diabetes was defined as glycosuria positivity and blood glucose levels ≥200 mg/dl under ad libitum dietary conditions. At 60 wk of age, body weight of each rat was measured after overnight fasting for 16 h and oral glucose tolerance test (OGTT) was performed. Glucose (2 g/kg body wt) in physiological saline was administered via the oral route, and blood samples were collected from the tail vein at 0 (fasting), 15, 30, 60, and 120 min. Blood glucose levels were measured by a portable glucose meter (ANTSENSE II, Bayer Medical, Tokyo, Japan). The area under the curve (AUC) of blood glucose was calculated according to the trapezoidal rule from the blood glucose levels at 0, 15, 30, 60, and 120 min during OGTT. The trapezoidal rule is a method for finding an approximate value for a definite integral.
Genomic DNA was extracted from the tail tip of each rat at 4 wk of age with the automatic DNA Isolation System (KURABO, Osaka, Japan). Primer sets of simple sequence length polymorphism (SSLP) markers used in this study have been described elsewhere (RATMAP, available at http://ratmap.gen.gu.se/; Rat Genome Database, available at http://rgd.mcw.edu/; Refs. 26, 50). Polymerase chain reaction (PCR) amplification was carried out with 200 nM sense and antisense primers, each dNTP at 200 μM, 12 ng of genomic DNA, and 0.3 U of Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany) in a total volume of 12 μl. The PCR product was mixed with loading buffer (0.03% bromphenol blue, 30% glycerol), electrophoresed on a 4% NuSieve 3:1 agarose gel (FMC BioProducts, Rockland, ME), and stained with ethidium bromide.
Before genetic analysis was performed, linkage maps were constructed for all autosomes and the X chromosome by genotyping all F2 rats with 173 SSLP markers. Linkage maps were constructed by Kosambi's map function in Map Manager QTX20b (25), and the average marker spacing was 9.6 cM. The physical map position of SSLP markers was based on Ensembl Genome Browser Rat (http://www.ensembl.org/Rattus_norvegicus/index.html) and the Rat Genome Database (http://rgd.mcw.edu/).
To identify genetic factors for diabetes, Yates’ 2 × 3 χ2-test was performed on genotype frequencies for all SSLP markers in autosomes and a 2 × 2 χ2-test in the X chromosome in diabetic F2 rats (n = 31) and nondiabetic F2 rats with AUC values of the lowest 20% (n = 33). Since AUC is thought to be the most efficient index of glucose tolerance, nondiabetic F2 rats with AUC values of the lowest 20% were used for comparison with diabetic F2 rats.
For QTL analysis, we performed a simple interval mapping method (10, 21) in Map Manager QTX20b and calculated the likelihood ratio statistic (LRS) value on linkage maps with the free regression model. Logarithm of odds (LOD) scores were obtained by dividing the LRS value by 4.605 (23). To evaluate statistical significance of the LOD scores obtained for each quantitative trait, we calculated genomewide threshold levels of LOD scores, i.e., suggestive (P < 0.63), significant (P < 0.05), and highly significant (P < 0.001). For example, when the LOD score obtained for the specific quantitative trait at the specific locus exceeds the highly significant threshold level, the locus is designated “a highly significant QTL.” The genomewide LOD score threshold levels were determined by permutation tests in 1-cM steps for 1,000 permutations (3). The designations “suggestive,” “significant,” and “highly significant” correspond to the 37th, 95th, and 99.9th percentiles of the permutation distribution, respectively. The 95% confidence interval (CI) for each QTL was determined with a 1.5-LOD drop from the QTL peak (32). The mode of inheritance of alleles at the QTL detected was determined by two statistical tests using Map Manager QTX20b (13). First, one of additive, dominance, and recessive regression models was contrasted with a no-QTL model. The LRS obtained was tested for significance at the experiment-wide 5% threshold estimated by 1,000 permutations of the corresponding model used. Second, if this test showed significance, the model was compared with a free model. When the difference in LRS between the two models exceeded the above threshold level, that model was rejected. In contrast, when the difference was within the threshold level, that model was accepted.
The pancreas was fixed in 10% neutral buffered formalin. The fixed specimens were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin for histopathological examination. Serial sections were viewed via light microscopy by an examiner blind to the experimental conditions of the animals.
One-way ANOVA and subsequent Student-Newman-Keuls (SNK) test were used to compare means among F2 rats with SDT homozygous genotype, SDT/F344 heterozygous genotype, and F344 homozygous genotype. Mann-Whitney U-test was used to compare means among SDT, F344, F1, and congenic rats. Differences for which the P value was <0.05 were regarded as significant. All statistical analyses were performed with ystat2006 (Igaku Tosho Shuppan, Tokyo, Japan).
