Genetic dissection of complex genetic factor involved in NIDDM of OLETF rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, noninsulin-dependent diabetes mellitus (NIDDM) in humans. NIDDM in this rat model was shown to be regulated by multiple genes. We have identified 14 quantitative trait loci (QTLs) responsible for NIDDM (Nidd1-14/of) on chromosomes 1, 5, 7, 8, 9, 11, 12, 14, 16, and 17 by a whole genome search in 160 F2 progenies obtained by mating the OLETF and the F344 rats. Among these loci, two QTLs, Nidd1 and 2/of, were declared significant loci at a genome-wide level. Nidd3, 8, 9, and 13/of exhibited heterosis: heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. We also found evidence for interaction (epistasis) between Nidd1/of and Nidd2/of, between Nidd1/of and Nidd10/of, between Nidd2/of and Nidd8/of, and between Nidd2/of and Nidd14/of. Furthermore, Nidd6 and 11/of showed linkage with body weight, and Nidd1, 2, 8, 9, 10, and 12/of had an interaction with body weight. These indicated that NIDDM in the OLETF would have a higher degree of genetic complexity. We suggest several interesting candidate genes located in rat genomic regions for Nidd1-14/of or the syntenic regions in human genome.

OLETF allele of hyperglycemic QTL Nidd3/of is dominant.

The OLETF rat is a well-established model for the study of type 2 diabetes associated with obesity and has been shown to possess multiple hyperglycemic alleles in its genome. Here we focused on and carefully characterized one of the previously reported congenic strains, F.O-Nidd3/of that carries the OLETF allele of the Nidd3/of locus (also known as Niddm21 in the Rat Genome Database) in the normoglycemic F344 genetic background. A prominent finding was that the F1 progeny between the congenic and the F344 stain, whose genotype is heterozygote at the Nidd3/of locus, showed mild hyperglycemia equal to the parental congenic rat, suggesting that the OLETF allele is dominant. To our knowledge, this is the first study in which a diabetic QTL has been directly demonstrated to be dominant by using congenic strains.

Pnlip encoding pancreatic lipase is a possible candidate for intramuscular fat accumulation QTL in OLETF rat.

A quantitative trait locus (QTL) responsible for intramuscular fat accumulation in Musculus longissimus of Otsuka Long-Evans Tokushima Fatty (OLETF) rat, Imfm, was previously mapped to the approximately 10-cM genomic region between D1Rat166 and D1Rat90 on chromosome 1 using Imfm congenic strain. In this study, we refined the Imfm region to a approximately 2.3-cM genomic region between D1Rat225 and D1Rat90, using 12 informative recombinants selected from 176 (Imfm congenic x F344) F, x Imfm congenic backcross progenies. Among 46 genes located within the approximately 2.3-cM region, pancreatic lipase gene, Pnlip, that is a possible candidate for obesity QTL, Nidd6/of, was thought to be the most prominent and physiologically relevant positional candidate for the Imfm QTL. It was previously showed that Pnlip possesses an OLETF allele-specific increase of mRNA levels in the pancreas, and that the OLETF allele is longer in variable number of tandem repeat (VNTR) within the 5'-flanking region than normal alleles. We found complete cosegregation of the Imfm QTL with Pnlip VNTR in the 12 informative recombinants, suggesting that Pnlip is also a possible candidate for the Imfm QTL.

Calpain 10 as a predictive gene for type 2 diabetes: evidence from a novel screening system using white blood cells of Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

The mRNA expression of type 2 diabetes-related genes in white blood cells (WBC) was examined before and after onset in Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The level of the calpain 10 (CAPN10) transcript was significantly decreased compared to control animals in WBC before and after onset. Significant decreases in this gene expression were also found in the major insulin-target tissues as well as WBC before onset. These results suggest that gene expression in WBC could be a useful screening system for predicting the incidence of type 2 diabetes before onset in OLETF rats, and that CAPN10 represents a potential candidate gene for predicting type 2 diabetes in human.

Examination of OLETF-derived non-insulin-dependent diabetes mellitus QTL by construction of a series of congenic rats.

