Diabetic modifier QTL, Dbm4 , affecting elevated fasting blood glucose concentrations in congenic mice

We have previously identified four significant quantitative trait loci (QTL) affecting plasma glucose concentrations on F 2 progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and non-diabetic A/J mice, one of which on chromosome 15 named Dbm4 (diabetic modifier QTL 4) was shown to affect fasting plasma glucose concentrations with a maximum LOD score of 6.17. To estimate the influence of Dbm4 itself to the diabetes-related phenotypes, we constructed congenic strain with heterozygous Ins2 mutation using the Akita allele as donor of Dbm4 locus in the A/J genetic background, and measured quantitative traits including plasma glucose concentrations in glucose tolerance test (GTT). In this study, we found the Akita allele in Dbm4 was associated with higher fasting plasma glucose concentrations as in previous QTL analysis. According to gene expression assay, key enzymes of hepatic gluconeogenesis were to the more increased degree in the liver of congenic mice compared to the A/J allele based control mice. Based on these results, we concluded that diabetic modifier gene(s) exist on Dbm4 locus affecting fasting plasma glucose concentrations via regulation of gluconeogenic gene expression in the hypoinsulinemic diabetic condition. Identification of the modifier gene responsible for Dbm4 would provide new drug development targets for human type 2 diabetes with hepatic insulin resistance.


INTRODUCTION
Type 2 diabetes is a multifactorial disorder caused by both genetic (Leiter, 1989(Leiter, , 1993 and environmental factors. To discover causative genetic factors in complex diseases, genetic analyses of rodents and humans have been conducted. Quantitative trait loci (QTL) analysis has been widely applied to several rodent models of type 2 diabetes to identify chromosomal loci for genetic modifiers, and is rather advantageous than genetic studies in humans in that rodents offer both reduced environmental variation and genetic heterogeneity. To determine if one QTL independently contribute to phenotypic trait, it is essential to produce congenic mice that differ only in QTL. Congenic mice are useful to assess the potential effects of the locus solely on traits related to diabetes, because the difference of quantitative trait may possibly be attributed to the association with modifier genes in the multiple QTLs (Kose et al., 2002). QTL analyses of diabetes have been performed with several murine models to identify chromosomal loci of genetic modifiers Togawa et al., 2006) and the effects of a sole QTL have been verified using congenic mice (Mizutani et al., 2006;Stoehr et al., 2004). Recently novel diabetic modifier gene like Sorcs1 on chromosome 19 was identified using QTL and congenic approach (Clee et al., 2006).
We previously performed QTL analysis using F 2intercross progeny derived from non-obese diabetic Akita and non-diabetic A/J mice (Takeshita et al., 2006). The Akita mouse is a spontaneous model of type 2 diabetes Edited by Toshihiko Shiroishi * Corresponding author. E-mail: mtsita@circus.ocn.ne.jp established from the C57BL/6 strain (Yoshioka et al., 1997), and a heterozygous Cys96Tyr mutation of the insulin 2 gene (Ins2) has been identified as the etiology for the hypoinsulinemic diabetes (Kayo and Koizumi, 1998;Wang et al., 1999), with severe and mild diabetes, respectively, in male and female mice (Yohioka et al., 1997). The A/J mouse, used in our crosses, is genetically different from the C57BL/6 strain as exemplified by its phenotypic resistance to high-fat diet-induced diabetes (Surwit et al., 1991). In our QTL analysis, we identified the Dbm4 locus on chromosome 15 affecting fasting plasma glucose concentrations with significant logarithm of odds (LOD) score of 6.17 in the hypoinsulinemic conditions derived from heterozygous Ins2 mutation (Ins2-Hetero). The Dbm4 also weakly affects plasma glucose concentrations after glucose administration in glucose tolerance tests (GTT) with LOD score of 3.24, 2.37, and 2.91 (30, 60, and 120 min, respectively), in hypoinsulinemic Ins2-Hetero mice, all of which were detected only in male mice showing more severe diabetes than female mice (Takeshita et al., 2006). Therefore we proposed potential modifier gene(s) on the Dbm4 affecting diabetic traits in Ins2-Hetero derived hyperglycemic condition.