Establishment of F344.SDT-Dmsdt1 congenic strain.
Female F344 rats were crossed with male SDT rats to produce F1 rats. Female F1 rats were backcrossed to male F344 rats to produce N2 generations. Male and female heterozygous carriers of the Dmsdt1 region on chromosome 3 were continuously backcrossed to F344 rats to produce the next generation by the speed congenic technique (43). In N5 generations heterozygous carriers of the Dmsdt1 region on chromosome 3 were checked by 173 SSLP markers, and confirmation that most other genomic regions were changed to the F344 genetic background was obtained. In N7 and N8 generations, two types of congenic rats were analyzed for histological changes in the pancreas: congenic F344.SDT-Dmsdt1 rats heterozygous for the SDT allele at the Dmsdt1 region between D3Wox9 and D3Arb20 [F344.SDT-Dmsdt1(hetero); n = 5], and littermates homozygous for the F344 allele at the Dmsdt1 region (F344-type control; n = 5). These two types of congenic rats along with F344 and F1 rats were examined for the number of islets with both hemosiderin deposition and fibrosis in two specimens at a distance of 200 μm. F344.SDT-Dmsdt1(hetero) rats were also analyzed for glucose tolerance for comparison with F344 rats.
Phenotypic characterization of SDT, F344, F1, and F2 rats.
To perform genetic analysis of diabetes in SDT rats, we produced (F344×SDT)F1 and F2 rats and compared the onset of diabetes in SDT, F344, F1, and F2 rats up to 60 wk of age (Fig. 1A). SDT rats developed diabetes at 14 wk of age or later and showed 100% incidence of diabetes at 32 wk of age, but neither F344 nor F1 rats developed diabetes. F2 rats developed diabetes at 25 wk of age or later, and the cumulative incidence of diabetes reached 19% (31/167) at 60 wk of age.
OGTT was done on each animal at 60 wk of age for genetic analysis of glucose intolerance (Fig. 1 and Supplemental Table S1).1 Consistent with the finding that all SDT rats developed diabetes, the blood glucose levels and AUC of blood glucose levels during OGTT were extremely high in all SDT rats. All of the traits of F1 rats were significantly higher than those of control F344 rats, indicating that dominantly acting SDT alleles are involved in the expression of these traits. All of the traits of F2 rats were widely distributed, ranging from normal to extremely high, suggesting the involvement of multiple genes in the expression of these traits. Since body weights were significantly influenced by the duration of diabetes, F2 rats exhibiting diabetes at 57 wk of age or earlier were excluded from the following QTL analysis for body weight.
The finding of glucose intolerance in F1 rats suggests a defect in insulin secretion due to pathological inflammatory changes in the pancreas similar to those in SDT rats. We therefore performed histological analysis of the pancreas of F1 rats (Fig. 2). At 12 wk of age, no or only slight changes were found in the pancreas of F1 rats (data not shown). At 15 wk of age, inflammation was found in and around pancreatic islets (Fig. 2A). Inflammation in the islets with hemosiderin deposition and fibrosis gradually increased (Fig. 2B), and the number of pancreatic β-cells decreased with age (Fig. 2C). However, the inflammatory changes in F1 rats were obviously weak compared with those of SDT rats, and the pancreatic β-cells of F1 rats were yet sufficient to control blood glucose levels under ad libitum dietary conditions. Indeed, none of the F1 rats developed diabetes up to 75 wk of age. These findings indicate that dominantly acting SDT alleles are involved in the pathological inflammatory changes.
Genetic mapping of a major locus for diabetes on chromosome 3.
To genetically map loci for diabetes, we performed a genomewide scan using a total of 167 male F2 rats. By χ2-test using diabetic F2 rats (n = 31) and nondiabetic F2 rats with AUC value of the lowest 20% (n = 33), we found significant deviations (P < 0.05) from expected Mendelian genotype frequencies at several markers on chromosomes 1, 3, 5, 8, 10, 14, and 20 (Table 1 and Supplemental Table S2). Among these, several markers on chromosome 3 showed the strongest deviations from expected Mendelian genotype frequencies (D3Arb18, χ2 [df = 2] = 19.85, P < 0.001), suggesting the presence of a major locus responsible for diabetes. We designated this locus Dmsdt1, for diabetes mellitus locus in the SDT rat no. 1. The strong bias to homozygosity or heterozygosity for the SDT allele in diabetic animals suggests that a dominantly acting SDT allele at Dmsdt1 is involved in the development of diabetes in F2 rats.
