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Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544
Correspondence: Address all correspondence and requests for reprints to: Alan D. Attie, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706-1544. E-mail: attie{at}biochem.wisc.edu
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Abstract |
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 Top Abstract I. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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I. Physiological Definition |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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Hyperglycemia may result from either a relative or absolute insulin deficiency. Absolute deficiency results from a loss of insulin production, e.g., the immunological destruction of ß-cells that occurs in type 1 diabetes. Relative deficiency, as seen in type 2 diabetes, may occur when there is an increased demand for insulin to compensate for a blunted tissue response to the hormone, termed insulin resistance. In this context, insulin production may be insufficient to maintain normoglycemia, despite a relatively robust insulin secretory response. Thus, diabetes can occur in an individual with higher than normal insulin levels. This failure of islets and/or individual ß-cells to maintain sufficient insulin production is an important component of type 2 diabetes in addition to insulin resistance. Thus, in our descriptions of the mouse models below, we have tried to distinguish models of insulin resistance from those that also exhibit ß-cell failure.
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II. The High Prevalence of Diabetes: Implications for Genetics |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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Approximately one half of the risk for type 2 diabetes is attributable to genetic variation. Twin studies have shown high concordance rates (>80%) for monozygous twins; however the rate is not 100%, demonstrating the effects of environmental factors in the etiology of the disease (6, 7, 8, 9). Individual phenotypes associated with type 2 diabetes, such as ß-cell function and insulin resistance, often have even greater heritabilities (7, 10, 11, 12, 13, 14, 15, 16, 17, 18). Despite the strong genetic component, however, genes with major effects do not account for the bulk of diabetes susceptibility alleles. Together, the genes underlying the rare monogenic forms of diabetes [e.g., maturity onset diabetes of the young (MODY)] account for only a small proportion of the genetic variation associated with type 2 diabetes (7, 19, 20). Instead, numerous genes each contribute a small increase in risk, although if present at high enough frequencies, they may contribute substantially to the population risk, e.g., the Pro12Ala substitution in PPARG (6, 21, 22).
The rapidity of the increase in diabetes onset, coinciding with the adoption of Western lifestyles, suggests that environmental stressors are unmasking prior genetic predisposition. The high incidence of a disorder with a large genetic component suggests that diabetogenic alleles might have experienced positive selection at previous times in human history. The "thrifty gene hypothesis" states that alleles that favor efficient fuel absorption and retention would experience positive selection in times of famine (23, 24). These same alleles would promote diabetes in times of nutritional excess. This hypothesis predicts that regions of the world with populations descended from those having experienced famine will experience a spike in diabetes as they adopt Western-style diets high in caloric density.
Identification of the genetic variants underlying the world diabetes epidemic is tantamount to a genetic-sensitized screen. Millions of people are being exposed to an unprecedented level of metabolic stress in the form of caloric excess. Alleles that were hitherto phenotypically silent are now leading to diabetogenic phenotypes. Although tragic from a public health standpoint, this worldwide gene environment experiment will likely yield new insights into the key bottlenecks in pathways that maintain glucose homeostasis.
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III. What Does Genetics Offer? |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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The correlation between biochemical measurements and disease suffers from the difficulty in establishing causality. A correlation can occur by chance, it can occur because of a common etiology, or there can be a true cause-effect relationship between the biochemical measure and disease. Genetics offers the power to establish unambiguous models of causality; genetic variation leads to phenotypic variation in a one-way direction. Linkage studies, demonstrating the coinheritance of a specific region of the genome along with the trait of interest, thus require analysis of related individuals and are limited in resolution primarily by the number of individuals. Cross-sectional population-based studies, where large cohorts can more easily be recruited, are necessarily correlational (i.e., showing a correlation between inheritance of a particular marker and phenotype), and do not establish direct causality.
Recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping have greatly increased the markers available for linkage analysis and fine-mapping and are allowing a better understanding of the haplotype structure of the genome (25). With this knowledge, it is possible to identify conserved haplotype blocks inherited from a more distant common ancestor that cosegregate with the trait of interest. As such, whole genome association studies with very dense SNP genotyping (hundreds of thousands of SNPs per individual) are being performed. This will allow interrogation of the genome at a heretofore unprecedented level of resolution. The assembly of very large, well-defined cohorts and collaborative efforts among multiple groups will also aid these efforts and, indeed, is already yielding success (26). These advances suggest that the pace of gene discovery in complex human disease will greatly accelerate. Several recent reviews of human diabetes genetics describe the progress to date (6, 7, 8, 9, 19, 21, 27, 28).
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IV. Advantages of Model Organisms such as the Mouse |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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Once a linkage region has been identified, one is able to deconstruct a complex trait, breaking it into individual components, through the generation of congenic strains wherein a QTL region from one strain background is introgressed into the other strain background. This creates individuals that are genetically identical except at the locus of interest. For example, a type 2 diabetes-susceptible strain may have separate loci affecting insulin secretion, insulin resistance, and ß-cell survival. Congenics for each of these loci allow each trait to be studied independently. In addition to fine-mapping, this process can also separate multiple QTL lying under the same initial logarithm of the odds (LOD) peak. Nowhere has this been better demonstrated than with the type 1 diabetes susceptibility loci in the nonobese diabetic (NOD) mouse, or type 2 diabetes susceptibility loci in the Goto Kakizaki (GK) rat (29, 30, 31, 32).
For complex diseases like type 2 diabetes, where numerous factors, both genetic and environmental, can affect the trait being studied, the strength of the correlation between variation at a particular gene locus and a phenotype is affected by how far removed the underlying gene is from the phenotype. For example, fasting insulin levels are influenced by insulin sensitivity, insulin clearance, and insulin secretion. Insulin secretion, in turn, is affected by the total ß-cell mass and by the ability of individual ß-cells to sense and respond to glucose. Similarly, numerous processes underlie insulin resistance, and so forth. Thus, although these traits are still complex, they have less genetic heterogeneity than a broader trait, such as plasma insulin levels (14). Studies that focus on these individual components of the phenotype are more likely to detect genetic linkages than studies that merely measure fasting insulin. Mapping sensitivity and fine-mapping may also be increased by studying more detailed phenotypes that are closer to the underlying etiology of the phenotype, thereby reducing the noise from other genetic and environmental impacts on more downstream phenotypes. Extremely detailed phenotypic refinement may even lead to identification of the specific process affected (e.g., glucose sensing vs. vesicle trafficking in models of insulin secretory defects). Animal models offer an advantage in that tissue samples are available for analysis to allow this greater phenotyping capability. In addition, phenotyping is performed in a controlled environment, reducing environmental noise. This also allows for greater flexibility in examining gene-environment (such as gene-diet) interactions on QTL.
The mouse strains together contain as much genetic diversity as the human population; however, much less of this diversity is represented in the commonly used inbred strains (33). Recent SNP analysis suggests that all the commonly used strains are derived from a limited number of founder strains, setting an upper limit to the number of ancestral alleles at any given locus (34, 35). Nonetheless, the haplotype patterns across the strains are complex (36, 37, 38, 39). The strains differ in their susceptibility to disease and in their response to environmental factors like diet. Strain background has been shown to influence most aspects of diabetes including insulin secretion, insulin resistance, and pancreatic characteristics such as ß-cell survival and islet number.
Through the years, many spontaneous mutations have arisen and been preserved as valuable new animal models. The obese (ob) mouse with a mutation in the gene for leptin (Lep) and diabetes with a mutation in the leptin receptor (Lepr) are prominent examples (40, 41, 42). Similarly, other mutations result in spontaneous obesity and severe insulin resistance, such as the fat mutation in carboxypeptidase E (Cpe), the tubby mutation, and the ectopic expression induced by the yellow (y) allele of agouti (Ay) (43, 44, 45, 46). However, the diabetic phenotype associated with these mutations is highly dependent upon the strain background in which they are studied (as described in the sections below). The Akita mouse represents the only spontaneous mutation in mouse models directly causing nonobese type 2 diabetes (47). We have summarized the features of this model later in this review.
Only approximately 300 genes exist in humans that were not identified in the initial mouse genome assemblies (48, 49). There are large regions of synteny between the mouse and human genomes (49), thus linkages are easily translated between genomes. Under contract from the National Institutes of Health, Perlegen, Inc., is sequencing the genomes of 15 commonly used and genetically diverse mouse strains to capture the spectrum of genetic variation that exists within these strains. Additional projects are under way to genotype hundreds of thousands of SNPs in numerous strains to allow for haplotype construction and inference of genotypes of strains other than those being sequenced (35). The Mouse Phenome Database contains quantitative phenotype information for most of the commonly used mouse strains, with the ability to correlate these phenotypes with the available SNP data (50, 51). These valuable resources will greatly aid positional cloning projects in the mouse. A listing of these useful web sites is provided in an appendix to this review.
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V. Origin of Inbred Mouse Strains |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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Most of the strains in use today derived from four subspecies of the house mouse (Mus musculus) that diverged approximately 1 million years ago: the domesticus subspecies that evolved in Western Europe; the musculus subspecies that evolved in Eastern Europe, Russia, and Northern China; the castaneus subspecies that evolved in Southeast Asia and Southern China; and the molossinus subspecies, which is a hybrid of the castaneus and musculus subspecies, from Japan (34, 53). The genealogies of the commonly used mouse strains have been described, and more recently, detailed strain haplotypes have been inferred from SNP analysis (33, 34, 36, 37, 38, 54, 55, 56, 57). This has confirmed that the majority of inbred strains arose from a limited number of founders, primarily of the domesticus and musculus subspecies (34), and has also demonstrated the complex haplotype patterns that exist across strains (39, 57, 58).
Each strain bears a unique combination of disease susceptibility and resistance loci, with the phenotype observed in each strain due to the composite effects of all these loci. Strains that are not diabetic may harbor diabetes-promoting alleles that are masked in an otherwise protective background, but the effects of these alleles may be observed when placed in the context of other diabetogenic genes. Thus, genetic analysis in animals whose genomes have been combined can reveal diabetogenic alleles contributed by the nondiabetic parental strain. Phenotypic differences across these strains exist for the majority, if not all, phenotypes that have been assessed, and this wealth of diversity has enabled genetic mapping of qualitative and quantitative trait loci controlling these differences.
In Section VI, we describe the genetic and genomic methods that can lead to gene discovery. In Section VII, we summarize the phenotypes of the mouse strains used in diabetes research and the linkages that have been described in these models. We identify examples where specific genes related to diabetes have been identified. We have avoided mention of the numerous insights that have been obtained through transgenic and knockout models, because these have been reviewed extensively elsewhere (59, 60, 61, 62), and we only refer to them where strain has been shown to modify the phenotype.
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VI. Experimental Strategies for Gene Mapping |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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When a trait is scored in a binary fashion (affected vs. unaffected), then every new individual is informative and helps to narrow the position of the gene responsible for the trait. In an experimental cross for a dichotomous trait, the mapping resolution is simply a function of the sample size. But, for a quantitative trait like blood glucose or insulin, one cannot score animals as affected or unaffected because their values fall on a continuum. The mapping resolution is largely limited by the effect size of the locus (63). QTL often have small effect sizes. Thus, even with infinitely dense markers, mapping resolution is still limited for most quantitative traits. This helps us to understand why only about 1% of genes underlying the more than 2,000 rodent QTL have been identified (64).
