type 2 diabetes ( t2d ) is a metabolic disorder characterized by high blood glucose due to insulin resistance and insulin deficiency .
t2d accounts for more than 90% of all diabetes , and the prevalence of t2d and prediabetes is rapidly rising worldwide .
better treatment and prevention of t2d and its complications are needed to overcome the disease .
to date , many animal models of t2d have been established such as the goto - kakizaki ( gk ) rat , otsuka long - evans tokushima fatty ( oletf ) rat , spontaneously diabetic torii ( sdt ) rat , sdt fatty rat , wistar fatty rat , zucker diabetic fatty ( zdf ) rat , db / db mouse , kk - a mouse , nagoya shibata yasuda ( nsy ) mouse , new zealand obese ( nzo ) mouse , tallyho ( th ) mouse , and the tsumura suzuki obese diabetic ( tsod ) mouse . among rat models of the disease ,
it is well recognized that a mutation in the leptin receptor gene ( lepr ) causes infertility in the homozygous state .
thus , rats homozygous for a missense mutation ( fatty , fa ) in lepr are infertile ; these include the sdt fatty rat , wbn / kob fatty rat , wistar fatty rat , zdf rat , and the zucker fatty ( zf ) rat , all of which harbor the fa mutation .
the infertility of fa / fa homozygous animals requires maintenance and production of the strains by mating between fa/+ heterozygous animals .
only one male of eight pups obtained by this type of mating is expected to be fa / fa homozygous and serve as research material .
we establish here a novel diabetic rat strain , zfdm , in which the fa / fa male rats have reproductive ability .
we performed initial characterization of the strain to compare their phenotypic characteristics with those of the original zf rats .
zfdm rats ( hos : zfdm - lepr ,  fa / fa and fa/+ ) were provided by hoshino laboratory animals , inc .
normoglycemic zf rats ( slc : zucker , fa / fa and + /+ ) were purchased from japan slc , inc .
all animals were maintained under specific pathogen free conditions at 23  2c and 55  10% relative humidity with a 12 h light - dark cycle and were provided with water and a commercial diet ce-2 ( clea japan , inc . ,
tokyo , japan ) at the animal facility of kobe biotechnology research and human resource development center of kobe university .
all animal experiments were approved by the committee on animal experimentation of kobe university and carried out in accordance with the guidelines for animal experimentation at kobe university .
the zfdm and zf rats were checked for body weight and nonfasting blood glucose level by a portable glucose meter ( antsense iii , horiba , ltd . ,
diabetes was defined as a blood glucose level equal to or higher than 300  mg / dl under ad libitum dietary conditions . at 6 , 8 , 12 , 16 , and 20 weeks of age ,
nonfasting whole blood samples were collected from the tarsal vein , and plasma samples were separated by centrifugation and stored at 80c for later insulin measurement . in different batches , zfdm rats at 8 , 12 , 16 , 20 , and 24 weeks of age were checked for body weight , body length ( head to anus ) , and nonfasting blood glucose level and were sacrificed for histological analysis .
fasting whole blood samples were collected from the heart , and serum samples were separated by centrifugation and stored at 80c for later leptin , adiponectin , and lipid parameter measurements .
body mass index ( bmi ) was calculated by dividing body weight ( in grams ) by body length ( in centimeters ) squared .
plasma insulin levels were measured by insulin elisa kit ( shibayagi co. , ltd . ,
serum leptin and adiponectin levels were measured by leptin elisa kit ( morinaga institute of biological science , inc . ,
serum lipid parameters were measured by hitachi automatic analyzer 7070 ( hitachi high - technologies corporation , tokyo , japan ) .  
the fixed specimens were embedded in paraffin , sectioned at 4  m , and stained with hematoxylin and eosin for histopathological examination .
differences in blood glucose level , body weight , body length , bmi , plasma insulin level , serum leptin level , serum adiponectin level , and serum lipid parameters were assessed using welch 's t - tests .
to improve the reproductive efficiency of our zf rat colony , we have been trying to obtain fa / fa animals having reproductive ability since 1992 ( figure 1 ) . in 1997 , we fortunately identified fa / fa males having reproductive ability and tried to maintain the colony by using these fa / fa males . in 2005
, we successfully established an outbred zf rat colony in which fa / fa males having reproductive ability were frequently and continuously obtained .
