Genetic variation in Glp1r expression influences the rate of gastric emptying in mice

We demonstrated previously that food intake traits map to a quantitative trait locus (QTL) on proximal chromosome 17, which encompasses Glp1r (glucagon-like peptide 1 receptor), encoding an important modulator of gastric emptying. We then confirmed this QTL in a B6.CAST-17 congenic strain that consumed 27% more carbohydrate and 17% more total calories, yet similar fat calories, per body weight compared with the recipient C57BL/6J. The congenic strain also consumed greater food volume. The current aims were to 1) identify genetic linkage for total food volume in F(2) mice, 2) perform gene expression profiling in stomach of B6.CAST-17 congenic mice using oligonucleotide arrays, 3) test for allelic imbalance in Glp1r expression, 4) evaluate gastric emptying rate in parental and congenic mice, and 5) investigate a possible effect of genetic variation in Glp1r on gastric emptying. A genome scan revealed a single QTL for total food volume (Tfv1) (log of the odds ratio = 7.6), which was confirmed in B6.CAST-17 congenic mice. Glp1r exhibited allelic imbalance in stomach, which correlated with accelerated gastric emptying in parental CAST and congenic B6.CAST-17 mice. Moreover, congenic mice displayed an impaired gastric emptying response to exendin-(9-39). These results suggest that genetic variation in Glp1r contributes to the strain differences in gastric emptying rate.     

Increased physical activity cosegregates with higher intake of carbohydrate and total calories in a subcongenic mouse strain

C57BL/6 J (B6) and CAST/EiJ (CAST), the inbred strain derived from M. musculus castaneus, differ in nutrient intake behaviors, including dietary fat and carbohydrate consumption in a two-diet-choice paradigm. Significant quantitative trait loci (QTLs) for carbohydrate (Mnic1) and total energy intake (Kcal2) are present between these strains on chromosome (Chr) 17. Here we report the refinement of the Chr 17 QTL in a subcongenic strain of the B6.CAST- ^{D17Mit19-D17Mit91} congenic mice described previously. This new subcongenic strain possesses CAST Chr 17 donor alleles from 4.8 to 45.4 Mb on a B6 background. Similar to CAST, the subcongenic mice exhibit increased carbohydrate and total calorie intake per body weight, while fat intake remains equivalent. Unexpectedly, this CAST genomic segment also confers two new physical activity phenotypes: 22% higher spontaneous physical activity levels and significantly increased voluntary wheel-running activity compared with the parental B6 strain. Overall, these data suggest that gene(s) involved in carbohydrate preference and increased physical activity are contained within the proximal region of Chr 17. Interval-specific microarray analysis in hypothalamus and skeletal muscle revealed differentially expressed genes within the subcongenic region, including neuropeptide W (Npw); glyoxalase I (Glo1); cytochrome P450, family 4, subfamily f, polypeptide 1 (Cyp4f15); phospholipase A2, group VII (Pla2g7); and phosphodiesterase 9a (Pde9a). This subcongenic strain offers a unique model for dissecting the contributions and possible interactions among genes controlling food intake and physical activity, key components of energy balance.     

Gene expression between a congenic strain that contains a quantitative trait locus of high bone density from CAST/EiJ and its wild-type strain C57BL/6J

Peak bone density is an important determining factor of future osteoporosis risk. We previously identified a quantitative trait locus (QTL) that contributes significantly to high bone density on mouse chromosome 1 from a cross between C57BL/6J (B6) and CAST/EiJ (CAST) mouse strains. We then generated a congenic strain, B6.CAST-1T, in which the chromosomal fragment containing this QTL had been transferred from CAST to the B6 background. The congenic mice have a significantly higher bone density than the B6 mice. In this study we performed cDNA microarray analysis to evaluate the gene expression profile that might yield insights into the mechanisms controlling the high bone density by this QTL. This study led to several interesting observations. First, approximately 60% of 8,734 gene accessions on GEM I chips were expressed in the femur of B6 mice. The expression and function of two-thirds of these expressed genes and ESTs have not been documented previously. Second, expression levels of genes related to bone formation were lower in congenic than in B6 mice. These data are consistent with a low bone formation in the congenic mice, a possibility that is confirmed by reduced skeletal alkaline phosphatase activity in serum compared with B6 mice. Third, expression levels of genes that might have negative regulatory action on bone resorption were higher in congenic than in B6 mice. Together these findings suggest that the congenic mice might have a lower bone turnover rate than B6 mice and raise the possibility that the high bone density in the congenic mice could be due to reduced bone resorption rather than increased bone formation. Electronic Publication     

Allelic variation on murine chromosome 11 modifies host inflammatory responses and resistance to Bacillus anthracis

Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36-74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.     