B. Backcross and intercross
In mouse genetics, the most common method for gene mapping is to breed together the two inbred strains in which a phenotypic difference has been detected. These F1 offspring are then bred to one or both of the parental stains (backcross) or to each other (intercross) to generate an F2 sample (Fig. 1
). Each time an F1 animal makes gametes, it generates recombinant chromosomes, with each gamete containing a unique distribution of the parental alleles. Thus, a backcross generates mice with discernible products of recombination on the haploid set of chromosomes inherited from the F1 parent, whereas in an intercross between two F1 animals, one can find recombinants on each transmitted chromosomal strand. The offspring of a backcross are either homozygous for one parental genotype or heterozygous. Thus, only alleles acting in a dominant fashion can be detected. In contrast, an F2 has all three possible genotypes at any locus, i.e., homozygotes for each parental allele and the heterozygote, and thus provides information on additivity and dominance. To obtain a picture of all the QTL, an F2 has a higher degree of power, requiring fewer individuals. However, for major dominant QTL, a backcross may have more power (65).
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C. Recombinant inbred strains
One disadvantage of backcross and F2 samples is that each test animal is unique; thus there is no possibility of biological replication. Recombinant inbred (RI) strains are produced by brother-sister mating of pairs of F2 mice and their offspring (Fig. 2). After 20 generations, each new line of animals is essentially inbred and has fixed in their genomes a unique mosaic of alleles from the two parental strains. Unlike an F2 sample, a RI panel is homozygous at all loci. RI strains were first used in genetic mapping in 1971 (52). Owing largely to the efforts of Benjamin Taylor, The Jackson Laboratory created several panels of RI strains from widely used strains differing in susceptibility to various diseases and infections. Mapping a trait simply requires scoring that trait in the panel of strains and comparing the strain distribution pattern of the trait with that of the marker genotypes. The panel need only be genotyped once, but because each strain is propagated, animals can be phenotyped on multiple occasions across time and even institutions. This allows all data obtained on an RI panel to be correlated to identify common loci affecting multiple phenotypes.
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A second design that maintains the reproducibility of RI strains but creates more genetic diversity is the RI intercross, which creates F1 animals from each pair of RI strains in the panel (74). Finally, additional backcrossing before inbreeding generates recombinant congenic strains (RCS) in which each strain carries randomly distributed congenic segments of donor genome, inversely proportional in amount to the number of backcrosses performed. For example, each strain from an N3 backcross (an F1 backcrossed twice), will have a random distribution of roughly 12.5% of the donor genome in the recipient background. Several RCS panels have been created, including a reciprocal set between the commonly used A/J and C57BL/6 strains (75).
D. Chromosome substitution (consomic) strains
Chromosome substitution strains (CSS) are created by introgressing an entire chromosome from a donor strain into a background strain. Nadeau and colleagues (76) created a panel of 22 strains that are genetically C57BL/6, except that each contains a single chromosome derived from the A/J strain, which differs from the C57BL/6 strain in many traits. A parallel effort is under way to produce two panels, in which the chromosomes of the Brown Norway strain are each being introgressed into either the Dahl salt-sensitive or the Fawn Hooded Hypertensive rat (77). Lusis and colleagues (78) have created a panel in which various large segments (not entire chromosomes, but overlapping large segments that span chromosomes) of DBA/2 have been introgressed into the C57BL/6 background and have shown that these strains differ in plasma glucose and insulin levels. Genome-tagged mice, in which overlapping segments spanning the entire genome of one strain are introgressed into a different strain have also been created in which DBA/2 or CAST/Ei segments have been introgressed into the C57BL/6 background (79). The power to detect QTL is higher in CSS panels than in an F2 intercross because there is less genetic variance introduced by background loci and because fewer markers need to be tested (80). However, epistatic interactions between donor-derived loci on different chromosomes cannot be detected, and extensive fine-mapping of the resultant loci is required to identify the underlying genetic variant(s).
E. Sensitized screens
A sensitized screen is one in which the animals are rendered more sensitive to a stress by some prior manipulation, and then the response of the animals to the stressor is mapped. In diabetes, this is commonly performed by feeding normally non- or mildly insulin resistant animals high-fat or high-carbohydrate diets, or by studying the response of the animals to a genetic defect that induces insulin resistance, to identify alleles that modify this phenotype. The most commonly used models have been the monogenic obesity mutations, obese (ob) and diabetes (db) in the leptin (Lep) and leptin receptor (Lepr) genes, respectively. The classical studies of Coleman and Hummel (81) were the first to show that strain background affects diabetes susceptibility in mice.
F. Outbred stocks
A powerful alternative to crosses between inbred strains is the generation of outbred stocks (Fig. 3). In analogy to human populations, many generations of outbreeding yield a mosaic that is substantially more genetically fragmented than an F2 intercross. One set, consisting of eight founder strains, was bred for more than 60 generations and now has an average recombination distance of just 1.7 cM (82). An association study can be performed on a sample of 1–2,000 mice derived from this stock and have a power to detect a QTL with a resolution of 0.5–1.0 cM. These studies require dense genotyping, but this is now being facilitated by various automation schemes and genomewide SNP panels. This is similar to an association study in a human population, with the added advantage of knowledge of the haplotype structure of the ancestral strains. An elegant variation of the association study is to cross outbred animals to an inbred strain to create an outbred-inbred F2 (83). Mapping can then be done in two steps, first at low-resolution with a small number of markers, then at higher resolution in selected regions of the genome showing evidence of linkage to the trait. Recent success with this approach has come in the positional cloning of a QTL for anxiety (84).
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VII. Mouse Strains Used in Diabetes Research |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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A. C57BL/6
The C57BL/6 strain was created by Little in 1921 from a mating of female no. 57 and male no. 52 from Lathrop’s stock. SNP analysis indicates that the C57BL/6 strain shares some Mus musculus molossinus genomic regions and is somewhat removed evolutionarily from the other inbred strains, except the other C57-derived strains and C58 (36, 37, 88). The C57BL/6 strain is the most widely used of all inbred strains, accounting for some 14% of all such uses. Several RI panels have been generated with C57BL/6 as one strain (72). C57BL/6 is the most commonly used reference strain in studies of diabetes.
There is some confusion about whether or not one should consider the C57BL/6 strain to be "diabetes-susceptible" or "diabetes-resistant." Both protective and diabetes susceptibility alleles/phenotypes have been observed in this strain. Thus, the phenotype is largely relative to the strain it is being compared with and may depend on the criteria by which diabetes has been defined. C57BL/6 may be best described as a strain of intermediate susceptibility. Indeed, data from the Mouse Phenome Database shows that glucose and insulin levels of this strain are intermediate to those of the other strains (2).
Several studies have provided evidence of increased diabetes susceptibility in the C57BL/6 strain. In parallel to studies of diet-induced atherosclerosis susceptibility, the C57BL/6 mouse emerged as susceptible to diet-induced obesity and hyperglycemia (89, 90, 91, 92). Studies from the Surwit laboratory showed that a diet enriched with fat and carbohydrate (mainly from disaccharides) produced a roughly 50% increase in fasting plasma glucose and a 10.4-fold increase in plasma insulin in C57BL/6 mice (91). By contrast, glucose remained nearly unaltered, and insulin rose only 2.6-fold in A/J mice, demonstrating enhanced diet-induced insulin resistance and hyperglycemia in C57BL/6. Kaku et al. (93) found C57BL/6 to be the least glucose tolerant of six strains fed a normal chow diet. More recently, Goren et al. (94) compared glucose and insulin tolerance of C57BL/6, C57BLKS/J, DBA/2, and 129X1. Consistent with the aforementioned study of Kaku et al. (93), C57BL/6 was inherently less glucose tolerant in a glucose tolerance test. But, in an insulin tolerance test, the two C57 strains were more sensitive to the exogenously added insulin than DBA/2 or 129X1, suggesting that a defect in insulin secretion is the likely cause of the glucose intolerance of the C57BL/6 mice. An in vivo study that assessed insulin sensitivity and insulin secretion during the early and late stages of feeding a high-fat diet to C57BL/6 mice concluded that after longer-term feeding, although total insulin secretion was sufficient to compensate for insulin resistance, defective acute insulin secretion in response to glucose is a major contributor to glucose intolerance (95).
Other investigators have also identified an insulin secretion defect in the C57BL/6 strain compared with other strains (93, 96, 97, 98, 99). In perifusion experiments, islets from high-fat, high-simple carbohydrate diet-fed C57BL/6 mice had markedly impaired secretion of insulin in response to high (27.7 mM) glucose compared with A/J (100, 101). This was primarily attributed to a defect in the second-phase insulin secretion. Furthermore, C57BL/6 islets have been shown to be unresponsive to intermediate glucose levels (10 mM), due to incomplete closure of their KATP channels (98).
These studies are consistent with the C57BL/6 mouse having a ß-cell defect in glucose responsiveness, which can, to a certain point in vivo, be offset through the generation of sufficient ß-cell mass. In vivo, ß-cells from this mouse strain have very high proliferative capacity (102). A study by Bock and co-workers surveyed the pancreatic architecture of several strains. In comparison to the others, lean C57BL/6 have a relatively low total islet mass and ß-cell mass when expressed per body mass. But, C57BL/6 had the highest number of islets, albeit the smallest islets, consistent with a large foundation from which ß-cell mass can expand when stressed (103). However, this relatively small existing ß-cell pool may also contribute to the observations that C57BL/6 is more sensitive than other strains of mice to the ß-cell destructive effects of streptozotocin (104, 105).
To identify genes involved in the glucose intolerance of the C57BL/6 strain, Toye et al. (98) created an F2 intercross from C57BL/6 and C3H/HeH. The mice were phenotyped for their response to glucose and insulin tolerance tests. Fasting insulin, postglucose insulin, and postinsulin glucose linked to distinct loci, confirming that variation in each of these parameters has a genetically distinct cause.
A QTL on chromosome 13 that was linked to postglucose insulin levels contains the nicotinamide nucleotide transhydrogenase (Nnt) gene. Sequencing of the cDNA for this gene revealed a M35T polymorphism within the mitochondrial leader sequence. The C57BL/6 allele also has an in-frame deletion of exons 6 through 11, leading to a transcript of approximately 508 instead of approximately 1,261 bp. The gene is expressed at a much lower level in C57BL/6 than in C3H/HeH islets. The syntenic region to the mouse Nnt locus on human chromosome 5p15 is one of three loci linked with a genetic modifier of age of onset of diabetes in families with HNF1
(MODY3) mutations (106).
Nnt is an inner mitochondrial membrane enzyme that catalyzes the exchange of reducing equivalents between nicotinamide-adenine dinucleotide (NAD+) and NAD phosphate (NADP+). The reaction is coupled to the transport of protons from the cytosol to the mitochondrial matrix: NADH + NADP+ + H+cytosolic 
NAD+ + NADPH + H+matrix.
Together with the QTL mapping study, a very strong case implicates Nnt as the gene responsible for impaired insulin secretion in the C57BL/6 mouse. Knockdown of Nnt expression in the Min6 insulinoma cell line abolished the insulin secretion normally observed in response to 10 mM glucose (107). The same group screened an N-ethyl-N-nitrosourea (ENU)-mutagenized DNA archive and found gametes from two mutant mice with different Nnt nonconservative missense mutations, one in the NAD+ binding domain and the other in a transmembrane domain. Mice expressing either of these mutant alleles had impaired glucose tolerance, and islets from these mice had impaired glucose-stimulated insulin secretion. In short, the mutant mice reproduced the insulin secretion phenotype of the C57BL/6 mouse. Finally, transgenic expression of the 129S6/SvEvTac allele of the Nnt gene rescued the insulin secretion phenotype of the C57BL/6 mouse (108).
It is noteworthy that this gene was identified and implicated in insulin secretion through genetic linkage rather than a biochemical hypothesis. How would loss of Nnt function reduce insulin secretion in response to glucose? Islets harboring the mutant Nnt allele produce a much higher amount of reactive oxygen species in response to 20 mM glucose (107), supporting the prior hypothesis that Nnt detoxifies reactive oxygen species (109). Because reactive oxygen species have been shown to increase uncoupling of oxidative phosphorylation, Nnt would be predicted to indirectly affect mitochondrial ATP yield and thus the extent of the closure of the ATP-sensitive K+ channels. This prediction is consistent with the reduced glucose utilization seen in islets from the mutant mice (107).