In this study, we report the phenotypic assessment of congenic mice with the Akita allele on Dbm4 locus in the A/J genetic background.

Mouse strains and construction of congenic mice
Male Akita mice and female A/J mice were purchased at 7 weeks of age from SLC Japan (Shizuoka, Japan). Mice were maintained with free access to food (CRF-1, purchased from Oriental Yeast, Tokyo, Japan) and water in a temperatureand humidity-controlled environment under a 12 h light-dark cycle. Congenic mice containing the homozygote for Akita mouse genotype as a donor of Dbm4 in the A/J recipient were constructed by repeated backcrosses and intercross as follows. The male Akita mice were crossed with the female A/J mice to produce first backcross progeny (BC1) which had the Akita-derived heterozygous mutation at the Ins2 locus and heterozygous Akita allele of Dbm4 locus estimated by genotyping as described below. BC2 progeny were produced by backcrossing male BC1 with female A/J mice. Male mice containing A/J background alleles (except Ins2 and the Dbm4 locus, and Y-chromosome) were selected and used for further backcrosses five or more times to obtain mice with purified A/J genetic background, which retained more than 95% of A/J alleles. Derived backcross progeny contained A/J genetic background with Akita alleles of Dbm4 and Ins2 loci, and then male and female progenies were intercrossed to obtain congenic strains which contained the homozygous Akita alleles of Dbm4 (Ak/Ak) with heterozygous mutation of Ins2 gene, and control strains which contained the homozygous A/J alleles of Dbm4 (Aj/ Aj) with Ins2 heretozygous mutation. Breeding procedures of congenic and control strains were supported by Oriental BioService, Inc (Kyoto, Japan). All animal experiments were approved by the Animal Ethical Committee of Astellas Pharma Inc.
Genetic diagnosis of the insulin 2 gene mutation and genome-wide genotyping Genomic DNA was extracted from the tail of each mouse at four weeks of age using DNeasy Tissue Kit (QIAGEN, Hilden, Germany). Ins2 genotype was determined to estimate the existence of Cys96Tyr missense mutation by RFLP analysis as previously described (Takeshita et al., 2006). PCR fragment length polymorphism analysis was performed for genomewide genotyping using primers for microsatellite markers that were polymorphic between Akita and A/J mice as previously described (Takeshita et al., 2006). All primers were purchased from Applied Biosystems (Foster City, CA, USA). Thirteen well-amplified microsatellite markers were used for genotyping the Dbm4 locus and 102 markers were used for assaying genetic background (information available on request).

Phenotyping of congenic mice
Male congenic and control mice were used for phenotypic characterisation. Each congenic or control mouse was weaned at 4 weeks of age. At 10 weeks of age, individual body weight (BW) was measured after overnight fasting for 16 hours, and glucose tolerance test (GTT) was performed as follows. After fasting for 16 hours, 2 g/kg BW of glucose in distilled water was administered orally, and blood sample was collected from the retro-orbital sinus using a capillary pipette before (designated as 0 min), 30, 60, and 120 min after glucose administration. Plasma samples were obtained by centrifugation to measure glucose concentrations by the mutarotase-glucose oxidase method using the Glucose CII Test WAKO (Wako, Osaka, Japan). Mice were anesthetically sacrificed and liver and epididymal white adipose tissue (EWAT) were isolated to measure the wet tissue weight and/or to estimate liver specific gene expression at 16 weeks of age.
Statistical analysis Data were presented as mean ± S.E.M. Statistical analysis was carried out by two-tailed unpaired Student's t test. Statistical significance was defined as p value less than 0.05.