Macronutrient diet selection in thirteen mouse strains

The strain distribution for macronutrient diet selection was described in 13 mouse strains (AKR/J, NZB/B1NJ, C57BL/6J, C57BL/6ByJ, DBA/2J, SPRET/Ei, CD-1, SJL/J, SWR/J, 129/J, BALB/cByJ, CAST/Ei, and A/J) with the use of a self-selection protocol in which separate carbohydrate, fat, and protein diets were simultaneously available for 26-30 days. Relative to carbohydrate, nine strains consumed significantly more calories from the fat diet; two strains consumed more calories from carbohydrate than from fat (BALB/cByJ, CAST/Ei). Diet selection by SWR/J mice was variable over time, resulting in a lack of preference. One strain (A/J) failed to adapt to the diet paradigm due to inadequate protein intake. Comparisons of proportional fat intake across strains revealed that fat selection/consumption ranged from 26 to 83% of total energy. AKR/J, NZB/B1NJ, and C67BL/6J mice self-selected the highest proportion of dietary fat, whereas the CAST/Ei and BALB/cByJ strains chose the lowest. Finally, epididymal fat depot weight was correlated with fat consumption. There were significant positive correlations in AKR/J and C57BL/6J mice, which are highly sensitive to dietary obesity. However, absolute fat intake was inversely correlated with epididymal fat in two of the lean strains: SWR/J and CAST/Ei. We hypothesize that the SWR/J and CAST/Ei strains are highly sensitive to a negative feedback signal generated by increasing body fat, but the AKR/J and C67BL/6J mice are not. The variation in dietary fat selection across inbred strains provides a tool for dissecting the complex genetics of this trait.     

High-Resolution Mapping of a Genetic Locus Regulating Preferential Carbohydrate Intake,Total Kilocalories,and Food Volume on Mouse Chromosome 17

The specific genes regulating the quantitative variation in macronutrient preference and food intake are virtually unknown. We fine mapped a previously identified mouse chromosome 17 region harboring quantitative trait loci (QTL) with large effects on preferential macronutrient intake-carbohydrate (Mnic1), total kilcalories (Kcal2), and total food volume (Tfv1) using interval-specific strains. These loci were isolated in the [C57BL/6J.CAST/EiJ-17.1-(D17Mit19-D17Mit50); B6.CAST-17.1] strain, possessing a ∼40.1 Mb region of CAST DNA on the B6 genome. In a macronutrient selection paradigm, the B6.CAST-17.1 subcongenic mice eat 30% more calories from the carbohydrate-rich diet, ∼10% more total calories, and ∼9% more total food volume per body weight. In the current study, a cross between carbohydrate-preferring B6.CAST-17.1 and fat-preferring, inbred B6 mice was used to generate a subcongenic-derived F₂ mapping population; genotypes were determined using a high-density, custom SNP panel. Genetic linkage analysis substantially reduced the 95% confidence interval for Mnic1 (encompassing Kcal2 and Tfv1) from 40.1 to 29.5 Mb and more precisely established its boundaries. Notably, no genetic linkage for self-selected fat intake was detected, underscoring the carbohydrate-specific effect of this locus. A second key finding was the separation of two energy balance QTLs: Mnic1/Kcal2/Tfv1 for food intake and a newly discovered locus regulating short term body weight gain. The Mnic1/Kcal2/Tfv1 QTL was further de-limited to 19.0 Mb, based on the absence of nutrient intake phenotypes in subcongenic HQ17IIa mice. Analyses of available sequence data and gene ontologies, along with comprehensive expression profiling in the hypothalamus of non-recombinant, cast/cast and b6/b6 F₂ controls, focused our attention on candidates within the QTL interval. Zfp811, Zfp870, and Btnl6 showed differential expression and also contain stop codons, but have no known biology related to food intake regulation. The genes Decr2, Ppard and Agapt1 are more appealing candidates because of their involvement in lipid metabolism and down-regulation in carbohydrate-preferring animals.     