Another way to metabolically stress mice to find strain differences in diabetes susceptibility is to induce insulin resistance by introgressing null alleles in genes required for insulin signaling. When C57BL/6, DBA/2, and 129X1/Sv mice were created that were double heterozygotes for null alleles at the insulin receptor and insulin receptor substrate loci, all developed severe insulin resistance, as expected (110). The C57BL/6 mice developed glucose intolerance and dramatic hyperinsulinemia, which correlated with a striking 22-fold increase in ß-cell mass. The DBA/2 mice also developed hyperinsulinemia, but did not have as severe glucose intolerance or as strong a ß-cell proliferative response as the C57BL/6 mice. Thus, it was the C57BL/6 mice that had a much higher prevalence of diabetes.
Despite the decreased insulin secretion and glucose intolerance of the C57BL/6 strain, C57BL/6 mice are relatively resistant to obesity-induced diabetes when compared with several other strains, as described below. This was revealed in the classic experiments of Douglas Coleman conducted over 30 yr ago. His experiments were the first to show that strain background affected the susceptibility of mice to obesity-induced diabetes. Coleman found that genetically obese mice (due to homozygosity for the ob mutation in the leptin gene; Lepob/ob) in the C57BLKS/J strain background are severely diabetic, whereas genetic obesity in the C57BL/6 background was associated with severe insulin resistance, but not the development of diabetes (111). In the C57BL/6 background, homozygosity for the Lepob mutation led to moderate (
220 mg/dl), transient hyperglycemia between 8 and 12 wk of age, but then glucose returned to near normal levels (81). This return to normal glucose levels is due to a marked increase in insulin arising from a dramatic expansion of ß-cell number in the C57BL/6 mice that is not sustained in the C57BLKS/J background (112), consistent with the aforementioned high ß-cell proliferative capacity of C57BL/6 mice. C57BL/6 mice are also relatively resistant to the development of diabetic nephropathy (113).
B. C57BLKS/J
The C57BLKS/J strain arose through the inadvertent genetic contamination of C57BL/6 (114). Although the non-C57BL/6 alleles are scattered across all the chromosomes and are primarily from the DBA/2 strain, recent SNP analysis indicates that there are contributions from at least three other strains, including C57BL/10, 129, and another unidentified strain that accounts for nearly 10% of the genome (36, 115, 116). As described above, the diabetes severity associated with homozygosity for the Lepob or Leprdb mutations is much greater on the C57BLKS/J than the C57BL/6 background (81). Because the genomic constitution of this strain derives primarily from C57BL/6 and this strain is more diabetes-prone than C57BL/6, we can presume that the diabetogenic alleles are derived from the contaminating strains or perhaps the interaction between these alleles and diabetogenic C57BL/6 alleles.
Lean C57BLKS/J are very sensitive to streptozotocin (117). C57BLKS/J mice have an approximate 50% reduction in ß-cell proliferative capacity compared with C57BL/6, and an increased ß-cell susceptibility to glucotoxicity (118, 119). Mean islet area is increased in male C57BLKS/J mice, but total pancreatic insulin content is reduced compared with C57BL/6 (119).
Mapping studies have been performed in C57BL/6 x C57BLKS/J Leprdb/db F2 mice. Coleman found concordance between the segregation of a malic enzyme regulatory locus on chromosome 12 and diabetes in a Leprdb/db F2 between C57BL/6 and C57BLKS/J, with the low activity allele associated with diabetes (111, 120). More recently, Paigen and colleagues (121) identified additional suggestive loci for increased glucose levels from C57BLKS/J alleles. A mapping study on C57BLKS/J x DBA/2 Leprdb/db F2 mice identified a locus on chromosome 9 associated with impaired glucose tolerance (122). The DBA/2 allele at this locus contributes to the increased glucose levels, suggesting that the C57BLKS/J allele is a C57BL/6-derived protective modifier and that the DBA/2 strain has diabetogenic loci in addition to those introgressed into the C57BLKS/J strain.
It is unclear, however, whether the effects of these alleles require the absence of effective leptin signaling. When the fat mutation in carboxypeptidase E (Cpe), which was originally identified on the HRS strain background, was introgressed onto the C57BLKS/J background, C57BLKS/J-Cpefat/fat males became diabetic despite reduced adiposity, whereas HRS-Cpefat/fat mice were more hyperinsulinemic and did not develop hyperglycemia (123). However, C57BLKS/J-Cpefat/fat mice were more insulin sensitive and showed less ß-cell/islet damage than C57BLKS/J-Leprdb/db mice (124). In fact, the hyperglycemia reached a plateau and eventually reversed in these animals, suggesting that the severe diabetes in C57BLKS/J-Leprdb/db mice results from an interaction between C57BLKS/J alleles and the Leprdb mutation. This could in part explain why high-fat, high-sucrose feeding showed C57BLKS/J to have a reduced response in terms of weight gain and glucose and insulin levels compared with C57BL/6 (125). A recent study in C57BLKS/J x DBA/2 F2 mice, either Leprdb/db or Leprdb/+, has shown that regulation of glucose levels is subject to complex interactions between sex and Leprdb genotype and epistatic interactions between loci (126).
C. DBA/2
The DBA strain was the first inbred strain created by Little, in 1909, deriving its name from its coat-color alleles dilute (d), brown (b), and non-agouti (a). Of the two substrains, it is the DBA/2 substrain that is commonly used in diabetes research. Given the strain differences between C57BLKS/J and C57BL/6 in diabetes susceptibility and the knowledge that C57BLKS/J and C57BL/6 differ genetically primarily due to the introgression of DBA/2-like alleles, this strain has been used as a model for genetic studies as well.
Like C57BLKS/J-Leprdb/db, DBA-Leprdb/db mice become severely diabetic (111). DBA/2 Lepob/ob mice are also severely diabetic; however, unlike C57BLKS/J obese mice, not all the mice undergo pancreatic decompensation (127). Because the C57BLKS/J strain is primarily a combination of the C57BL/6 and DBA/2 genomes, this suggests that either non-DBA/2 non-C57BL/6 alleles in C57BLKS/J or interactions between DBA/2 and C57BL/6 alleles promote the severe decompensation seen in the C57BLKS/J strain. However, as described above, there are also likely additional diabetogenic alleles in the DBA/2 strain not present in C57BLKS/J (122, 126). The DBA/2 strain is also susceptible to diabetic nephropathy (113).
In contrast to genetic obesity, lean DBA/2 mice that are fed a high-fat diet are more glucose tolerant than C57BL/6 (98). In a survey of nine inbred strains, DBA/2 had the lowest glucose levels on high-fat feeding (92), and data from the Mouse Phenome Database show that DBA/2 mice have among the highest insulin levels (2). When insulin resistance is induced through double heterozygosity for insulin receptor (Insr) and insulin receptor substrate-1 (Irs1) knockout alleles, DBA/2 mice have reduced insulin resistance and a slower onset of hyperglycemia than C57BL/6 (110). DBA/2 mice have higher insulin levels in the fed state than C57BL/6 and other strains (93), which suggests that DBA/2 may have increased insulin secretion. Indeed, compared with C57BL/6, DBA/2 hypersecrete insulin in response to glucose from birth, which has been attributed to increased islet glucose utilization and GLUT2 levels (97). In a survey of pancreatic structure across seven mouse strains, DBA/2 also had the most islet and ß-cell mass of all the strains (103).
Combined, these data suggest that DBA/2 ß-cells are acutely sensitive to obesity. The DBA/2 strain is genetically distinct from the other strains used in diabetes research (36, 37) and may provide important insight into factors underlying obesity-induced ß-cell failure. A set of very large insert congenic strains spanning the genome in which DBA/2 alleles have been introgressed into the C57BL/6 background have been developed (78) and will be an excellent resource for mapping these differences.
D. 129
The 129 strain has a complicated breeding history, which has resulted in numerous distinct substrains in existence today. Outcrossing among these substrains has led to several genetic differences between them, which have been extensively characterized, and a standardized nomenclature has been developed (128, 129). The majority of ES cell lines used in generating gene-targeted mice are derived from substrains of the 129/Sv strain, a heterogeneous strain which is the ancestor of many of today’s substrains (128, 129). Thus, most knockouts are congenic strains in which the targeted allele and some amount of flanking sequence have been introgressed into other strain backgrounds. Depending on the amount of remaining flanking sequence, it is possible that nearby 129/Sv-derived alleles may contribute to the observed phenotype. For simplicity, here we refer to all substrains as 129, however it should be noted that the phenotypic effects may differ between the various substrains.
On both chow and high-fat-diet feeding, the 129 strain maintains low insulin and is more glucose tolerant than other strains (2, 130, 131). This suggests that 129 mice have increased insulin sensitivity and is consistent with the observation that wild-type 129 mice have low ß-cell mass (103, 110). In a C57BL/6x 129 F2 study, five loci for hyperglycemia or hyperinsulinemia have been identified (130). At each of these loci, the 129 allele was associated with the lower insulin or glucose phenotype.
Thus, the 129 strain is diabetes-resistant in the context of genetic obesity. Alleles from the 129 strain are capable of conferring protection to C57BLKS/J- Leprdb/db mice (132). Similarly, mice homozygous for a db allele (db3J) found on this strain have very mild and transient hyperglycemia, marked hyperinsulinemia, and dramatically elevated pancreatic insulin content. Although this is accompanied by progressive hyperglucagonemia, the mice ultimately develop hypoglycemia leading to sudden death (133).
Similarly, lean 129 mice with genetically induced defects in insulin signaling due to double heterozygosity for Irs1 and Insr mutations have only mild hyperinsulinemia with little insulin resistance and do not increase ß-cell mass compared with wild-type 129 mice (110). This is in contrast to C57BL/6 and DBA/2 mice bearing the same mutations, which develop marked hyperinsulinemia with much more severe insulin resistance and resulting in diabetes (110, 131). In these double-heterozygous mice, linkages for plasma glucose levels have been identified on chromosomes 12 and 14, where C57BL/6 alleles are diabetogenic (131).
In contrast to the insulin resistance evoked by Irs1-Insr double heterozygosity, heterozygosity for only a null allele of the Insr results in more severe hyperinsulinemia in the 129 than C57BL/6 background (134). When loci were mapped in an Insr+/– C57BL/6x 129 F2 population, five loci (significant and suggestive) affecting plasma insulin levels were identified; however, at four of these loci, C57BL/6 alleles were linked to the higher serum insulin concentrations. Nonetheless, this suggests that the 129 strain may harbor at least one diabetogenic locus.
Additional evidence of diabetes promoting alleles in the 129 strain come from other knockouts in the insulin signaling pathway. The insulin receptor substrate-2 (Irs2) gene is important for the normal survival and development of ß-cells (135). In a C57BL/6x 129/sv mixed background, Irs2–/– mice have reduced Pdx1 expression and develop diabetes (136). However, mice with this mutation on the pure C57BL/6 background preserve the expression of the Pdx1 transcription factor and its downstream genes (137). This suggests that alleles from the 129 background contribute to the down-regulation of Pdx1.
E. BTBR
The black and tan, brachyuric (BTBR) mouse strain was developed by L. C. Dunn from an outbred stock he obtained from Nadine Dobrovolskaia-Zavadskaia (138). The strain was maintained by Dorothea Bennett after 1962, and then by her graduate students, Flaherty and Artzt, who passed it on to Jean-Louis Guenet at the Pasteur Institute. Guenet maintained the strain for 45 generations, then passed it to Dove’s laboratory at the University of Wisconsin. During its breeding history, it was bred with 129 mice and selected for good breeding performance (139). Thus, it is genetically most related to the 129 strain (36, 57). However, in contrast to 129, BTBR is a diabetes-susceptible strain.
