Dose Effect and Mode of Inheritance of Diabetogenic Gene on Mouse Chromosome 11

The quantitative trait locus (QTL) mapping in segregating crosses of NSY (Nagoya-Shibata-Yasuda) mice, an animal model of type 2 diabetes, with nondiabetic strain C3H/He mice has identified diabetogenic QTLs on multiple chromosomes. The QTL on chromosome 11 (Chr11) (Nidd1n) showing the largest effect on hyperglycemia was confirmed by our previous studies with homozygous consomic mice, C3H-11NSY, in which the NSY-derived whole Chr11 was introgressed onto control C3H background genes. C3H-11NSY mice also showed a streptozotocin (STZ) sensitivity. In the present study, we constructed heterozygous C3H-11NSY mice and the phenotypes were analyzed in detail in comparison with those of homozygous C3H-11NSY and C3H mice. Heterozygous C3H-11NSY mice had significantly higher blood glucose levels and STZ sensitivity than those in C3H mice. Hyperglycemia and STZ sensitivity in heterozygous C3H-11NSY mice, however, were not as severe as in homozygous C3H-11NSY mice. The body weight and fat pad weight in heterozygous C3H-11NSY mice were similar to those in C3H and homozygous C3H-11NSY mice. These data indicated that the introgression of Chr11 of the diabetes-susceptible NSY strain onto diabetes-resistant C3H caused marked changes in the glucose tolerance and STZ susceptibility even in a heterozygous state, and suggested that the mode of inheritance of a gene or genes on Chr11 for hyperglycemia and STZ sensitivity is additive.


Introduction
Patients with type 2 diabetes have a complex phenotype with impaired insulin secretion, insulin resistance both in the liver and peripheral tissues, and increased hepatic glucose production, all of which contribute to the development of overt hyperglycemia [1]. Genetic dissection of type 2 diabetes in humans is difficult because of the genetic heterogeneity, multigenicity, and environmental variation. One way to overcome this complexity in the human population is to use well-de�ned animal models of type 2 diabetes.
e NSY (Nagoya-Shibata-Yasuda) mouse is an inbred animal model with a spontaneous development of type 2 diabetes [2,3]. e animal is moderately obese, and both impaired insulin response to glucose and insulin resistance contribute to diabetes [2,3]. Quantitative trait locus (QTL) mapping in segregating crosses of NSY with the nondiabetic strain C3H/He has identi�ed diabetes-related QTLs on multiple chromosomes [4,5]. Using the consomic strategy, we previously dissected a quantitative trait locus (QTL), Nidd1n, on chromosome 11 (Chr11) affecting glucose-related phenotypes [6,7]. e constructed strain, C3H-11 NSY , which carries the homozygous NSY-derived diabetes-susceptible Chr11 on C3H-derived diabetes-resistant background genes, showed hyperglycemia [6] and streptozotocin (STZ) sensitivity [7] compared with control C3H strain. Although these data 2 Journal of Diabetes Research clearly demonstrated the presence of the diabetogenic gene(s) on mouse Chr11, the dose effect and the mode of inheritance of the gene(s) responsible for each diabetes-related phenotype are still unclear. In the present study, we constructed heterozygous C3H-11 NSY mice, and the phenotypes were analyzed in detail in comparison with those of homozygous C3H-11 NSY and C3H mice.

Materials and Methods
2.1. Animals. NSY mice [2] were originally obtained from the Branch Hospital of the Nagoya University School of Medicine. C3H/He mice were purchased from Charles River Laboratories (Kanagawa, Japan). ese strains were maintained by a brother-sister mating. Maintained male C3H/He mice were used in this study ( for Study 1, for Study 2).
e consomic strain, C3H-11 NSY , was previously constructed using a marker-assisted method [6,7]. Shortly, F1 male mice were obtained by mating (NSY × C3H/He). ese males were then mated with C3H/He females, and their male progeny, heterozygous for Chr11, were used for the next generation. is process was repeated until all the markers for background typing became homozygous for the C3H genotype (N6 or N7), at which point a heterozygous consomic strain was obtained and the male mice were used in this study (heterozygous C3H-11 NSY : for Study 1). Mice heterozygous for Chr11 were intercrossed to obtain mice homozygous for Chr11. Constructed homozygous C3H-11 NSY mice were maintained by brother-sister mating. To analyze male heterozygous C3H-11 NSY mice in this study ( for Study 2), male homozygous C3H-11 NSY were mated with female C3H/He mice.
All mice had free access to tap water and a standard diet (CRF-1: Oriental Yeast, Tokyo, Japan) in an air-conditioned room (22-25 ∘ C) with a 12 h light-dark cycle (6 : 00-18 : 00 h) in the animal facilities of the Osaka University Graduate School of Medicine. e experiment was approved by the Osaka University Graduate School of Medicine Ethics Committee.