RESULTS AND DISCUSSION
We had previously identified four major QTLs affecting diabetic traits. The most significant linkage with plasma glucose concentrations was detected on Dbm4 located on chromosome 15 with a maximum LOD score of 6.17 for fasting plasma glucose concentrations (Takeshita et al., 2006), which is the QTL overlapped with the previous report in the TSOD mouse strain (Hirayama et al., 1999). To discover diabetic modifier located on Dbm4, we constructed congenic mice, A/J.Akita, by backcrossing Akita to A/J mice, which retained the Akita mousederived Ins2-Hetero mutation on chromosome 7 and the homozygous Akita allele of Dbm4 locus (Ak/Ak) on chromosome 15 between the microsatellite markers D15Mit182 and D15Mit2 in the A/J allele genetic background (Fig. 1). We analyzed phenotypes of congenic mice to compare to the A/J allele control mice with Ins2-Hetero mutation and homozygote for A/J allele of the Dbm4 locus (Aj/Aj).
To estimate the effect of Dbm4 locus solely on diabetic traits, we performed GTT in male congenic mice as we had done on the F 2 intercross mice in the QTL analysis Fig. 1. LOD score plots of modifier QTL Dbm4 on chromosome 15 and construction of congenic mice. LOD score plots for fasting plasma glucose concentrations of male Ins2-Hetero mice in our previous QTL analysis. The horizontal solid line indicates the threshold LOD score value of 2.8 for a suggestive linkage (Dbm4). The short bold line shows a genetic distance of 10 cM. The congenic strain constructed by backcross recombination retains the homozygous Akita allele of the gray-shadowed regions indicated below the LOD plots, which was included by the flanking marker of D15Mit182 and D15Mit2. (Takeshita et al., 2006). In congenic mice (Ak/Ak), fasting plasma glucose concentrations were significantly higher than in control mice (Aj/Aj) (187.51 ± 14.42 vs. 145.97 ± 8.09 mg/dL) and there were no significant differences in plasma glucose concentrations at any other time points in GTT (Fig. 2), in spite that the Akita allelederived increases in plasma glucose concentrations had been observed at any time points during GTT in our previous QTL analysis (Takeshita et al., 2006). As for plasma insulin concentrations, the values were quite low in the hypoinsulinemic Ins2-Hetero condition and no change was observed between the groups (Supplementary Table S1). Our current congenic study reproduced the result of QTL analysis only in fasting plasma glucose concentrations, however it may be considered that the phenotypes of congenic mice actually attributed to the genetic differences between Ak/Ak and Aj/Aj alleles in Dbm4, compared to the results in QTL analysis which included additional effect of whole genome association. It may also be suggested by the QTL analysis result with much weaker association of Dbm4 locus with plasma glucose concentrations in GTT at 30, 60, or 120 min rather than fasting plasma glucose concentrations (LOD scores of 3.24, 2.37, and 2.91 for 30, 60, and 120 min in GTT, rather than 6.17 for fasting plasma glucose). Therefore these data suggested that Ak/Ak alleles of diabetic modifier gene(s) located on Dbm4 is associated with severe diabetes compared to the Aj/Aj allele through increased fasting plasma glucose concentrations in the hypoinsulinemic condition.