Lean male F1 hybrids between C57BL/6 and BTBR are more glucose intolerant than either parental strain, suggesting that interactions between C57BL/6 and BTBR alleles can further promote diabetes susceptibility (140). Insulin-stimulated glucose uptake into isolated adipocytes from BTBR (141) and the BTBR x C57BL/6 F1 mice is severely defective (142). Isolated skeletal muscles from the F1 mice also have defective insulin-stimulated glucose transport, although a much milder defect is observed in muscles from lean BTBR mice (140). In vivo, glucose uptake into muscle and adipose tissue in male BTBR mice under hyperinsulinemic clamp conditions is less than C57BL/6, although insulin-mediated suppression of hepatic glucose output is unaffected in BTBR (J. B. Flowers and A. D. Attie, unpublished observations). Islets isolated from BTBR mice hypersecrete insulin at low glucose concentrations and in response to nonglucose secretagogues (143).
Similar to C57BLKS/J mice, when the Lepob mutation is bred into BTBR background, BTBR Lepob/ob mice develop severe diabetes (141, 144). Young BTBR Lepob/ob mice are hyperinsulinemic compared with C57BL/6 Lepob/ob. However, as these mice age, insulin levels decline, resulting in marked hyperglycemia (141). By 3 months of age, pancreatic insulin content is dramatically reduced, and islet architecture is severely disrupted in BTBR Lepob/ob mice (our unpublished observations). We have used this model for several genetic and genomic studies (144, 145, 146, 147) and in a cross between BTBR and C57BL/6 mice, we have mapped several diabetes-related QTL in Lepob/ob F2 animals (144).
Using overlapping interval-specific congenic (65) and subcongenic strains, Clee et al. (148) recently positionally cloned one of these loci on chromosome 19. The gene underlying the t2dm2 QTL was found to be SorCS1 (148), a receptor expressed in islets that binds to platelet-derived growth factor-BB (149). Platelet-derived growth factor-BB is required for pericyte recruitment to the islet microvasculature, a step critical for vessel formation and stability (150, 151). Thus, we believe that SorCS1 may play a role in the development or maintenance of the islet vasculature, which would be important for expanding ß-cell mass in response to insulin resistance. Interestingly, the diabetogenic allele at this locus was contributed from the C57BL/6 strain (144, 148) and was associated with reduced insulin secretion in vivo, suggesting that alterations in SorCS1 may also contribute to the impaired insulin delivery to the bloodstream of the C57BL/6 strain.
F. A/J
The A/J strain is the classic "diabetes-resistant" mouse strain and is among those with the lowest glucose levels (2). When fed a high-fat, cholesterol-containing diet, A/J mice maintain very low glucose and insulin levels. Compared with the C57BL/6 strain, A/J mice are resistant to high-fat, high-carbohydrate feeding-induced obesity, insulin resistance, and glucose intolerance (89, 90, 91, 125), in part due to increased insulin secretion and a sustained second-phase response (100, 101). Thus, A/J mice have reduced glucose even on chow (91, 152) and do not expand ß-cell mass when fed high-fat diets (100). However, even this resistant strain may harbor some alleles promoting glucose intolerance (153, 154, 155). It is a common progenitor in RI and recombinant congenic panels.
G. BALB/c
The BALB/c strain is genetically most related to A/J (36, 37). Consistent with this, it is more glucose tolerant than C57BL/6 (98) and more resistant to the glucotoxic effects of streptozotocin (104, 117). BALB/c mice have a relatively high ß-cell mass due to a large number of islets (103).
In the BALB/c background, Lepob/ob mice have increased insulin levels in the fed state compared with C57BL/6 Lepob/ob (156). Interestingly, BALB/c Lepob/ob mice have reduced adiposity, which correlates with increased thermogenesis. In contrast to other strains, BALB/c mice homozygous for the Lepob mutation remain fertile (156).
H. C3H
C3H/HeJ is a diabetes-resistant strain. This strain is related to BALB/c, A/J, and AKR (36). It is highly glucose tolerant and has a robust insulin secretory response, which is sufficient to overcome its slight insulin resistance (93, 98). C3H mice are resistant to the ß-cell toxic effects of streptozotocin, possibly due to the effects of a single gene (105, 117). ß-Cell mass in C3H is derived from relatively few islets of large size (103), resulting in increased ß-cell mass compared with C57BL/6, but less than other strains such as DBA/2 and BALB/c. As described above, mapping in F2 and F3 samples derived from C3H and C57BL/6 has identified loci on chromosomes 2 and 13 associated with improved glucose tolerance relative to C57BL/6 (99). In contrast, a C3H-derived allele associated with impaired glucose tolerance has recently been mapped in a cross with C57BLKS/J-db mice (157).
I. AKR
AKR mice are closely related to the LG strain, described below, and are also related to C3H, BALB/c, and A/J (36). The AKR strain is very sensitive to diet-induced obesity (92), and adipocytes from this strain exhibit increased insulin-stimulated glucose uptake compared with SWR/J mice (158). Unlike its related strains described above, when fed a high-fat diet, AKR mice are more hyperinsulinemic than others (2). They are more insulin resistant than C57BL/6, primarily due to reduced glucose uptake into adipose tissue (96). Despite this, AKR are more glucose tolerant and have lower plasma glucose levels than C57BL/6 because of increased insulin secretion during a glucose challenge (96). AKR mice are relatively resistant to the effects of streptozotocin (117).
J. CAST/Ei
The CAST/Ei strain is an inbred line derived from wild Mus musculus castaneus mice from Thailand. Compared with other strains with data in the Mouse Phenome Database, CAST/Ei mice have relatively high glucose levels (2). The Lusis group (159) has made extensive use of this strain in studies of obesity and atherosclerosis. CAST/Ei mice are very lean, but, on a high-fat diet, hyperinsulinemic relative to C57BL/6. In an F2 derived from CAST/Ei and C57BL/6, three independent loci on chromosome 2 segregate with body weight loci and exhibit suggestive linkage to plasma insulin levels (159). Differences in glucose and insulin levels have been observed in congenic strains where CAST/Ei alleles have been transferred into the C57BL/6 background (160). In a recent study, a new selectively inbred strain, the Horio-Niki diabetic (HND) mouse, was derived from C57BL/6 and M. m. castaneus parents (161). These nonobese mice have impaired insulin secretion in vivo, and an altered response to 2-adrenergic stimulation, suggesting that this may be a model of impaired neurological control of insulin secretion (162).
K. Nonobese diabetic (NOD) and nonobese nondiabetic (NON)
These strains have a complicated history. Originally, a mouse with spontaneous development of cataracts was observed in a closed outbred colony of Imperial Cancer Research (ICR) mice. This mouse was selected and inbred to develop the CTS (Cataract-Shiongi) line, however none of the offspring developed cataracts (29, 163). As cataracts can be a complication of diabetes, at the F6 generation, two lines were split, one with euglycemia (A) and one with mild hyperglycemia (B). At the F20 generation, a spontaneously diabetic mouse was observed in the euglycemic A line. The offspring of this mouse were inbred for several generations with selection for spontaneous diabetes to derive the NOD line, whereas mice from strain B did not develop spontaneous diabetes, and became the NON control line (163).
The NOD strain is a model of type 1 autoimmune diabetes, with a complex genetic etiology. The genetics of this strain have been extensively studied and involve at least 20 different loci, none of which independently result in diabetes (29). The genetic complexities of this strain are also evident in that several of the mapped Idd loci have ultimately been dissected into multiple distinct loci (29, 164). Analysis of these loci often involves haplotype comparisons across several diabetes susceptible and resistant strains, in congenics where the genotype at several other loci is controlled (164, 165). Interestingly, alleles that further promote diabetes in NOD mice derived from the nondiabetic control strains, e.g., C57BL/6, have also been observed (166), and alleles associated with the sensitivity of this strain to streptozotocin have been mapped (167). We will not focus on the models of type 1 diabetes in this review, but point the reader to other reviews of the subject (168, 169, 170, 171, 172).
Of more relevance to type 2 diabetes is the NON strain, which has been shown to have alleles promoting type 2 diabetes (173, 174, 175). This should not be particularly surprising, given its selection for mild hyperglycemia during its breeding history and that several other models of ß-cell dysfunction and type 2 diabetes have been derived from the outbred ICR mice (see descriptions of the NSY and ALR and ALS strains below), suggesting that this stock has numerous alleles promoting diabetes (29, 163). As the genes are identified, it will be interesting to observe whether there is any overlap between type 1 and type 2 diabetes susceptibility alleles (29). NON mice are glucose intolerant and have impaired glucose-stimulated insulin secretion, but they do not transition to overt diabetes even when fed an 11% high-fat diet (176). With 45% fat and simple carbohydrates, the mice become transiently hyperglycemic and very obese, but eventually go into remission from the hyperglycemia (E. H. Leiter, personal communication).
The genetic mapping studies that mapped diabetes in the NON strain used an F2 intercross between this strain and NZO, described below. In contrast to either parental strain, F1 mice exhibit severe fasting hyperglycemia, and nearly 100% of males develop diabetes (173). Two NON-derived diabetogenic loci were identified in the F2 sample (173), and a third suggestive NON-derived locus was identified in a (NZOxNON) x NON backcross (174).
L. New Zealand obese (NZO)
The New Zealand obese (NZO) strain was derived from an outbred stock from the Imperial Cancer Research (ICR) laboratory in England. It was originally selected for agouti coat color, along with the New Zealand Black (NZB) and New Zealand White (NZW) strains (177), and is a model of spontaneous polygenic obesity and insulin resistance. Leptin receptor polymorphisms have been detected in this strain; however, they do not appear to directly cause the obesity of the strain, because the related NZB strain shares these polymorphisms and is not obese (178). Furthermore, studies mapping obesity-related genes in this strain have not observed linkage to this region (179, 180). It is possible, however, that these polymorphisms may confer leptin resistance or impaired leptin transport across the blood-brain barrier (181) and interact with other obesity-promoting alleles to enhance the obesity of these mice (178, 180).
The phenotype of NZO mice resembles the metabolic syndrome (182). Compared with the related NZB and NZC strains and the lean Swiss Jackson Laboratory (SJL) strain, NZO mice are markedly hyperinsulinemic, with males developing hyperglycemia (182, 183). The mice have reduced insulin-stimulated glucose uptake into muscle and adipose tissue, along with increased hepatic glucose production (183). Serum triglycerides and blood pressure are also increased in these mice (182). First-phase insulin secretion in response to a glucose bolus is impaired in NZO mice (183). After initial ß-cell hyperplasia, decompensation in males is accompanied by reduced ß-cell mass, degranulation, and ultrastructural changes (184). However, in various NZO substrains, not all NZO males decompensate, apparently needing to exceed a body weight threshold before diabetes develops (173, 174, 176).
Like NZB mice, NZO mice have an autoimmunity phenotype; they have autoantibodies against the insulin receptor, which may contribute to their insulin resistance (185). Leukocyte infiltration of the pancreas has also been observed in NZO mice, and diabetes is worsened in NZO x NZB F1 mice (177, 184). However, later studies have shown that this autoimmunity is not essential for the development of diabetes in these mice (176).
Loci contributing to obesity and diabetes susceptibility in this strain have been detected on nearly every chromosome (175). Genetic mapping studies have also been performed in a (NZO x SJL) x SJL backcross panel (179). Interestingly, these studies identified a major locus on chromosome 4 from the SJL background that contributes to the development of diabetes (hypoinsulinemia and hyperglycemia) in these obese mice (179, 186). This locus, Nidd/SJL, is associated with hyperglycemia and relative hypoinsulinemia, due to a loss of ß-cells and islet disorganization in high-fat-fed mice (186). A strong interaction between this locus and one controlling obesity accelerates the progression of diabetes in these mice. Subsequent mapping in female mice demonstrated that NZO-derived alleles contribute to obesity and hyperinsulinemia (180).