Analysis of Hyperglycemia-Related Phenotypes in Heterozygous C3H-11
and C3H (Study 1). Glucose tolerance was assessed by intraperitoneal glucose tolerance test (ipGTT) (2 g glucose/kg body weight) in overnight-fasted mice at 48 weeks of age, and blood glucose levels were measured at 0, 30, 60, 90, and 120 min using Glutest E (Kyoto Daiichi Kagaku, Kyoto, Japan). e area under the glucose curve (gAUC) was calculated according to the trapezoid rule from the glucose measurements at 0, 30, 60, 90, and 120 min.
Insulin secretion in response to glucose was assessed by intraperitoneal glucose tolerance test (ipGTT) (2 g glucose/kg body weight) in overnight-fasted mice at 52 weeks of age, and blood glucose levels and plasma insulin levels were measured at 0, 15, and 30 min. Plasma insulin level was measured using an enzyme-immunosorbent assay (ELISA) kit (Morinaga, Yokohama, Japan). Insulin values in micrograms per liter obtained by ELISA were converted to picomoles per liter by multiplying by a factor of 174. Incremental glucose (ΣΔ glucose) and incremental insulin (ΣΔ insulin) were calculated from the measurements at 0, 15, and 30 min. Insulinogenic index was calculated as ΣΔ insulin divided by ΣΔ glucose. Insulin resistance was assessed by HOMA-IR, which was calculated from the basal insulin and glucose concentrations.
Anatomical analysis was performed at 54 weeks of age. Aer body weight measurement, mice were killed under sevo�urane. e liver, epididymal fat pads, and mesenteric fat pads were dissected and weighed.

Analysis of Streptozotocin Sensitivity in Heterozygous C3H-11
(Study 2). e mice received a single injection of STZ at a dose of 175 mg/kg body weight at 12 weeks of age. STZ was dissolved in sodium citrate buffer (Wako Pure Chemical Industries, Ltd., Osaka, JAPAN) and immediately injected intraperitoneally. Blood glucose level and body weight were measured on days 0, 1, 2, 4, 5, 7, and 8 aer injection. Mice with a blood glucose level higher than 11.1 mmol/L were considered hyperglycemic. Homozygous C3H-11 NSY and C3H data have been reported previously [7] and reanalyzed in this study. Although data of a small number of heterozygous C3H-11 NSY have been described [7], we constructed a large number of heterozygous C3H-11 NSY ( ).

Statistical Analysis.
All values are expressed as mean ± SEM. Statistical analysis was performed by Mann-Whitney test. Survival curves were analyzed with the log-rank test. Statistical tests were performed using PRISM soware (Graphpad Prism).
was considered to indicate a statistical signi�cance.

Phenotypes of Heterozygous C3H-11
Mice (Study 1). Heterozygous C3H-11 NSY mice showed signi�cantly higher blood glucose levels aer fasting ( ) and at all time points aer a glucose challenge ( ) than those in C3H mice (Figure 1), indicating that the introduction of a single dose of NSY-Chr11 converted normoglycemic C3H mice to hyperglycemic mice. Compared to homozygous C3H-11 NSY mice [6], however, heterozygous C3H-11 NSY mice had lower blood glucose levels aer fasting ( ) and at all time points aer a glucose challenge ( ) (Figure 1), suggesting that NSY-Chr11 in�uences glucose tolerance in an additive manner.
As shown in Table 1, insulin secretion in response to glucose as assessed by insulinogenic index tended to be lower  in heterozygous C3H-11 NSY than in C3H mice and was similar to that in homozygous C3H-11 NSY mice. Fasting insulin level (0 min in Table 1) and HOMA-IR in heterozygous C3H-11 NSY tended to be higher than those in C3H mice.
In the anatomical analysis, body weight in heterozygous C3H-11 NSY mice was not signi�cantly different from that in C3H mice (Table 2). Fat pad weight and percent fat pad weight/body weight were not signi�cantly different either. Compared to that in homozygous C3H-11 NSY mice in our previous study [6], body weight was not signi�cantly different. Fat pad weight and percent fat pad weight/body weight were not signi�cantly different either. ose in heterozygous C3H-11 NSY mice, however, tended to be lower than those in homozygous C3H-11 NSY mice and similar to those in C3H mice. . Heterozygous C3H-11 NSY mice showed a higher blood glucose level aer streptozotocin injection than C3H mice (Figure 2(a)), and life table analysis demonstrated a signi�cant difference in survival curves between C3H-11 NSY and C3H mice ( ) (Figure 2(b)), indicating that the introgression of a single dose of Chr11 from STZ sensitive NSY mice converted STZ resistant C3H mice to STZ sensitive. Compared with homozygous C3H-11 NSY mice, however, heterozygous C3H-11 NSY mice showed signi�cantly lower blood glucose levels (Figure 2(a)), and life table analysis demonstrated a signi�cant difference in survival curves between homozygous and heterozygous C3H-11 NSY mice ( ) (Figure 2(b)), suggesting that NSY-Chr11 in�uences STZ sensitivity in an additive manner.