From our result that the Ak/Ak alleles in Dbm4 locus was suggested to be associated with higher fasting glu-cose concentrations, diabetic modifier located on Dbm4 may possibly influence hepatic gluconeogenesis, because overnight fasting is well-known to lead to increased activity of gluconeogenic key enzymes (Anderson, 1974). To assess this possible hypothesis, we conducted gene expression analysis of G6Pase and PEPCK, which is two major rate-limiting enzyme for hepatic gluconeogenesis. The expression levels of both enzyme genes were significantly elevated in congenic mice compared to control mice, to the extent of 3.4 and 2.0 fold for G6Pase and PEPCK, respectively (Fig. 3). Regulation of hepatic gluconeogenesis is considered to be achieved mainly through control of the expression of these key enzymes (Radziuk and Pye, 2001;Takashima et al., 2010), although actual enzyme activities were not measured in our assay. Therefore our study suggested that the Ak/Ak allele might contribute to upregulate hepatic gluconeogenesis compared to Aj/Aj allele in Ins2-Hetero derived hypoinsulinemic diabetic condition. Diabetic modifier in Dbm4 locus is considered not to control the insulin secretion or insulin action itself due to the result of no difference in plasma insulin concentration between congenic and control groups. Therefore, it may possibly have an action on gluconeogenetic pathway directly in liver or contribute to hepatic insulin sensitivity or resistance. Based on our genetic search, 115 of Ensembl genes identified on Dbm4 locus were listed in Supplementary Table S2. Some of these may have potential diabetes-related information by investigating co-citation relation between each gene and diabetes, however, there is no strong suggestion that candidate gene(s) located on Dbm4 is directly associated with gluconeogenesis or insulin sensitivity in hepatocyte from Fig. 2. Higher fasting plasma glucose concentrations in congenic mice. GTT was performed in male Ins2-Hetero mice at 10 weeks of age. Open circles denote the plasma glucose concentrations at each time point in control mice whose genotypes are homozygous for A/J allele on Dbm4 (Aj/Aj, N = 34), and closed circles denote those in congenic mice whose genotypes are homozygous for Akita allele (Ak/Ak, N = 13). Data are shown as means ± S.E.M. Statistical significance was shown for p < 0.05 (*) between the congenic and control groups. the previous information. There is some possible transcriptional factors on Dbm4 locus such as zinc finger proteins (Zhx2,Zhx1,Zfp406,Zfp41,Zc3h3,Zfp623,Zfp707,Scrt1,Zfp251,Zfp7), forkhead box protein (Foxh1), and nuclear receptor binding protein (Nrbp2). We suggest a possibility that a key diabetic modifier on Dbm4 may act as an transcriptional regulator of hepatic gluconeogenesis according to our result, even though actual relationship remains unclear. These genes, therefore, also seem to be possible candidate modifier genes. In addition, previous report suggested that Myc, which is an oncogene located on Dbm4, regulate liver metabolism indirectly (Riu et al., 2002), so that this is also considered as a candidate modifier gene.
Besides the diabetes-related trait, body weight and some wet tissue weights were compared between congenic and control groups, even though any wet tissue weight had not been determined in our previous QTL analysis. As for body weight, there was no statistical difference between the groups as we had no significant linkage in our previous QTL analysis. In contrast, statistically higher liver weight was observed in congenic mice compared to control mice (p < 0.05, for liver weight per body weight ratio), in spite of no difference in EWAT-weight (Table 1). The change in other tissue weights (i.e., heart, spleen) of congenic mice were insignificant compared to that of control mice (Supplementary Table S3). These findings suggest that phenotypic difference between congenic and control groups is mainly due to functional changes in liver including glucose metabolism (gluconeogenesis) and possibly also lipid metabolism. This suggestion is also supported by the previous report of QTL on chromosome 15 overlapped with Dbm4 that some genes including candidate responsible gene Sqle and Dgat1 mainly function in liver as a regulator of lipid metabolism (Stylianou et al., 2005).
To discover genuine modifier genes, additional genetic approach as with producing sub-congenic mice is necessary to narrow the region responsible for trait variation (i.e. gluconeogenesis). Moreover it is essential for gene identification to develop integrated approach including expression analysis, polymorphism search, functional analysis of candidate genes and also detailed etiological analysis using congenic mice. The identification of novel modifiers in the mouse will apply to mechanistic analysis of human type 2 diabetes.
Based on these, we concluded that diabetic modifier gene(s) existing on Dbm4 locus affect fasting plasma glucose concentrations possibly via regulation of gluconeogenic gene expression in the hypoinsulinemic diabetic condition. Identification of the modifier gene responsible for Dbm4 would provide new drug development targets for human type 2 diabetes with hepatic insulin resistance.
This study was supported by a grant from Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER). The authors thank both Dr. Youzou Takehisa and Dr. Takahiro Takehisa in Setagaya Memorial Hospital for kind consideration in preparing this article.
The current affiliation for Shigeru Takeshita: Clinical Pharmacology, Development, Astellas Pharma Inc., Tokyo, Japan. Data are shown as means ± S.E.M, n = 9 for control and n = 6 for congenic mice groups. Statistical significance between control and congenic mice groups is shown for p < 0.05 (*), according to Student's-t test. *