QTL analysis of glucose tolerance and body weight.
By QTL analysis, we mapped four significant QTLs affecting glucose tolerance on chromosomes 3, 8, 13, and 14 and one significant QTL affecting body weight on chromosome 3 (Fig. 3 and Supplemental Table S3).
A highly significant QTL affecting blood glucose levels at 60 and 120 min during OGTT was mapped on the Dmsdt1 region on chromosome 3, between D3Arb18 and D3Mit12 (Fig. 3A). The region near D3Mit12 (57 cM) had maximum LOD scores of 5.23 and 5.30 for blood glucose levels at 60 and 120 min, respectively, and accounted for 12% and 13% of the phenotypic variances, respectively (Supplemental Table S3). The mean values for blood glucose levels at 60 and 120 min of F2 rats with the SDT/F344 heterozygous genotype (S/F) at D3Mit12 were comparable to those of F2 rats with the SDT homozygous genotype (S/S) but were significantly higher than those of F2 rats with the F344 homozygous genotype (F/F) (Fig. 4A and Supplemental Table S4). These data indicate that a dominantly acting SDT allele at Dmsdt1 is involved in the higher blood glucose levels in F2 rats.
Two significant QTLs, designated Dmsdt2 and Dmsdt3, affecting blood glucose levels during OGTT and fasting blood glucose level were mapped on chromosomes 8 and 13, respectively (Fig. 3, B and C). Recessively acting SDT alleles at these loci are involved in the higher blood glucose levels in F2 rats (Fig. 4, C–F, and Supplemental Table S4).
A highly significant QTL, designated Dmsdt4, affecting blood glucose levels at 60 and 120 min and AUC during OGTT was mapped on chromosome 14 at D14Arb18 (17 cM) (Fig. 3D). This region showed maximum LOD scores of 5.56, 5.84, and 5.08 and accounted for 12%, 13%, and 11% of the phenotypic variances, respectively (Supplemental Table S3). Mean values for blood glucose levels at 60 and 120 min and AUC of F2 rats with the SDT/F344 heterozygous genotype (S/F) at D14Arb18 were significantly lower than those of F2 rats with the SDT homozygous genotype (S/S) but were significantly higher than those of F2 rats with the F344 homozygous genotype (F/F) (Fig. 4, G and H, and Supplemental Table S4). These data indicate that an additively acting SDT allele at Dmsdt4 is involved in the higher blood glucose levels in F2 rats.
For body weight, we mapped a significant QTL on chromosome 3 near D3Wox9 (29 cM) (Fig. 3A). This region had a maximum LOD score of 4.32 and accounted for 8% of the phenotypic variance (Supplemental Table S3). We designated this QTL Bwsdt1, for body weight QTL in the SDT rat no. 1. A dominantly acting SDT allele at this locus is involved in the higher body weight in F2 rats (Supplemental Table S4).
In addition to the four significant QTLs affecting glucose tolerance described above, suggestive evidence of linkage was found on chromosomes 1, 5, 7, 10, and 20 (Supplemental Fig. S1). At these five regions, except for chromosome 1, the SDT alleles are involved in the higher blood glucose levels and AUC (data not shown).
Congenic analysis of the major diabetogenic locus Dmsdt1.
The phenotypic analysis of F1 rats described above indicates that dominantly acting SDT alleles are involved in glucose intolerance and pathological inflammatory changes in the pancreas. In addition, genetic analysis shows that Dmsdt1 has the strongest effect on the onset of diabetes in which a dominantly acting SDT allele is involved. Because SDT rats exhibit pathological inflammatory changes in the pancreas accompanied by a decrease in the number of pancreatic β-cells (7, 8), a dominantly acting SDT allele at Dmsdt1 might well induce such changes in the pancreas associated with the development of diabetes. Accordingly, we produced a congenic strain for Dmsdt1, termed F344.SDT-Dmsdt1. At 35 wk of age, blood glucose levels during OGTT of congenic rats heterozygous for the SDT allele at the Dmsdt1 region [F344.SDT-Dmsdt1(hetero)] were somewhat higher than those of littermates homozygous for the F344 allele at the Dmsdt1 region (F344-type control), but there was no statistically significant difference (Fig. 5D). At the same age, inflammation in and around pancreatic islets and hemosiderin deposition and fibrosis in the islets were frequently found in congenic F344.SDT-Dmsdt1(hetero) rats (Fig. 5A), which is similar to those found in F1 rats (Fig. 5B). In contrast, there were few pancreatic islets with hemosiderin deposition or fibrosis in F344-type control rats (Fig. 5C). Although the relative abundance of pancreatic islets having hemosiderin deposition and fibrosis in congenic F344.SDT-Dmsdt1(hetero) rats was small compared with that in F1 rats (Fig. 5C), these findings indicate that a dominantly acting SDT allele at Dmsdt1 induces the pathological inflammatory changes in the pancreas.