The Otuska Long-Evans Tokushima Fatty (OLETF) rat is one of the well-characterized animal models for the study of type 2 diabetes. Our previous QTL mapping identified 11 loci responsible for non-insulin-dependent diabetes mellitus (NIDDM) susceptibility in the OLETF rat. Here we generated a series of congenic animals by individually introgressing all 11 OLETF-derived NIDDM loci into a normoglycemic F344 background. Subsequent oral glucose tolerance test revealed that the congenic strains for Nidd1/of, Nidd2/of, Nidd3/of Nidd4/of, Nidd7/of, and Nidd10/of showed significantly higher levels of blood glucose in comparison with parental host strain F344. Furthermore, simultaneously made heterozygote animals for Nidd1/of and Nidd2/of did not increase blood glucose levels, indicating that these loci are recessively inherited as predicted by the QTL analysis. Congenic strains for the other five loci-Nidd5/of, Nidd6/of, Nidd8/of, Nidd9/of, and Nidd11/of-were apparently normoglycemic, presumably owing to heterosis or because the effect of these loci may not be detected unless interactions with other OLETF genes exist. We believe that these congenic strains should provide useful agents for decomposing complex diabetic traits and for positional cloning.

Enhanced expression of adhesion molecules of the retinal vascular endothelium in spontaneous diabetic rats.

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are a spontaneously diabetic strain with clinical features resembling those of human noninsulin-dependent diabetes mellitus. These rats show increased leukocyte entrapment in the retina. The present study was designed to investigate the immunolocalization of intercellular adhesion molecules-1 (ICAM-1) and P-selectin in the retinas of OLETF rats. Four 72-week-old male OLETF rats and 4 age- and sex-matched Long-Evans Tokushima Otsuka (LETO) control rats were used. Western blot analysis and immunohistochemical study were performed using an anti-P-selectin monoclonal antibody (mAb) and an anti-ICAM-1 mAb. Western blot analysis showed increased expression of both ICAM-1 and P-selectin in the retinas of OLETF rats. Immunohistochemically, OLETF rats expressed greater amounts of ICAM-1 and P-selectin in the retinal vascular endothelium than did LETO rats. These findings demonstrated the upregulation of ICAM-1 and P-selectin in the retina of OLETF rats. The enhanced expression of these adhesion molecules might participate in the pathogenesis of early diabetic microangiopathy in the retina.

Detection of a quantitative trait locus for intramuscular fat accumulation using the OLETF rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese type 2 diabetes. We showed that the OLETF rat exhibits higher levels of intramuscular fat content in Musculus longissimus as compared to the Fischer-344 (F344) rat. Our investigation was designed to identify quantitative trait loci (QTLs) contributing to the increased levels of intramuscular fat content by performing a whole-genome search using 108 F2 intercross obtained by mating the OLETF and the F344 rats. We identified one QTL responsible for intramuscular fat accumulation on rat chromosome 1 with a maximum lod score of 3.4, which accounts for 5% of the total variance. As expected, the OLETF allele corresponds to the increased levels of intramuscular fat content.

Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats.

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is an animal model of type 2 diabetes, characterized by abdominal obesity, insulin resistance, hypertension, and dyslipidemia. To elucidate the underlying molecular mechanism of obesity and its related complications, we used representational difference analysis and identified the genes more abundantly and specifically expressed in the visceral adipose tissue (VAT) of obese OLETF rats compared with the diabetes-resistant counterpart, that is, Long-Evans Tokushima Otsuka (LETO) rats. By Northern blot analysis, we confirmed the differential expression of 13 genes, including 3 novel genes. The upregulated expression of well-characterized lipid metabolic enzymes, such as lipoprotein lipase, phosphoenolpyruvate carboxykinase, and cholesterol esterase, were observed in VAT of OLETF rats. We demonstrated the differential expression of secreted proteins in VAT of OLETF rats, such as thrombospondin 1 and contrapsin-like protease inhibitor. In contrast to lipid enzymes, the secreted proteins revealed exclusive mRNA expression and they were not detected in VAT of LETO rats. Furthermore, the novel genes OL-16 and OL-64 were also expressed specifically in VAT of OLETF rats and were absent in that of LETO rats and other tissues, including subdermal and brown adipose tissues. The C-terminal partial amino acid sequence of OL-64 revealed that it showed approximately 40% homology with alpha(1)-antitrypsin and it seemed to be a new member of the serine proteinase inhibitor (SERPIN) gene family. VAT of OLEFT rats had a unique gene expression profile, and the accumulated VAT-specific known and novel secreted proteins may play a role(s) in the pathogenesis of obesity and its related complications.