A genome-wide set of congenic mouse strains derived from CAST/Ei on a C57BL/6 background

We previously reported the construction of two sets of heterozygous congenic strains spanning the mouse genome. For both sets, C57BL/6J was employed as the background strain while DNA from either DBA/2 or CAST/Ei was introgressed to form the congenic region. We have subsequently bred most of these strains to produce homozygous breeding stocks. Here, we report the characterization of the strain set based on CAST/Ei. CAST/Ei is the most genetically distant strain within the Mus mus species and many trait variations relevant to common diseases have been identified in CAST/Ei mice. Despite breeding difficulties for some congenic regions, presumably due to incompatible allelic variations between CAST/Ei and C57BL/6, the resulting congenic strains cover about 80% of the autosomal chromosomes and will be useful as a resource for the further analysis of quantitative trait loci between the strains.     

Mx1 causes resistance against influenza A viruses in the Mus spretus-derived inbred mouse strain SPRET/Ei

Inbred SPRET/Ei mice, derived from Mus spretus, were found to be extremely resistant to infection with a mouse adapted influenza A virus. The resistance was strongly linked to distal chromosome 16, where the interferon-inducible Mx1 gene is located. This gene encodes for the Mx1 protein which stimulates innate immunity to Orthomyxoviruses. The Mx1 gene is defective in most inbred mouse strains, but PCR revealed that SPRET/Ei carries a functional allele. The Mx1 proteins of M. spretus and A2G, the other major resistant strain derived from Mus musculus, share 95.7% identity. We were interested whether the sequence variations between the two Mx1 alleles have functional significance. To address this, we used congenic mouse strains containing the Mx1 gene from M. spretus or A2G in a C57BL/6 background. Using a highly pathogenic influenza virus strain, we found that the B6.spretus-Mx1 congenic mice were better protected against infection than the B6.A2G-Mx1 mice. This effect may be due to different Mx1 induction levels, as was shown by RT-PCR and Western blot. We conclude that SPRET/Ei is a novel Mx1-positive inbred strain useful to study the biology of Mx1.     

Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei x C57BL/6J)

Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with "panhandle"-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated.The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.     

Genome-wide screening for genetic loci associated with noise-induced hearing loss

Noise-induced hearing loss (NIHL) is one of the more common sources of environmentally induced hearing loss in adults. In a mouse model, Castaneous (CAST/Ei) is an inbred strain that is resistant to NIHL, while the C57BL/6J strain is susceptible. We have used the genome-tagged mice (GTM) library of congenic strains, carrying defined segments of the CAST/Ei genome introgressed onto the C57BL/6J background, to search for loci modifying the noise-induced damage seen in the C57BL/6J strain. NIHL was induced by exposing 6-8-week old mice to 108 dB SPL intensity noise. We tested the hearing of each mouse strain up to 23 days after noise exposure using auditory brainstem response (ABR). This study identifies a number of genetic loci that modify the initial response to damaging noise, as well as long-term recovery. The data suggest that multiple alleles within the CAST/Ei genome modify the pathogenesis of NIHL and that screening congenic libraries for loci that underlie traits of interest can be easily carried out in a high-throughput fashion.     

Food restriction-like effects of dietary dehydroepiandrosterone. Hypothalamic neurotransmitters and metabolites in male C57BL/6 and (C57BL/6 x DBA/2)F1 mice