In the present study, we attempted to identify genetic factors for diabetes, islet inflammation, and fibrosis in SDT rats. For this purpose, we produced (F344×SDT)F2 rats and examined the onset of diabetes, glucose tolerance, and histology of the pancreas. Genetic analysis of diabetes identified a major locus, Dmsdt1, on chromosome 3, at which a dominantly acting SDT allele was involved. By producing a congenic strain for Dmsdt1, we found that the dominantly acting SDT allele induces islet inflammation and fibrosis, and is therefore likely to be involved in the development of the disease. To our knowledge, Dmsdt1 is the first genetic factor for islet inflammation and fibrosis that has been found by genetic linkage and congenic analyses in spontaneous animal models.
There are several differences between our previous and present studies. In the previous study (26), we concentrated on detecting recessively acting genetic factors by use of a backcross (N2 cross) and identified three QTLs. However, in that process, unmapped dominantly acting genetic factors were found to be involved in islet inflammation and fibrosis, which is thought to be the main cause of β-cell decrease in the pathogenesis of diabetes in SDT rats. We therefore attempted to identify the dominantly acting genetic factors by use of the F2 cross in the present study. Indeed, the previously identified three QTLs were not mapped in the present study (Supplemental Fig. S1, A, B, and G), and the four QTLs identified in this study, excepting Dmsdt2, were not detected in the previous study. In addition, the age of phenotyping was different in the two studies. The previous study evaluated glucose tolerance at 20 wk of age, and none of the backcrossed rats had developed diabetes at that age. In the present study, we phenotyped the animals up to 60 wk of age and obtained a considerable number of diabetic animals, permitting linkage analysis to directly identify genetic factors involved in the development of diabetes. Finally, the control rats used for producing the cross differed in the two studies: BN rats were used in the previous study, and F344 rats were used in the present study.
It was found that glucose intolerance and the onset of diabetes were highly correlated with each other in the previous study (26), and all of the diabetic F2 rats showed severely impaired glucose tolerance in this study (AUC of diabetic F2 rats, mean ± SE, 68,565.97 ± 2,177.88, n = 31), suggesting strongly that genes involved in glucose intolerance are also involved in the development of diabetes. We performed QTL analysis for glucose tolerance, using 167 F2 rats and 173 SSLP markers with an average marker spacing of 9.6 cM (maximum spacing of 31.9 cM on chromosome X). Since the numbers of F2 animals and the average marker spacing in the present study are thought to be sufficient to perform ordinary QTL analysis, it is unlikely that there are genes with stronger effect than that of Dmsdt1 in the F2 cross. However, there is a possibility that other unmapped genes with minor effects might be involved. Nevertheless, Dmsdt1 and the other loci mapped in this study are the major genetic factors involved in glucose intolerance and onset of diabetes in this cross.
In SDT rats, most pancreatic β-cells disappear along with pathological inflammatory changes in the pancreas (27, 38). Pathological changes such as fibrosis and hyperplasia in the islet region have been observed in many obese animal models including leptin-deficient ob/ob mice, leptin-resistant db/db mice (2, 5), and leptin-resistant Zucker diabetic fatty rats (33). In these models, insulin resistance induced hyperplasia in islets. Because the SDT rat is a nonobese model of diabetes, the pathological changes in the islet region of SDT and SDT-derived congenic rats differ from those of obese models. While there are several nonobese animal models of pancreatic β-cell death such as the Akita mouse, in which insulin 2 mutation causes endoplasmic reticulum stress in β-cells (31), and the reductase Ncb5or-deficient mouse, in which the redox status is dysregulated in β-cells, resulting in insulin-deficient diabetes (22, 47). In addition, it has been suggested that reactive oxygen species overproduction leads to manifestations of oxidative stress and apoptosis in β-cells in transgenic mouse models (6, 7). Recently, Shimada et al. (35, 36) reported that allogeneic pancreas transplantation under immunosuppression can improve diabetes in SDT rats and the regeneration of β-cells in the native SDT pancreas. In these reports, they suggested that the amelioration of glucose toxicity improved the regeneration of the β-cells. Thus it is possible that β-cell stress and glucose toxicity contribute in part to β-cell death in SDT rats.