In an F2 derived from NZO and NON, Leiter et al. (173) identified three NZO-derived diabetogenic alleles. Two of these were suggestive loci affecting blood glucose concentrations. Interestingly, they were dependent on the direction of the original cross, i.e., whether the parental female or male was NZO. A second study, in an F1 x NON backcross, identified a major NZO-derived diabetogenic QTL (174). This locus influenced body weight, plasma glucose, and early hyperinsulinemia. This study also detected complex interactions between various loci (e.g., those affecting obesity and plasma glucose levels), which may contribute to the increased diabetes in F1 males compared with either parental strain. Interactions were also detected between various loci and the maternal genotype (environment), illustrating the complexity of factors that may influence diabetes-related QTL (174).
Remarkable follow-up studies by this group have attempted to reconstitute the diabetes susceptibility of the F1 animals using RCSs containing various combinations of the diabetogenic QTL (175, 176). When numerous epistatic interactions are evident, this approach has an advantage over creating single-region congenics, which do not allow for interaction among multiple loci. These studies have elegantly demonstrated that the development of diabetes depends on the inheritance of unique combinations of these QTL. Some strains are obese without diabetes, whereas the prevalence of diabetes increases in strains bearing an increasing number of diabetogenic QTL (175). As predicted, these strains differ in numerous characteristics, including islet pathology (176).
M. FVB
Although there is relatively little information on lean FVB mice, sensitized screens show that the strain harbors diabetogenic alleles, particularly relating to peripheral insulin resistance. Glucose levels are higher in this strain than many others, accompanied by relatively low insulin levels (2). In the FVB background, Leprdb/db mice are more insulin resistant and have more severe hyperinsulinemia and hyperglycemia than C57BL/6 Leprdb/db mice. FVB-Leprdb/db mice have enlarged islets; however, islet destruction is not observed (127, 187), suggesting that these strains primarily differ in alleles promoting insulin resistance and not ß-cell failure. Similarly, a comparison of FVB Lepob/ob and C57BL/6 Lepob/ob mice revealed increased hyperglycemia and peripheral insulin resistance in FVB Lepob/ob, although hepatic insulin resistance was increased in the C57BL/6 Lepob/ob mice (188). An FVB x C57BL/6-Leprdb/db F2 intercross mapped a locus on chromosome 5 where FVB alleles were associated with increased insulin. Congenic Leprdb/db mice inheriting C57BL/6 alleles in this region on the FVB background are unable to maintain insulin production and undergo pancreatic decompensation (187). This suggests that the insulin-resistant FVB background unmasks a C57BL/6-derived allele promoting ß-cell failure, which is otherwise silent in the C57BL/6 background.
A second leptin-deficient model is a mouse made lipoatrophic by adipose expression of a dominant-negative protein, AZIP, that interferes with the C/EBP and Jun transcription factors (189). In the C57BL/6 background, the AZIP mice had transient hyperglycemia, returning to normal by 4 months of age. In striking contrast, in the FVB strain background, the mice were severely diabetic with fed glucose levels of 600 mg/dl. Like the Lepob/ob mice, C57BL/6-AZIP mice had more severe liver insulin resistance, whereas FVB-AZIP mice have increased muscle insulin resistance (190). Under hyperinsulinemic conditions, the C57BL/6 AZIP mice had 40% higher glucose uptake into muscle, suggesting that the difference in glucose between these strains was likely due to a difference in peripheral insulin sensitivity (188).
N. KK
The KK strain was derived from wild-derived ddY mice in Japan in 1957 by Kondo (see Ref. 191). SNP analysis suggests that it is closely related to the NZO and NON strains (36). Because several breeding colonies of these mice have been maintained, there are now several substrains [T-KK (or Toronto-KK, KK/Upj), KK/HlLt, KK/Ta, KK/San]. For this review, we will refer to them all as KK.
KK mice are obese and hyperleptinemic. A leptin receptor amino acid variant has been identified in this strain that may account for differences in adiposity in females, although this has not been observed in all studies (192, 193). Compared with its ddY progenitor, KK mice have increased glucose and HbA1c levels and impaired glucose tolerance (194, 195, 196). Similarly, compared with C57BL/6 or BALB/c, KK have increased body weight, accompanied by glucose intolerance (191, 195). KK mice develop severe hyperinsulinemia, comparable to the severely insulin resistant C57BLKS/J Leprdb/db mice (2, 192, 195). KK mice have both muscle and adipose insulin resistance (197). Total pancreatic insulin content is increased, associated with islet hypertrophy and hyperplasia but marked degranulation (reduced number and size of granules) (191). Dilated islet capillaries and increased vascular endothelial cells have also been noted in these mice (191). The KK strain is also susceptible to diabetic nephropathy (113). With the introgression of the Ay mutation in agouti, they exhibit further reduced glucose tolerance and become more severely diabetic (194, 196). KK-Ay mice are hyperinsulinemic, have increased HbA1c, and exhibit early stage nephropathy (196). Numerous linkages for diabetes-related traits have been observed in several crosses with KK mice (193, 194, 195, 198, 199).
O. TallyHo
The TallyHo mouse strain is a naturally occurring model of obesity and type 2 diabetes. The strain derived from two Theiler Original male mice with polyuria and hyperglycemia, which were selectively inbred for hyperglycemia (200). Mice from this strain have increased body weight and body fat and are hyperlipidemic and hyperinsulinemic. Hyperglycemia, accompanied by hypertrophied and degranulated islets, develops only in male mice (201). Several loci influencing plasma glucose levels and body weight have been mapped in this strain in crosses with either CAST/Ei or C57BL/6 (200, 202).
P. Nagoya-Shibata-Yasuda (NSY)
The NSY strain was developed by selective inbreeding for glucose intolerance from outbred JcI:ICR mice, the same stock from which NOD and NON mice were derived (54, 203). Nearly all males have impaired glucose tolerance, which also develops in one third of females. Compared with the diabetes-resistant C3H strain, NSY males have increased fasting insulin levels and increased postprandial, but not fasting, glucose levels. These mice have impaired glucose tolerance and markedly impaired glucose-stimulated insulin secretion in vivo, which may account for the increased glucose levels in the fed state (203). NSY mice have increased pancreatic insulin content without an increase in mean islet size (203). Genetic analysis of this strain has mapped three primary loci [Nidd1nsy (Nidd1n), Nidd2nsy (Nidd2n), and Nidd3nsy (Nidd3n)] and one suggestive locus [Nidd4nsy (Nidd4n)] affecting insulin sensitivity and/or insulin secretion (204). Interestingly, the Nidd1n and Nidd2n loci overlap with regions contributing to ß-cell destruction or impaired regeneration in NOD mice derived from the same outbred population, suggesting that there may be common loci affecting susceptibility to both type 1 and type 2 diabetes (29, 205). Furthermore, this strain shares a locus affecting sensitivity to streptozotocin with the NOD strain (206).
Q. ALR/Lt and ALS/Lt
The ALS/Lt and ALR/Lt inbred strains were also derived from CD-1 (ICR) outbred mice by selecting for susceptibility or resistance to alloxan (free-radical)-induced diabetes, respectively (207). These strains are related to the NOD and its related strains (36). The ALR/Lt and ALS/Lt strains also have differential susceptibility of the mice to streptozotocin and of their isolated islets to peroxide. ALR/Lt mice have reduced markers of oxidative stress, likely due to a single genetic difference (207). The effect is an intrinsic property of the islets because the phenotype is maintained in cultured islets (207, 208). In the absence of an oxidative challenge, ALS/Lt males are hyperinsulinemic, hyperglycemic, and have impaired glucose tolerance, ultimately developing diabetes (209). ALS/Lt mice also have abnormal insulin secretion and develop diabetes when the agouti Ay mutation is introgressed into the strain (210). Because this occurs in the absence of alloxan, we might infer that genes protecting ß-cells from oxidative damage under normal conditions may be important mediators of type 2 diabetes.
Recently, Mathews et al. (211) found that diabetes in the ALS/Lt strain was reduced when mitochondrial DNA was derived from the ALR/Lt strain. They identified a single SNP differing between the ALS/Lt and ALR/Lt mitochondrial genomes; a L276M polymorphism in the NADH dehydrogenase 2 gene, which they propose contributes to resistance to both chemical and autoimmune diabetes (211). The same mutation in humans is also correlated with protection from type 1 diabetes (212).
R. M16
The M16 mouse strain was created through long-term selection of ICR outbred mice for body weight gain (213). Compared with the parental ICR stock, these mice have increased body weight and fat, due to increased food intake. Males and females are both hyperinsulinemic, but only males from this strain develop moderate hyperglycemia. Using an F2 comprised of nearly 1200 animals, Pomp and co-workers (214) identified numerous QTL affecting these phenotypes in an F2 cross with randomly selected ICR stock.
S. LG and SM
The LG (large) and SM (small) strains were independently derived, selecting for body size. LG was created in the 1930s by selection for large body size from a pool of albino mice (54), and it appears most closely related to AKR (36). SM was generated in the 1940s from a pool of mice derived from four crosses between seven strains, including dilute brown (dba), silver chocolate (sv ba), black and tan (at), pink-eyed short-eared dilute brown (psedba), albino (c), cinnamon spotted (bs), and agouti (a) (215). Thus, this strain has ancestors in common with both the DBA and BTBR strains, and it appears most closely related to DBA/2 (36).
The LG and SM strains have been used for extensive genetic analysis of traits related to type 2 diabetes. The strains differ in many traits, including obesity, plasma glucose, glucose tolerance, and response to high-fat feeding (216). Compared with LG, SM mice have increased serum glucose on a high-fat diet. A RI strain panel has been created from these strains (215) and shows variation across strains in the development of hyperglycemia and hyperinsulinemia. Several QTL for insulin, glucose, and glucose tolerance have been identified in the LGXSM RI strains (217, 218). Interestingly, the majority of loci affecting glucose levels are distinct from those affecting obesity and are only weakly correlated with those affecting insulin, suggesting that the loci primarily affecting glucose levels are not the same as those that affect insulin resistance (218).
The SM strain has been used in another RI panel, the SMXA RI panel, derived from SM and A/J. The RI strains show differences in body weight and plasma insulin levels (219). When compared with A/J, SM mice fed a high carbohydrate diet have reduced body weight and improved glucose tolerance, which is also observed in response to high-fat feeding (153, 220). Although neither parental strain is diabetic, high-carbohydrate-fed F1 animals have further impaired glucose tolerance, which appears to result from the interaction of at least three loci, two from A/J and one from SM (153). These RI strains have been characterized for other diabetes-related phenotypes, and it has been suggested that one strain of the panel, SMXA-5, may be a useful diabetes model because it displays insulin resistance accompanied by increased ß-cell mass (220, 221, 222). To identify further the A/J-derived loci that contribute to these phenotypes, an F2 sample derived from the SMXA-5 and the parental SM strain was created. This identified several loci affecting plasma glucose and insulin levels and glucose tolerance (154). A major locus on mouse chromosome 2 was identified where alleles from A/J contribute to impaired glucose tolerance. The phenotype was verified by a congenic strain in which A/J alleles were introgressed into the SM background (154).