Dehydroepiandrosterone (DHEA) is a precursor of sex hormones in mammals. Dietary DHEA serves to prevent or inhibit various diseases and also lengthens life spans of animals. Moreover, dietary DHEA inhibits food intake in certain strains of mice. We administered DHEA (0.45% w/w of food) to C57BL/6 (B6) and (B6 x DBA/2)F1 (BDF1) mice for 5 weeks. Food intake was inhibited in both strains of mice during the first week. Thereafter, B6, but not BDF1, mice consumed less food. Because hypothalamic serotonin and/or dopamine regulate appetite, satiety and other behaviors, the hypothesis tested was that hypothalamic concentration of serotonin, dopamine and/or their metabolites are affected differentially in B6 and BDF1 mice fed DHEA. In another study, mice were fed the AIN-76A diet with or without DHEA for 1 and 7 days or were pair-fed to DHEA-fed mice for 7 days. On Day 1 of DHEA feeding (acute effects) hypothalamic levels of serotonin, dopamine, and metabolites were unchanged in B6 mice, but levels of dopamine were increased and levels of dopamine metabolites were decreased in BDF1 mice. On Day 7 of DHEA feeding, levels of serotonin were increased in BDF1 but not B6 mice. On Day 7 of pair-feeding there were decreased levels of hypothalamic dopamine metabolites in BDF1 but not B6 mice. Paraventricular nuclei of BDF1 mice had decreased levels of serotonin but not of dopamine in all groups. Serum levels of DHEA and its metabolite, 5-androstene-3beta,17beta-diol, correlated significantly only with serotonin concentrations in BDF1 mice. The salient findings of these experiments are that DHEA inhibits food intake to a greater extent in B6 than in BDF1 mice. However, alterations of hypothalamic neurotransmitters were greater in BDF1 than in B6 mice. Because BDF1 and B6 mice share B6 genes, relevant gene(s) derived from DBA/2 mice might mediate the different responses detected.     

Genome-tagged mice (GTM): two sets of genome-wide congenic strains

An important approach for understanding complex disease risk using the mouse is to map and ultimately identify the genes conferring risk. Genes contributing to complex traits can be mapped to chromosomal regions using genome scans of large mouse crosses. Congenic strains can then be developed to fine-map a trait and to ascertain the magnitude of the genotype effect in a chromosomal region. Congenic strains are constructed by repeated backcrossing to the background strain with selection at each generation for the presence of a donor chromosomal region, a time-consuming process. One approach to accelerate this process is to construct a library of congenic strains encompassing the entire genome of one strain on the background of the other. We have employed marker-assisted breeding to construct two sets of overlapping congenic strains, called genome-tagged mice (GTMs), that span the entire mouse genome. Both congenic GTM sets contain more than 60 mouse strains, each with on average a 23-cM introgressed segment (range 8 to 58 cM). C57BL/6J was utilized as a background strain for both GTM sets with either DBA/2J or CAST/Ei as the donor strain. The background and donor strains are genetically and phenotypically divergent. The genetic basis for the phenotypic strain differences can be rapidly mapped by simply screening the GTM strains. Furthermore, the phenotype differences can be fine-mapped by crossing appropriate congenic mice to the background strain, and complex gene interactions can be investigated using combinations of these congenics.     

Genetic studies of flavivirus resistance in inbred strains derived from wild mice: evidence for a new resistance allele at the flavivirus resistance locus (Flv).

Studies of genetic resistance to flavivirus infection in laboratory mice have led to the development of a single model in which resistance is conferred by an autosomal dominant gene designated Flvr. Because of evidence suggesting that wild mice carry virus resistance genes which are not present in laboratory mice, we compared flavivirus resistance in the inbred strains CASA/Rk, CAST/Ei, and MOLD/Rk, which are derived directly from wild mice, and the congenic strains C3H/RV (Flvr/Flvr) and C3H/HeJ (Flvs/Flvs). Resistance to the Murray Valley encephalitis virus strain OR2 and the 17D vaccine strain of yellow fever virus was assessed by determining the lethality of intracerebral infection and by measuring virus replication in the brain. The resistance of the CASA/Rk and CAST/Ei strains resembled the resistance of C3H/RV mice, whereas the resistance of the MOLD/Rk strain was intermediate between those of C3H/RV and C3H/HeJ mice. Genetic analyses showed that resistance in both the CASA/Rk and MOLD/Rk strains is conferred by single autosomal dominant alleles at the Flv locus. Our data indicate that flavivirus resistance in the CASA/Rk strain is due to a gene which is similar or identical to Flvr, whereas resistance in the MOLD/Rk strain is due to a previously undescribed gene which we designate Flvmr to indicate minor resistance to flavivirus infection. Since genetic resistance to flaviviruses is rare in laboratory mice, the CASA/Rk and MOLD/Rk strains will be valuable for further investigation of this phenomenon.     