By using a congenic strategy, we found that a dominantly acting SDT allele at Dmsdt1 was sufficient for induction of pathological inflammatory changes in the pancreas, suggesting that Dmsdt1 is involved in the development of diabetes in SDT rats. However, the degree of inflammatory change in the congenic rats was remarkably mild compared with that in SDT and F1 rats, suggesting the involvement of other genes, including the three other QTLs identified in this study. Nevertheless, Dmsdt1 is one of the strongest genetic factors for islet inflammation and fibrosis reported so far. The Dmsdt1 region is orthologous to human chromosome 2q23.3–32.1, 11q12.1–p14.2, 15q14–21.1, and 20q13–11.21 and to mouse chromosome 2. Kovács et al. (19) detected a QTL affecting blood glucose levels at 60 min and AUC during intraperitoneal glucose tolerance test (IPGTT) on rat chromosome 3, near D3Mit3 in the Wistar Ottawa Karlsburg W (WOKW) rat. This region contains various transcription factors involved in the development and function of pancreatic β-cells, including NeuroD (14, 20, 24, 28), Pax6 (29, 34, 49), Nkx2-2 (41), HNF-3β (Foxa2) (40, 51), HNF4α (48), and IB2 (TCF7L2) (9, 11). In addition, there are two potent mediators of inflammation and immunity, Il1a and Il1b, in this region. Since these cytokines are important in the control of inflammatory response, the genes are potential candidates for Dmsdt1.
The region on rat chromosome 8 harboring Dmsdt2 affecting blood glucose levels during OGTT is orthologous to human chromosome 7p14.2–14.3, 11q22.3–25, and 15q21.2–21.3 and to mouse chromosome 9. This region is identical to the position of suggestive linkage for glucose tolerance detected in our previous study (26). At nearly the same position of Dmsdt2, Niddm21, a QTL affecting postprandial glucose levels and AUC, was mapped in the OLETF rat (18, 45). In addition, Nidd/gk5, a QTL affecting insulin secretion in response to glucose in the GK rat, was mapped around Dmsdt2 (8). It is unclear at present whether the same gene is responsible for these QTLs.
The chromosome 13 region harboring Dmsdt3 affecting fasting blood glucose level is orthologous to human chromosome 1q31 and 2q14–22 and to mouse chromosome 1. No QTL in this region has been reported to be associated with diabetes-related traits.
The region on rat chromosome 14 harboring Dmsdt4 affecting blood glucose levels during OGTT is orthologous to human chromosome 4q12–21.21 and to mouse chromosome 5. In this region, Obs5, a QTL for retroperitoneal fat pad weight, was mapped in the OLETF rat (30). The quantitative trait affected by Obs5 is different from that affected by Dmsdt4, suggesting that the same gene is not responsible for these QTLs. Interestingly, Pia.6, a QTL associated with arthritis, was mapped to the Dmsdt4 region in the DA rat (42). Analysis of a congenic strain revealed that the DA allele at Pia.6 changes acute arthritis into chronic arthritis (46). Since inflammation in the pancreas is observed in SDT rats, the same gene may be responsible for these QTLs and thus be involved in acceleration of inflammation in the pancreas of SDT rats. To examine the functional role of Dmsdt4, a congenic strain for Dmsdt4 must be investigated.
The region on rat chromosome 3 harboring Bwsdt1 affecting body weight is orthologous to human chromosome 2q23.3–32.1 and to mouse chromosome 2. This region contains several QTLs for body weight including Bw24, 31, and 36 (1, 39) and for body fat amount such as Niddm46 (44) in other strains.
In conclusion, we have identified a major diabetogenic locus, Dmsdt1, on rat chromosome 3, at which a dominantly acting SDT allele is involved. A congenic analysis revealed that an SDT allele at Dmsdt1 induces islet inflammation and fibrosis. Further characterization of Dmsdt1 is required to identify the genes responsible for islet inflammation and fibrosis in the pathogenesis of diabetes.
This study was supported by Grants-in-Aid for Scientific Research and Specially Promoted Research and a grant for the 21st Century Center of Excellence program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
We express our appreciation and thanks to the late Prof. K. Komeda (Tokyo Medical University) and also thank Drs. S. Toyokuni (Kyoto University) and Y. Seino (Nagoya University) for their helpful advice during the course of this study.
↵1 The online version of this article contains supplemental material.
Address for reprint requests and other correspondence: S. Seino, Div. of Cellular and Molecular Medicine, Dept. of Physiology and Cell Biology, Kobe Univ. Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan (e-mail:).
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