Identification of novel non-insulin-dependent diabetes mellitus susceptibility loci in the Otsuka Long-Evans Tokushima fatty rat by MQM-mapping method.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. We have previously identified 11 quantitative trait loci (QTLs) responsible for NIDDM susceptibility on Chromosomes (Chrs) 1, 5, 7, 8, 9, 11, 12, 14, and 16 (Nidd1-11/of for Non-insulin-dependent diabetes 1-11/oletf) by using the interval mapping method in 160 F(2) progenies obtained by mating the OLETF and the Fischer-344 (F344) rats. MQM-mapping, which was applied for QTL analysis based on multiple-QTL models, is reported to be more powerful than interval mapping, because in the process of mapping one QTL the genetic background, which contains the other QTLs, is controlled. Application of MQM-mapping in the F(2) intercrosses has led to a revelation of three novel QTLs on rat Chrs 5 (Nidd12/of), 7 (Nidd13/of), and 17 (Nidd14/of), in addition to Nidd1-11/of loci. The three QTLs, together with the Nidd1-11/of, account for a total of approximately 70% and approximately 85% of the genetic variance of the fasting and postprandial glucose levels, respectively, in the F(2). While the OLETF allele corresponds with increased glucose levels as expected for Nidd12 and 14/of, the Nidd13/of exhibits heterosis: heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. There is epistatic interaction between Nidd2 and 14/of. Additionally, our results indicated that the novel QTLs could show no linkage with body weight, but Nidd12/of has an interaction with body weight.

Mapping and characterization of quantitative trait loci for non-insulin-dependent diabetes mellitus with an improved genetic map in the Otsuka Long-Evans Tokushima fatty rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type, non-insulin-dependent diabetes mellitus (NIDDM) in humans. We have previously reported four quantitative trait loci (QTLs) responsible for NIDDM on Chromosomes (Chrs) 7, 14, 8, and 11 (Nidd1-4/of for Non-insulin-dependent diabetes1-4/oletf) by a whole-genome search in 160 F2 progenies obtained by mating the OLETF and the Fischer-344 (F344) rats. Our present investigation was designed to identify and characterize novel QTLs affecting NIDDM by performing a genome-wide linkage analysis of genes for glucose levels and body weight and analysis for gene-to-gene and gene-to-body-weight interactions on an improved genetic map with a set of 382 informative markers in the 160 F2 progenies. We have identified seven novel QTLs on rat Chrs 1 (Nidd5 and 6/of), 5 (Nidd7/of), 9 (Nidd8/of), 12 (Nidd9/of), 14 (Nidd10/of) and 16 (Nidd11/of) which, together with the Nidd1-4/of, account for a total of approximately 60% and approximately 75% of the genetic variance of the fasting and postprandial glucose levels, respectively, in the F2. While the OLETF allele corresponds with increased glucose levels as expected for the novel QTLs except Nidd8 and 9/of, the Nidd8 and 9/of exhibit heterosis: heterozygotes showing significantly higher glucose levels than OLETF or F344 homozygotes. There are epistatic interactions between Nidd1 and 10/of and between Nidd2 and 8/of. Additionally, our results indicated that the Nidd6 and 11/of could also contribute to an increase of body weight, and that the other five QTLs could show no linkage with body weight, but Nidd8,9, and 10/of have an interaction with body weight.

Identification of a quantitative trait locus influencing plasma insulin levels after 70% pancreatectomy using the OLETF rat.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for obese-type non-insulin-dependent diabetes mellitus (NIDDM) in humans. The OLETF rat has poor capacity for proliferation of pancreatic beta-cells after partial pancreatectomy, which may be the critical pathogenetic event in NIDDM development. The poor pancreatic beta-cell proliferation in this model is characterized by reduction in beta-cell mass and decrease in insulin content in the remnant pancreas. Our investigation was designed to identify quantitative trait loci (QTLs) responsible for beta-cell mass and plasma insulin levels after partial pancreatectomy by performing a genome-wide scan in an F2 intercross obtained by mating the OLETF and the Fischer-344 (F344) rats. We have identified a suggestive QTL for the plasma insulin levels, near D20Mgh5 on rat chromosome 20, with a maximum lod score of 3.75 which accounts for 20% of the total variance, while no QTLs were detected for beta-cell mass. This chromosome 20 QTL, whose OLETF allele is associated with low plasma insulin levels through acting in an incompletely recessive manner, may affect insulin secretion itself rather than beta-cell proliferation.