Rapid vascular responses to anthrax lethal toxin in mice containing a segment of chromosome 11 from the CAST/Ei strain on a C57BL/6 genetic background

Host allelic variation controls the response to B. anthracis and the disease course of anthrax. Mouse strains with macrophages that are responsive to anthrax lethal toxin (LT) show resistance to infection while mouse strains with LT non-responsive macrophages succumb more readily. B6.CAST.11M mice have a region of chromosome 11 from the CAST/Ei strain (a LT responsive strain) introgressed onto a LT non-responsive C57BL/6J genetic background. Previously, B6.CAST.11M mice were found to exhibit a rapid inflammatory reaction to LT termed the early response phenotype (ERP), and displayed greater resistance to B. anthracis infection compared to C57BL/6J mice. Several ERP features (e.g., bloat, hypothermia, labored breathing, dilated pinnae vessels) suggested vascular involvement. To test this, Evan's blue was used to assess vessel leakage and intravital microscopy was used to monitor microvascular blood flow. Increased vascular leakage was observed in lungs of B6.CAST.11M mice compared to C57BL/6J mice 1 hour after systemic administration of LT. Capillary blood flow was reduced in the small intestine mesentery without concomitant leukocyte emigration following systemic or topical application of LT, the latter suggesting a localized tissue mechanism in this response. Since LT activates the Nlrp1b inflammasome in B6.CAST.11M mice, the roles of inflammasome products, IL-1β and IL-18, were examined. Topical application to the mesentery of IL-1β but not IL-18 revealed pronounced slowing of blood flow in B6.CAST.11M mice that was not present in C57BL/6J mice. A neutralizing anti-IL-1β antibody suppressed the slowing of blood flow induced by LT, indicating a role for IL-1β in the response. Besides allelic differences controlling Nlrp1b inflammasome activation by LT observed previously, evidence presented here suggests that an additional genetic determinant(s) could regulate the vascular response to IL-1β. These results demonstrate that vessel leakage and alterations to blood flow are part of the rapid response in mice resistant to B. anthracis infection.     

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene

 Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4. Received: 15 May 1999 / Revised: 25 September 1999     

Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11^A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11.     

Gene doubling increases glyoxalase 1 expression in RAGE knockout mice

BackgroundThe receptor for advanced glycation end-products (RAGE) is a multifunctional protein. Its function as pattern recognition receptor able to interact with various extracellular ligands is well described. Genetically modified mouse models, especially the RAGE knockout (RAGE-KO) mouse, identified the amplification of the immune response as an important function of RAGE. Pro-inflammatory ligands of RAGE are also methylglyoxal-derived advanced glycation end-products, which depend in their quantity, at least in part, on the activity of the methylglyoxal-detoxifying enzyme glyoxalase-1 (Glo1). Therefore, we studied the potential interaction of RAGE and Glo1 by use of RAGE-KO mice.MethodsVarious tissues (lung, liver, kidney, heart, spleen, and brain) and blood cells from RAGE-KO and wildtype mice were analyzed for Glo1 expression and activity by biochemical assays and the Glo1 gene status by PCR techniques.ResultsWe identified an about two-fold up-regulation of Glo1 expression and activity in all tissues of RAGE-KO mice. This was result of a copy number variation of the Glo1 gene on mouse chromosome 17. In liver tissue and blood cells, the Glo1 expression and activity was additionally influenced by sex with higher values for male than female animals. As the genomic region containing Glo1 also contains the full-length sequence of another gene, namely Dnahc8, both genes were duplicated in RAGE-KO mice.ConclusionA genetic variance in RAGE-KO mice falsely suggests an interaction of RAGE and Glo1 function.General significanceRAGE-independent up-regulation of Glo1 in RAGE-KO mice might be as another explanation for, at least some, effects attributed to RAGE before.     