Discussion
is study clearly demonstrated that the introgression of Chr11 of the diabetes-susceptible NSY strain onto the diabetes-resistant C3H strain caused marked changes in glucose tolerance and STZ susceptibility, even in a heterozygous state. Blood glucose levels of heterozygous C3H-11 NSY mice were intermediate between those of homozygous C3H-11 NSY and C3H mice, suggesting that the mode of inheritance of glucose intolerance is additive.
Our previous studies with reciprocal F1 crosses of NSY and C3H mice showed a different mode of inheritance depending on the phenotypes studied [8]. Hyperglycemia and fasting plasma insulin showed autosomal dominant inheritance, while impaired insulin secretion and epididymal fat accumulation showed an autosomal recessive mode of inheritance [8]. Since F1 mice are heterozygous for all autosomes, the mode of inheritance observed in our previous study with F1 mice is the combined effect of all chromosomes responsible for each phenotype. In contrast, heterozygous C3H-11 NSY mice in the present study were heterozygous for only Chr11, while all other chromosomes were homozygous for control C3H mice, making it possible to clarify the dose effect and mode of inheritance of Chr11 independent of other chromosomes. e data in the present study indicated an additive mode of inheritance for hyperglycemia, which is different from the dominant inheritance of hyperglycemia observed in F1 mice [8]. e differences in mode of inheritance observed in this study with heterozygous C3H-11 NSY mice and in our previous study with F1 mice suggest a genetic interaction of Chr11 with other chromosomes. To clarify this, studies with double and triple consomic strains, in which different combinations of chromosomes from NSY mice were introgressed onto C3H background genes, are now underway.
Our previous studies with homozygous C3H-11 NSY mice demonstrated that introgression of two doses of Chr11 from NSY mice onto control C3H mice converted STZ resistant C3H mice to STZ sensitive [7]. STZ sensitivity in heterozygous C3H-11 NSY mice in the present study was intermediate between that of homozygous C3H-11 NSY and that of C3H mice, suggesting a dose effect of Chr11 on STZ sensitivity and an additive mode of inheritance for this phenotype.
Although the present study with C3H-11 NSY mice made it possible to clarify the effect of Chr11 independent of other chromosomes, the additive mode of inheritance of glucose intolerance and STZ sensitivity observed in the present study does not necessarily re�ect the effect of a single gene, but rather could result from the combined effect of multiple genes on Chr11. Further studies with congenic and subcongenic strains are necessary to clarify whether or not each phenotype observed in heterozygous C3H-11 NSY mice is based on a single gene or the combined effect of multiple genes. Values are total number or mean ± SEM. Heterozygous C3H-11 NSY were compared with C3H and homozygous C3H-11 NSY by Mann-Whitney U test, but no signi�cant difference was observed. † Data of homozygous C3H-11 NSY mice were already reported [6].  Our previous studies with homozygous C3H-11 NSY mice demonstrated that impaired insulin secretion in NSY mice was accounted for mostly by Chr11 and that insulin resistance, which is independent of adiposity and obesity, was accounted for partly by Chr11 [6]. In the present study, impaired insulin secretion and insulin resistance in heterozygous C3H-11 NSY mice were similar to those in homozygous C3H-11 NSY mice, although not statistically signi�cantly different from those in C3H mice probably due to the small number of mice analyzed. In fact, when heterozygous C3H-11 NSY mice in the present study were compared with our previous data of C3H mice [6], insulin secretion was signi�cantly impaired and insulin resistance was marginally stronger in heterozygous C3H-11 NSY mice ( and , resp.; Mann-Whitney test). Further studies with a larger number of mice are necessary to clarify the underlying mechanisms of glucose intolerance in heterozygous C3H-11 NSY mice, in particular the contribution of impaired insulin secretion and insulin resistance to glucose intolerance. Such studies are now underway.

Conclusions
ese data indicated that the introgression of Chr11 of the diabetes-susceptible NSY strain onto diabetes-resistant C3H caused marked changes in glucose tolerance and STZ susceptibility, even in heterozygous state, indicating that Chr11 harbors gene(s) responsible for hyperglycemia and STZ susceptibility, which is effective even in a single dose. Hyperglycemia and STZ sensitivity in heterozygous C3H-11 NSY mice, however, was not as severe as in homozygous C3H-11 NSY mice, suggesting that the mode of inheritance of a gene or genes on Chr11 for hyperglycemia and STZ sensitivity is additive. e difference in the dose effect and mode of inheritance for some phenotypes between the present study and our previous study with F1 mice suggests the genetic interaction between Chr11 and other chromosomes in determining the phenotypes. e consomic strains constructed in our studies will facilitate �ne mapping and identi�cation of responsible genes for diabetes-related phenotypes, as well as gene-gene and gene-environment interactions.

Con�ict of �nterests
e authors declare that they have no con�ict of interests.