T. Tsumura, Suzuki, Obese Diabetes (TSOD)
Like KK mice, the TSOD strain was derived as an inbred obese diabetic strain from the outbred Japanese ddY stock (223). Beginning in 1984, mice were selected for obesity and urinary glucose, then inbred, along with a nonobese/nondiabetic control line (TSNO) derived from sibs (223). Compared with their parental ddY strain, the TSNO strain, or BALB/c, these mice are obese and insulin resistant, which results in hyperglycemia and hyperinsulinemia in males (223, 224). Severe diabetes does not develop because TSOD mice can increase ß-cell mass; however, there appears to be a defect in insulin secretion in response to an in vivo glucose challenge (223). Older mice from this strain develop islet hypertrophy, and also have evidence of neuropathy and nephropathy (225). Loci affecting plasma glucose and insulin levels and glucose intolerance have been mapped in a cross between this strain and BALB/c (224). Interestingly, a suggestive locus for increased glucose levels during an ip glucose tolerance test (IPGTT) from the BALB/c strain was also observed. Replication of the effects of the Nidd5 locus from this strain on body weight has been obtained in congenic mice, although these mice do not display the differences in insulin levels that were also mapped to this marker (226), suggesting that multiple genes may be responsible for the Nidd5 phenotype.
U. The Akita mouse
The Akita mouse strain derived from a C57BL/6 mouse with a spontaneous mutation causing severe insulin-dependent diabetes (47). The trait was mapped to telomeric chromosome 7 in a backcross with C3H and an F2 intercross with M. m. castaneus (47). The hypoinsulinemia in these mice correlates with a reduced ß-cell mass. The phenotype of this strain is caused by a C96Y mutation in the insulin 2 (Ins2) gene, corresponding to the seventh amino acid on the insulin A chain (227). The mutation prevents formation of one of the disulfide bonds between the A and the B chains of the insulin molecule. This leads to reduced processing of proinsulin and a severe reduction in mature insulin.
It was not immediately obvious how a mutation in the insulin gene would lead to a reduction in ß-cell mass. The answer to this conundrum came from an understanding of the way in which cells confront an overload of misfolded proteins in the ER, i.e., "ER stress." Failure to properly respond to ER stress through activation of the unfolded protein response is the cause of several known inherited diseases. In the unfolded protein response, cells adapt by reducing the rate of synthesis of most proteins while increasing the synthesis of chaperonins (228). The inhibition of protein synthesis occurs when the ER kinase PERK phosphorylates translation initiation factor eIF2. PERK deficiency or a mutation abolishing eIF2 phosphorylation leads to a phenotype resembling that of the Akita mouse (229, 230). Although most protein synthesis is reduced upon eIF2 phosphorylation, the translation of the transcription factor ATF4 is increased, leading to increased expression of C/EBP homologous protein (CHOP). CHOP promotes cell death, presumably by reversing the attenuation of translation and by promoting excessively oxidizing conditions in the ER (231). A key observation is that deletion of the Chop gene delays the onset of diabetes in the Akita mouse, showing that at least in the earliest phase, this gene mediates the lethality of the protein folding defect (232). A recent study has mapped modifiers of the Akita phenotype in a cross with A/J mice and identified both A/J and C57BL/6-derived alleles that are associated with further glucose intolerance (155).
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VIII. Linkage Hot Spots |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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, we have summarized the linkages for glucose and insulin-related traits that have been identified in these mouse models. We have included the position of the peak marker but have not attempted to specify confidence intervals, unless these have been determined through a congenic strain. We also include a notation regarding whether the locus reached the genomewide significance threshold. Because several study design-related factors can influence whether a significant linkage is obtained, we have included all loci with at least nominal evidence of linkage, as they may still be real loci with smaller effects or effects that the study was underpowered to detect. Indeed, several of these loci overlap with each other and with other significant QTL. Additional loci identified only through analysis of epistatic interactions are described in Table 2.
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We observe four clusters (highlighted in Table 1
), where QTL from at least four independent strains have peak markers residing within 20 Mb of each other. On chromosome 9, between 36–49 Mb, are loci for increased fasting glucose and glucose or insulin levels during an IPGTT in crosses involving the NZO, C57BL/6, DBA/2, and KK strains (98, 122, 126, 173, 194). On chromosome 11 from 62–71 Mb, five loci have been identified from the NZO, C57BLKS/J, M16, SM, and NSY strains (123, 154, 173, 204, 214). Between 79–99 Mb on chromosome 12 are loci from the KK, NZO, 129, and C57BL/6 strains (131, 134, 173, 195). Finally, at the telomeric end of chromosome 19, from 41–55 Mb, four loci have been described from the HRS, NZO, TallyHo, and C57BL/6 strains (123, 144, 148, 180, 200). These "hot spots" of linkage are described more in the next section.
Because there are several regions of the mouse genome harboring multiple QTL, we asked whether these also correspond to diabetes-related QTL in rat models and examined their relationship to human diabetes QTL. To obtain a complete picture of the linkages observed in rodents that correspond to these loci, we have tabulated the diabetes-related QTL that have been observed in the rat (Supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://edrv.endojournals.org), and compiled a list of all loci for type 2 diabetes and glucose and insulin-related traits mapped in various human populations, along with the positions of the MODY genes and the genes for which associations with type 2 diabetes have been validated by meta-analysis (Supplemental Table 2). We have presented all loci, whether they reach genomewide significance or not, because there are often numerous individual studies all pointing to the same area, which on their own do not meet strict statistical criteria but when taken together provide stronger evidence for a QTL in that region. The data are for illustrative purposes only, and the reader is strongly encouraged to read the original papers for details on the significance of the QTL, their confidence intervals, and details of the study design and analysis methods, because it is beyond the scope of this review to include these details here. Consistent with our analysis of the mouse data, we have also avoided inclusion of obesity-related loci, because these are extensively reviewed in the Obesity Gene Map (233).
Examination of these data has revealed several hot spots containing multiple linkages across the species. Differences in the phenotypes mapped to these regions may either reflect differences in the assessed phenotypes, pleiotropic effects of the underlying gene, or be indicative of multiple genes in the region. Indeed, it may often be the case that the strength of a QTL is increased if there are multiple closely linked genes affecting the phenotype, especially if the mapped phenotype was broad, such as type 2 diabetes. Here, we have only cross-referenced the regions directly syntenic to the LOD peak observed in mice. Thus, overlap of the entire confidence intervals may include additional loci not emphasized here (perhaps also on different chromosomes if there is a break in the syntenic block). Despite the narrow limits of our assessment, the data strongly suggest that one or more important genes for diabetes susceptibility reside in these locations and highlight the relevance of the rodent linkages to humans.
A. Mouse chromosome 19, rat chromosome 1, and human chromosome 10q
Arguably, the most success in type 2 diabetes genetics to date has come from studies of mouse chromosome 19, rat chromosome 1, and human chromosome 10q. Four studies have mapped diabetes-related traits to distal chromosome 19 in the mouse. We identified T2dm2 in a cross between C57BL/6 and BTBR mice homozygous for the ob mutation (144). At this locus, the C57BL/6 strain contributes an allele associated with reduced insulin levels due to defective insulin secretion in vivo. As described above, we have recently identified the SorCS1 gene as underlying T2dm2 (148). The Tanidd1 locus, associated with increased glucose levels, was identified in the TallyHo mouse strain, in crosses between this strain and both the C57BL/6 and CAST/Ei strains (200). In the NZO strain, a locus associated with increased insulin levels was identified in an F2 cross between this strain and the Swiss/Jackson Laboratory (S/JL) strain (180). Most recently, a cross between the C57BLKS/J and HRS strains homozygous for the Cpefat mutation identified a locus, Find1, associated with increased plasma glucose levels (123).
In rats, the distal end of chromosome 1 is syntenic to this region. The GK rat strain was derived by selective inbreeding of Wistar rats with the highest glucose levels (234). Although not obese, GK rats have hyperglycemia, insulin resistance, defective insulin secretion, a deficit of ß-cells, and diabetic complications, i.e., retinopathy, neuropathy, and later in life (12–18 months), nephropathy (235). In an F2 cross between GK and nondiabetic Fisher 344 (F344) rats, Galli et al. (236) mapped multiple loci linked to fasting and postprandial glucose levels. The strongest linkage they observed, Niddm1, was on the distal region of chromosome 1, with a peak LOD of 11.0 for glucose at 60 min, at marker D1Mit7. Gauguier et al. (237) also mapped diabetes-related traits in the GK strain in a cross with the Brown Norway (BN) strain. They identified a peak linkage at D1Wox10, approximately 20 Mb centromeric to that of Galli and colleagues, for fasting glucose levels, postprandial glucose levels, insulin secretion, and adiposity, which they named Nidd/gk1.
Galli et al. (30) dissected the chromosome 1 locus by creating congenic strains in which segments of the GK chromosome 1 were introgressed into the F344 background. A congenic strain with a 52-cM insert, Niddm1a, recapitulated the impaired postprandial glucose tolerance detected in the original F2 study. Two nonoverlapping subcongenic strains, Niddm1b and Niddm1i, had distinct phenotypes, indicating that at least two distinct loci can autonomously contribute to this phenotype. Interestingly, both strains are insulin resistant, and adipocytes from both subcongenic strains had defective insulin-stimulated glucose incorporation to lipids (30). In addition, the strain with the more telomeric insertion, NIDDM1i, has defective postprandial insulin secretion in vivo (30) and severely defective glucose-stimulated insulin secretion in isolated islets (31). Additional fine-mapping studies by Gauguier and colleagues (238) have also identified an insulin secretion defect in this region. Very recently, Granhall et al. (32) have reported that the NIDDM1i locus itself contains multiple loci affecting plasma glucose concentrations. The SorCS1 gene is located within the NIDDM1i congenic strain, suggesting that it may be a candidate for the defective insulin secretion phenotype, although we do not see differences in insulin resistance in our mice. This gene is also the only gene contained within one of the glucose loci identified by Granhall et al. (32).
The Niddm1b locus has been further fine-mapped with subcongenic strains to a region of approximately 2 Mb (239). This region contains the insulin degrading enzyme gene (Ide). The GK allele has two coding differences from the F-344 allele, H18R and A890V (239). COS cells transfected with the GK allele degraded insulin 31% less actively than those expressing the F344 variant. The results were replicated in transfected fibroblasts (240). Neither of the amino acid substitutions individually reduced the activity of the IDE protein (239). Mice deficient in IDE have a 2.8-fold increase in fasting insulin and are glucose intolerant (241). Hence, the Ide gene is a likely diabetes susceptibility gene within the Niddm1b locus in the GK rat.
Linkage to this region has also been detected in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a strain that is derived from a spontaneously diabetic Long-Evans rat. Mice from this strain have mild obesity with early hypertriglyceridemia and impaired glucose tolerance, followed by hyperglycemia, pancreatic decompensation, and renal pathologies (242). Wei and colleagues (243, 244) identified a locus, Nidd6/of, affecting fasting and postprandial glucose levels and body weight in this region of chromosome 1 in an F2 intercross between OLETF and F344 rats. The authors so far have been unable to replicate the phenotype in a congenic strain (245).
Independently, in F2 crosses between OLETF and both the BN and F344 strains, the Dmo1 locus, affecting body weight along with fasting and postprandial glucose levels was mapped to the same marker, D1Rat90 (246), the telomeric boundary of the Niddm1i subcongenic strain. Watanabe and colleagues (247, 248, 249) created congenic strains in which segments from the BN strain have been introgressed into the OLETF background. Their detailed fine-mapping of this region to an interval of approximately 500 kb recently led to the identification of Gpr10, a G protein-coupled receptor, as most likely the gene underlying this Dmo1 locus (248, 249). The OLETF strain has a mutation in the start codon, causing translation to initiate at one of two downstream ATG codons, only one of which is in-frame. However, the primary phenotype in rats with this mutant receptor appears to be increased food intake; effects on glucose tolerance are secondary to this defect (249), and thus it is most specifically an obesity QTL. The authors also identified this mutation in GK rats and Zucker Diabetic Fatty (ZDF) rats, suggesting that it may play a role in the development of insulin resistance secondary to body weight gain in several strains. Notably, this gene is likely contained within the Niddm1i strain; however, it is unknown at this point whether this gene contributes to the insulin resistance phenotypes of this strain. Gpr10 resides outside the body weight region recently fine-mapped within Niddm1i (32).