Gene-environment interaction: a significant diet-dependent obesity locus demonstrated in a congenic segment on mouse Chromosome 7

We have previously reported suggestive evidence for a locus on Chromosome (Chr) 7 that affects adiposity in F₂ mice from a CAST/Ei × C57BL/6J intercross fed a high-fat diet. Here we characterize the effect of a high-fat (32.6 Kcal% fat) diet on male and female congenic mice with a C57BL/6J background and a CAST/Ei-derived segment on Chr 7. Adiposity index (AI) and weights of certain fat pads were approximately 50% lower in both male and female congenic mice than in control C57BL/6J mice, and carcass fat content was significantly reduced. The reduction of fat depot weights was not seen, however, in congenic animals fed a low-fat chow diet (12 Kcal% fat). The congenic segment is approximately 25 cM in length, extending from D7Mit213 to D7Mit41, and includes the tub, Ucp2, and Ucp3, genes, all of which are candidate genes for this effect. Some polymorphisms have been found on comparing c-DNA sequences of the Ucp2 gene from C57BL/6J and CAST/Ei mice. These results suggest that one or more genes present in the congenic segment modulate the susceptibility to fat deposition on feeding a high-fat diet. We were unable to show any significant difference between the energy intakes of the congenic and the control C57BL/6J mice on the high-fat diet. Also, measurements of energy expenditure in male mice at 6 weeks of age, during the first 2 weeks of exposure to the high-fat diet, failed to show any differences between control and congenic animals. Received: 30 September 1998 / Accepted: 22 December 1998     

Fine map of the Gct1 spontaneous ovarian granulosa cell tumor locus

The spontaneous development of juvenile-onset, ovarian granulosa cell (GC) tumors in the SWR/Bm (SWR) inbred mouse strain is a model for juvenile-type GC tumors that appear in infants and young girls. GC tumor susceptibility is supported by multiple Granulosa cell tumor (Gct) loci, but the Gct1 locus on Chr 4 derived from SWR strain background is fundamental for GC tumor development and uniquely responsive to the androgenic precursor dehydroepiandrosterone (DHEA). To resolve the location of Gct1 independently from other susceptibility loci, Gct1 was isolated in a congenic strain that replaces the distal segment of Chr 4 in SWR mice with a 47 × 10⁶-bp genomic segment from the Castaneus/Ei (CAST) strain. SWR females homozygous for the CAST donor segment were confirmed to be resistant to DHEA- and testosterone-induced GC tumorigenesis, indicating successful exchange of CAST alleles (Gct1 ^CA ) for SWR alleles (Gct1 ^SW ) at this tumor susceptibility locus. A series of nested, overlapping, congenic sublines was created to fine-map Gct1 based on GC tumor susceptibility under the influence of pubertal DHEA treatment. Twelve informative lines have resolved the Gct1 locus to a 1.31 × 10⁶-bp interval on mouse Chr 4, a region orthologous to human Chr 1p36.22.     

Genome-wide microarray expression analysis of CD4+ T Cells from nonobese diabetic congenic mice identifies Cd55 (Daf1) and Acadl as candidate genes for type 1 diabetes

NOD.Idd3/5 congenic mice have insulin-dependent diabetes (Idd) regions on chromosomes 1 (Idd5) and 3 (Idd3) derived from the nondiabetic strains B10 and B6, respectively. NOD.Idd3/5 mice are almost completely protected from type 1 diabetes (T1D) but the genes within Idd3 and Idd5 responsible for the disease-altering phenotype have been only partially characterized. To test the hypothesis that candidate Idd genes can be identified by differential gene expression between activated CD4+ T cells from the diabetes-susceptible NOD strain and the diabetes-resistant NOD.Idd3/5 congenic strain, genome-wide microarray expression analysis was performed using an empirical Bayes method. Remarkably, 16 of the 20 most differentially expressed genes were located in the introgressed regions on chromosomes 1 and 3, validating our initial hypothesis. The two genes with the greatest differential RNA expression on chromosome 1 were those encoding decay-accelerating factor (DAF, also known as CD55) and acyl-coenzyme A dehydrogenase, long chain, which are located in the Idd5.4 and Idd5.3 regions, respectively. Neither gene has been implicated previously in the pathogenesis of T1D. In the case of DAF, differential expression of mRNA was extended to the protein level; NOD CD4+ T cells expressed higher levels of cell surface DAF compared with NOD.Idd3/5 CD4+ T cells following activation with anti-CD3 and -CD28. DAF up-regulation was IL-4 dependent and blocked under Th1 conditions. These results validate the approach of using congenic mice together with genome-wide analysis of tissue-specific gene expression to identify novel candidate genes in T1D.