These loci (mouse chromosome 19 and rat chromosome 1) are syntenic with human chromosome 10q, where several diabetes-related loci have also been mapped. In fact, 10q is one of the well-replicated human type 2 diabetes susceptibility regions (6, 7, 9). A study of Mexican-Americans in San Antonio found significant linkage for age of onset and risk of type 2 diabetes to a broad region, with a peak LOD at 150 Mb on chromosome 10q (10). Significant linkage of diabetes with the same region on chromosome 10q was also detected in a study of 719 Finnish sib pairs (250). Several other groups have also mapped loci affecting diabetes risk to this broad region, including loci in the Pima Indians, British, African-American, Icelandic, Dutch, and French cohorts (251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263). Using a very high density of markers across this region, Grant et al. (26) have recently identified the TCF7L2 gene with a remarkably strong association with type 2 diabetes risk in Icelandic individuals. TCF7L2 (also known as TCF4) is a transcription factor involved in the Wnt signaling pathway through interactions with ß-catenin, and it plays an important role in the development of the enteroendocrine system (264, 265).
Thus, in addition to a gene affecting food intake, three potential type 2 diabetes susceptibility genes have been identified from these studies: SorCS1 in the mouse, Ide in the rat, and TCF7L2 in the Icelandic population (26, 148, 239). The corresponding Mb positions of these genes are: Ide (mouse chr19:37 Mb, human chr10:94 Mb, rat chr1:242 Mb), SorCS1 (mouse chr19:50 Mb, human chr10:109 Mb, rat chr1: 256 Mb), and Tcf7l2 (mouse chr19:56 Mb, human chr10:115 Mb, rat chr1:262 Mb). The IDE locus has been extensively surveyed in human genetic studies of diabetes. Several association studies offer the IDE locus as a candidate for diabetes susceptibility (266, 267, 268). However, a recent report that included both linkage and association studies in over 4000 individuals failed to implicate IDE in diabetes (269). Thus, the role of this gene in human type 2 diabetes susceptibility remains unclear. TCF7L2 and SorCS1 have both been reported recently, and very recently several studies have confirmed association of TCF7L2 SNPs and type 2 diabetes (270, 271, 272, 273, 274).
B. Mouse chromosome 9, rat chromosome 8, and human chromosome 11
Mouse chromosome 9 contains several diabetes-related loci between 36 and 49 Mb. Four studies have identified this region as associated with increased plasma glucose or increased glucose levels during an IPGTT. QTL for increased glucose were identified from the NZO strain in a cross with the NON strain (173) and in a cross between KK mice with the Ay agouti allele and C57BL/6 (194). Glucose intolerance QTL were mapped in crosses between C57BLKS/J and DBA/2 mice homozygous for the Leprdb mutation, whereas decreased insulin levels during a glucose tolerance test were mapped in a cross between C57BL/6 and C3H mice (98, 122, 126).
The syntenic region in rat is chromosome 8 (38–53 Mb). Two rat models have identified linkages in this region. Moralejo et al. (275) identified a linkage to glucose levels during an oral glucose tolerance test in the OLETF rat. Interestingly, heterozygotes at this locus had the highest glucose levels, suggesting an interaction between the OLETF and F344 alleles. Linkage to postchallenge glucose levels was also observed in the Spontaneously Diabetic Torii (SDT) rat, a model of nonobese type 2 diabetes with severe retinopathy (276, 277).
This region corresponds to human chromosome 11 (113–125 Mb). The peak markers of five different studies fall within this region. Hanson et al. (278) identified a significant linkage to type 2 diabetes susceptibility in the Pima Indians at 123 Mb. Putative linkages for type 2 diabetes were also observed in Caucasian families from Utah and the Netherlands (253, 279). Suggestive linkage for increased plasma glucose levels was detected in this region in individuals from the Framingham study (280), and studies of Mexican-Americans from San Antonio have identified a QTL for plasma insulin levels (281).
C. Mouse chromosome 11, rat chromosome 10, and human chromosome 17p
The 62- to 71-Mb region of mouse chromosome 11 harbors five loci for diabetes-related traits, identified in a diverse set of strains. A locus for decreased plasma insulin levels was identified in the NZO strain (173), whereas the M16 strain has a QTL for increased insulin levels in this region (214). Phenotypes related to glucose intolerance (increased glucose levels and area under the curve) were identified in studies of the SMXA-5 RI strain (154), and also in the NSY strain (204). Finally, a cross between C57BLKS/J and HRS mice homozygous for the fat mutation found a locus affecting plasma glucose levels in this region (123).
The syntenic region of the rat genome is chromosome 10, approximately 48–58 Mb. The Niddm3 QTL for traits related to fasting glucose levels and glucose intolerance in the GK strain has been observed in a broad region that overlaps this syntenic region (236).
This region is syntenic to the tip of the p-arm of human chromosome 17 (5–20 Mb), where several diabetes-related linkages have also been detected. Studies of Finnish sib pairs from the FUSION group have identified significant QTL affecting fasting insulin, the insulin/glucose ratio, 2-h insulin levels during a glucose tolerance test, and insulin sensitivity in this region (282). A meta-analysis of four European genome scans has identified significant evidence of a type 2 diabetes susceptibility locus at approximately 14 Mb (283). A significant QTL for leptin levels that interacts with a locus on chromosome 3 affecting insulin levels and the insulin/glucose ratio has also been found in this region (284). Six linkages that did not reach the genomewide significance threshold have been identified in this region in cohorts from Japan, the United Kingdom, the United States, Finland, the Netherlands, and West Africa (253, 263, 285, 286, 287, 288), providing additional evidence of the importance of this region in diabetes susceptibility.
D. Mouse chromosome 12, rat chromosome 6, and human chromosome 14
Mouse chromosome 12 (79–99 Mb) also harbors several QTL for diabetes-related traits. Loci for increased glucose in the KK strain (195) and insulin levels in the NZO strain have been detected (173). A locus for increased fed insulin levels in 129 mice was detected in a cross of C57BL/6 x 129 Insr+/– mice (134), whereas a C57BL/6-derived locus for increased fed glucose and diabetes was observed in a cross of Insr+/– Irs1+/– mice from these strains (131). It is unclear at this time whether these represent different descriptions of the same locus or are distinct loci.
The corresponding region in the rat is chromosome 6, from approximately 110–125 Mb. Only one QTL on rat chromosome 6 has been detected: a locus affecting glucose intolerance in the SDT rat (276). The peak marker for this QTL is D6Mgh1 (at 136 Mb). Although this peak is not specifically in the region syntenic to the LOD peaks from the mouse studies, it is very likely that there is at least some overlap of the confidence intervals between these studies. Thus, it is possible that this represents a locus corresponding to those observed in mice.
Five studies have identified linkages in the corresponding region of human chromosome 14 (70–90 Mb). A significant QTL for fasting insulin levels was detected in Finnish sib pairs (282). This region also houses a significant linkage to type 2 diabetes in the Pima (278), although a second study in this cohort suggesting linkage with maternal inheritance at this region did not reach statistical significance (289). Other studies, which did not reach genomewide significance levels, included a linkage to type 2 diabetes in Ashkenazi Jews (290), a QTL for glucose utilization in African-Americans and Mexican-Americans from the Insulin Resistance Atherosclerosis Study (13), and a complex metabolic syndrome trait that includes HOMA as a component in the National Heart, Lung and Blood Institute Family Heart Study (291).
E. Other type 2 diabetes susceptibility genes identified in the rat
Although not major hot spots in the mouse, two other potential diabetes susceptibility genes have been identified in rat studies. Work by Aitman et al. (292) has demonstrated that Cd36 accounts for part of the adipocyte insulin resistance locus mapped near D4Bro1 on rat chromosome 4 in the Spontaneously Hypertensive Rat (SHR). CD36 is a class B scavenger receptor and fatty acid translocase with numerous functions (293). The defect in the SHR rat results from a complex gene duplication/deletion event resulting in absent expression of the wild-type gene, and the phenotype could be rescued by transgenic overexpression of wild-type CD36 in the mutant strain (294). The importance of this gene was initially questioned because not all SHR strains have this Cd36 mutation, and the overall phenotype of the strains is similar, but the SHR/Izm colony lacking the mutation shows little evidence of an insulin resistance QTL on this region of chromosome 4, consistent with the idea that Cd36 is an insulin resistance gene in some SHR strains (295, 296).
In rats, this locus is associated with reduced insulin-stimulated glucose uptake into adipocytes and decreased catecholamine-stimulated free fatty acid secretion (292). Overexpression of Cd36 in these rats improved oral glucose tolerance and increased muscle glucose uptake (294). These findings predict that loss of Cd36 should be associated with insulin resistance. However, CD36-deficient mice have decreased, not increased, fasting (overnight) glucose and insulin levels, and on a chow diet, CD36 knockouts are more insulin sensitive (297, 298). Whole body glucose uptake is increased, primarily due to increased uptake in muscle; however, insulin fails to inhibit hepatic glucose production, and ketone bodies are elevated (299). These data are consistent with increased glucose utilization in muscle in the presence of impaired fatty acid uptake by this tissue, diverting excess fatty acids to the liver for oxidation and storage. In response to high-carbohydrate feeding, however, the knockout mice are more insulin resistant (298). None of these studies have examined adipocytes. Muscle-specific overexpression in mice was associated with increased fasting glucose and insulin (300). Thus, the differences with the rat may relate to tissue-specific effects of loss of CD36, differences in dietary conditions, or perhaps species differences in the relative utilization of free fatty acid and glucose. Nonetheless, these data support a role of Cd36 in the determination of insulin sensitivity.
Mouse Cd36 is found at 17 Mb on chromosome 5. Given the apparent species differences in the effects of this gene, it is perhaps not surprising that no QTL have been mapped to this region in the mouse. CD36 is located on human chromosome 7, at approximately 80 Mb, where several QTL for diabetes-related traits have been observed (12, 262, 301, 302). Some, but not all, studies have shown increased plasma glucose levels, insulin resistance, and/or a relationship to diabetes in humans with CD36 deficiency (303, 304, 305, 306, 307). Recently, Corpeleijn et al. (308) have described the first association of SNPs in this gene with insulin resistance, although these findings have not yet been replicated. As suggested from the rodent data, the role of CD36 in the development of insulin resistance may be dependent upon environmental factors such as diet.
The cholecystokinin A receptor (Cckar, Cck1r) is another potential diabetes susceptibility gene that has been identified in the rat. Hirashima et al. (309) first reported a linkage for impaired glucose tolerance in the OLETF rat to chromosome 14, near marker D14Mit4, which they called Odb2. This linkage was independently replicated, and this region was also shown to be associated with reduced pancreatic proliferative capacity after pancreatectomy (243, 310). About the same time, it was discovered that the OLETF strain has an approximately 7-kb deletion that removes the promoter and first two exons of the nearby (17-Mb distal) Cckar gene (311). A role for Cckar genotype in glucose intolerance was suggested by showing that F2 rats homozygous for the deleted Cckar have impaired glucose tolerance compared with the cohort of F2 animals with the intact gene, but in comparing F2 to backcross rats both homozygous for the disrupted Cckar, it was also determined that additional OLETF alleles contribute to the glucose intolerance of this strain (312). Functionally, signaling through the Cckar is associated with secretion of insulin, somatostatin, and leptin, also with gastric emptying and gastric and intestinal motility, and with the acute inhibition of food intake, all of which might be related to the development of glucose intolerance (313). These data suggest that the Cckar is a candidate gene for this QTL, and indeed numerous phenotypes observed in the OLETF strain have been attributed to the absence of the Cckar in these rats.
Although intriguing, these studies do not provide genetic validation that the Cckar is the gene underlying this QTL or conclusively show which phenotypes in the OLETF rat are due to the absence of this receptor. Consistent with the F2 vs. backcross comparison, numerous additional QTL affecting body weight and glucose tolerance have been detected in this strain, including at least three others on rat chromosome 14 (233, 243, 244, 245, 246, 275, 314, 315, 316). Furthermore, the phenotype of Cckar knockout mice does not replicate that of OLETF rats (317, 318). Consistent with its role in food intake, Cckar-deficient mice fail to decrease food intake in response to cholecystokinin (CCK); however, they show no difference in body weight, glucose levels, or pancreatic morphology (318). These differences may be due to species-specific effects or to interactions with other OLETF alleles, but it is also possible that the OLETF phenotype mapped to this region is not entirely due to the Cckar mutation. Notably, the Ppargc1a gene is less than 3 Mb from the Cckar gene and would also be an excellent candidate.
In addition, because the Cckar mediates food intake, could the glucose intolerance phenotype be secondary to differences in obesity? Wei et al. (243) mapped body weight in their study and did not detect significant linkage to body weight here (Nidd10/of), although they did detect an interaction of this locus with body weight in determining postchallenge glucose levels. A large-insert congenic strain has been created, in which a greater than 40-Mb region of OLETF chromosome 14 has been introgressed into the F344 background (245). This F.O-Nidd10/of congenic strain maintains differences in postchallenge glucose levels but does not show differences in body weight, suggesting that the glucose intolerance phenotype is independent of body weight differences.
The Cckar gene is located on mouse chromosome 5, at approximately 53 Mb. QTL near this region have been detected in the NZO (173) and FVB (187) strains. In humans, the CCKAR is located on chromosome 4 (26 Mb), and as described above is very near the PPARGC1A gene, which has been associated with type 2 diabetes susceptibility (21). A QTL for HbA1c levels in the Old Order Amish overlaps this region (12), and a type 2 diabetes QTL was observed nearby in Dutch individuals (253). A single study has suggested associations of SNPs in the promoter region of CCKAR with increased body fat and plasma insulin levels (319), and another study has detected amino acid variants in this gene in obese individuals (320). Thus, at this point, perhaps it is best to describe the Cckar as a candidate for diabetes susceptibility; however, additional evidence, either through further genetic studies or by restoring Cckar expression in OLETF rats, would greatly strengthen the argument that this is a diabetes susceptibility gene.
F. Hot spots in human linkage studies
What about the other major regions of human type 2 diabetes susceptibility genes on chromosomes 1q, 2q, 3p, 3q, 8p, 12q, 18, and 20q? Have any of these regions also been detected in the mouse? In short, the answer is yes. Mouse genetics has identified linkages in the syntenic regions corresponding to all these areas, with the 1q and 2q loci particularly evident.
Nearly 50 QTL have been mapped on human chromosome 1q in the region from approximately 150–250 Mb (11, 12, 251, 252, 254, 256, 257, 258, 260, 261, 262, 263, 278, 279, 280, 282, 283, 289, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337). In mouse, this corresponds to regions of chromosome 3, (90 Mb), chromosome 1 (130–194 Mb), chromosome 8 (123–125 Mb), and chromosome 13 (13 Mb). Six QTL for glucose or insulin levels in the regions of mouse chromosome 1 have been detected in the TSOD, KK, A/J, LG, and the wild-derived PERA/Ei strains (155, 193, 198, 218, 224, 338), as has another for glucose intolerance in the small segment of chromosome 13 (67). Regions of rat chromosomes 2 (180–190 Mb), 13 (40–111 Mb), 17 (70 Mb), and 19 (55–56 Mb) are syntenic to human chromosome 1q. The Nidd/gk2, Niddm2, and Nidd/gk6 QTL in the GK rat (236, 237), the Ir2 QTL in the SHR strain (339), and the Insul1 QTL identified in a cross between the Lyon Hypertensive (LH) and normotensive (LN) strains are located within these regions (340).
Human chromosome 2q (110–240 Mb) has roughly 25 QTL for diabetes and diabetes-related traits (12, 257, 258, 262, 263, 279, 281, 285, 291, 301, 321, 322, 327, 337, 341, 342, 343, 344, 345). This region contains the CAPN10 gene, identified as a type 2 diabetes susceptibility gene through positional cloning, and for which growing evidence favors its role in this disease (21, 346, 347, 348). In addition, this region contains the NEUROD gene, which has been implicated in MODY (349), and the IRS1 gene, where SNPs have been significantly associated with type 2 diabetes in a meta-analysis (350). This broad region has syntenic regions primarily on mouse chromosomes 1 (48–94 Mb) and 2 (45–82 Mb), where several diabetes-related traits have been mapped in the KK, LG, SM, C57BL/6, TSOD, CAST/Ei, and A/J strains (67, 134, 154, 159, 160, 194, 214, 217, 219, 224, 226). In the rat, the majority of this region is syntenic to chromosomes 3 and 9 (20–70 and 45–90 Mb, respectively). The region on chromosome 9 contains the Nidd8/of QTL from the OLETF strain (243, 244, 245). Capn10 is within this region, and a Gly
Ser mutation at amino acid 195 has been identified in the OLETF strain, although the functional consequences of this variant are unclear (351).
Regions on both arms of human chromosome 3 have numerous QTL (10, 11, 252, 253, 256, 258, 261, 262, 280, 282, 284, 302, 328, 331, 332, 333, 341, 342, 344, 352, 353, 354, 355, 356, 357). The p-arm (80 Mb), which contains PPARG, is syntenic to regions of mouse chromosomes 6, 9, 14, and 16. On mouse chromosome 6, a QTL for plasma glucose levels in the A/J strain has been detected (153, 155), and there is a potential overlap with the Nidd3/nsy locus (204). A QTL from C57BL/6 has been found in the syntenic region on chromosome 14 (130, 131). In the rat, this region has synteny to chromosomes 4, 8, 9, 15, and 16. A locus for increased postprandial glucose levels in the OLETF rat near D4Mgh7 may overlap with this region (246), and a QTL on chromosome 16, identified in a cross of Wistar and Zucker Leprfa/fa rats, is also in the corresponding region (358). The q-arm (140–200 Mb) corresponds to parts of mouse chromosomes 3, 9, and 16. Again, only a single mouse QTL associated with increased insulin in the SM strain is situated in the major syntenic regions (126). Segments of rat chromosomes 2, 8, and 11 are syntenic to this region. The Nidd4/of QTL for plasma glucose levels in the OLETF strain is contained within the corresponding region of chromosome 11 (244, 245, 275). Additional QTL have been detected in the central region of human chromosome 3. Corresponding regions are found on mouse chromosomes 6, 9, 14, and 16.
Human chromosome 8p (0–40 Mb), with roughly a dozen QTL (251, 253, 254, 255, 258, 263, 279, 285, 301, 322, 328, 341, 353, 359, 360), has syntenic regions on mouse chromosomes 8 and 14. Three QTL for glucose intolerance have been detected in the KK, DBA/2 (chromosome 8), and C57BL/6 (chromosome 14) strains (126, 130, 194, 198). Rat chromosomes 15 and 16 harbor the syntenic regions; however, no QTL have been detected in the corresponding areas.
Several loci reaching genomewide significance have been detected around approximately 70 Mb, and others near 120 Mb on human chromosome 12 (291, 352, 361, 362, 363, 364), along with many other suggestive QTL in these regions (13, 252, 257, 259, 263, 283, 286, 287, 301, 321, 328, 335, 337, 352, 359). In the mouse, the primary syntenic regions are chromosome 10 (100–120Mb) and chromosome 5 (115–130 Mb). Diabetes-related QTL have been found in both these locations from the SM, B6, and C57BLKS/J strains (123, 134, 153, 154, 155, 219). Chromosomes 7 (50–60 Mb) and 12 (40 Mb) in the rat are syntenic to these loci; however, no QTL have been found in either of these areas.
Linkages have been observed spanning human chromosome 18 (12, 252, 253, 255, 258, 259, 279, 280, 302, 323, 324, 332, 333, 334, 337, 343, 344, 345, 352, 354, 359). There is some evidence of mouse QTL in the corresponding syntenic regions that are primarily located on chromosomes 17 and 18 (153, 173). Rats have syntenic regions on chromosomes 9, 13, and 18, but we have not found any QTL reported in these locations. Thus, in contrast to the other hot spots, the QTL on human chromosome 18 have not been detected in the rodent models.
Finally, human chromosome 20q is another hot spot containing several significant QTL. This region is entirely syntenic to mouse chromosome 2 (150–180 Mb). Somewhat surprisingly given the number of significant linkages in this region, and in contrast to the well-replicated regions on chromosomes 1q and 2q, only three mouse diabetes-related QTL have been observed in this region (99, 159, 214). Two of these are loci detected in the C57BL/6 strain, in crosses with either C3H or CAST/Ei, whereas the other was identified in the M16 strain. On the syntenic region of rat chromosome 3 is a QTL for response to pancreatectomy (365). The p-arm of human chromosome 20 also has several QTL (250, 253, 260, 261, 287, 290, 324, 337, 366). No QTL have been detected in the syntenic region of mouse chromosome 2, but in the rat, two additional QTL on chromosome 3 are syntenic to this region (339, 367).
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IX. Summary/Conclusion |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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X. Appendix: Useful Web Site Resources for Mouse Genetics Research |
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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Ensembl genome assembly: http://www.ensembl.org/
Mouse Genome Informatics: http://www.informatics.jax.org/
Mouse Phenome Database: http://www.jax.org/phenome
National Center for Biotechnology Information (NCBI) mouse genome resources: http://www.ncbi.nlm.nih.gov/genome/guide/mouse/
dbSNP for mouse SNP data: http://www.ncbi.nlm.nih.gov/SNP/MouseSNP.cgi
Mouse-human-rat homology maps: http://www.ncbi.nlm.nih.gov/Homology/
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online October 3, 2006
Abbreviations: AIL, Advanced intercross line; BN, Brown Norway; CCK, cholecystokinin; Cpe, carboxypeptidase E; GK, Goto Kakizaki; ICR, Imperial Cancer Research; IPGTT, ip glucose tolerance test; LG, large strain; LH, Lyon hypertensive; LN, Lyon normotensive; LOD, logarithm of the odds; MODY, maturity onset diabetes of the young; NAD, nicotinamide-adenine dinucleotide; NADP, NAD phosphate; NOD, nonobese diabetic; NSY, Nagoya-Shibata-Yasuda; NZB, New Zealand Black; NZO, New Zealand obese; NZW, New Zealand White; OLETF, Otsuka Long-Evans Tokushima Fatty; QTL, quantitative trait loci; RCS, recombinant congenic strain; SDT, spontaneously diabetic Torii; SHR, spontaneously hypertensive rat; SJL, Swiss Jackson Laboratory; SM, small strain; SNP, single nucleotide polymorphism; TSOD, Tsumura, Suzuki, Obese Diabetes.
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TopAbstractI. Physiological DefinitionII. The High Prevalence...III. What Does Genetics...IV. Advantages of Model...V. Origin of Inbred...VI. Experimental Strategies for...VII. Mouse Strains Used...VIII. Linkage Hot SpotsIX. Summary/ConclusionX. Appendix: Useful Web...References |
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25). Maximum genetic diversity is maintained if each breeding male and female contributes a single male and female to the next breeding generation.