we have been maintaining this colony by mating between fa/+ females and 10- to 15-week - old fa / fa males . in 2008 , during maintenance of the above - mentioned zf rat colony , we incidentally identified one 25-week - old fa / fa male rat exhibiting diabetes ( figure 1 ) .
afterward , we measured blood glucose levels of this fa / fa male 's descendants at 10 weeks of age and selected the animals showing relatively high blood glucose levels to maintain the colony . consecutive maintenance by this type of selective breeding for more than 10 generations resulted in establishment of an outbred diabetic rat colony , which we designated hos : zfdm - lepr ( zfdm ) .
it is most noteworthy that the fa / fa males in this zfdm rat colony are fertile and diabetic .
we then compared body weight and nonfasting blood glucose level of fa / fa and fa/+ male rats in the zfdm strain from 5 to 21 weeks of age ( figures 2(a ) and 2(b ) ) .
differences in body weight between fa / fa and fa/+ rats were evident as early as 6 weeks of age ( fa / fa 158.4  4.5  g versus fa/+130.6  4.8  g , p = 0.0004 ) and were increased gradually until 21 weeks of age ( fa / fa 448.5  15.3  g versus fa/+  375.3  5.8  g , p = 0.0004 ) .
differences in nonfasting blood glucose level between fa / fa and fa/+ rats were evident as early as 8 weeks of age ( fa / fa 128.2  4.7  mg / dl versus fa/+  104.7  2.1  mg / dl ,  p = 0.0003 ) and were increased markedly until 21 weeks of age ( fa / fa 464.2  30.0  mg / dl versus fa/+  118.6  2.1  mg / dl , p < 0.0001 ) . nonfasting
plasma insulin level was significantly higher in fa / fa rats than that in fa/+ rats at 6 weeks of age ( fa / fa 0.89  0.12  ng / ml versus fa/+  0.56  0.04  ng / ml , p = 0.026 ) , and that in fa / fa rats was increased until 16 weeks of age and then decreased but remained significantly higher than that of fa/+ rats at 20 weeks of age ( fa / fa 1.23  0.18  ng / ml versus fa/+  0.62  0.03  ng / ml , p = 0.006 ) ( figure 2(c ) ) . the fa / fa rats developed diabetes ( nonfasting blood glucose levels  300  mg / dl ) as early as 10 weeks of age and the cumulative incidence of diabetes reached 100% by 21 weeks of age ( figure 2(d ) ) . in contrast , none of the fa/+ rats developed diabetes . in different batches of the experiment , we compared body weight , nonfasting blood glucose level , body length , and bmi of fa / fa and fa/+ male rats in the zfdm strain at 8 , 12 , 16 , 20 , and 24 weeks of age ( figure 3 ) . body weight and
nonfasting blood glucose level of both animals showed similar tendencies to those described above ( figures 3(a ) and 3(b ) ) .
body length was significantly larger in fa/+ rats than that in fa / fa rats at 16 weeks of age and later ( 16 weeks of age : fa / fa 22.9  0.2  cm versus fa/+  23.9  0.2  cm , p = 0.007 ) ( figure 3(c ) ) .
differences in bmi between fa / fa and fa/+ rats were evident at 8 weeks of age ( fa / fa 0.59  0.01  g / cm versus fa/+  0.54  0.01  g / cm , p = 0.028 ) , were increased markedly by 12 weeks of age , and were maintained until 24 weeks of age ( fa / fa 0.84  0.02  g / cm versus fa/+  0.65  0.01  g / cm , p < 0.0001 ) ( figure 3(d ) ) .
most of the lipid parameters and leptin levels were higher in fa / fa rats than those in fa/+ rats .
in contrast , the adiponectin level in fa / fa rats was decreased with age and was lower than that in fa/+ rats at 24 weeks of age ( figure 4(h ) ) . to compare the phenotype of the zfdm strain and that of the normoglycemic zf strain , we measured body weight and nonfasting blood glucose level of fa / fa and + /+ rats in the zf strain from 5 to 21 weeks of age ( figures 5(a ) and 5(b ) ) .
differences in body weight between fa / fa and + /+ rats in the zf strain were evident as early as 5 weeks of age ( data at 6 weeks of age for comparison with the zfdm strain : fa / fa 161.8  3.0  g versus + /+  129.2  2.3  g , p < 0.0001 ) and were gradually increased until 21 weeks of age ( fa / fa 558.8  7.4  g versus + /+  367.0  6.9  g , p < 0.0001 ) ( figure 5(a ) ) . while the body weights of fa / fa rats in both zfdm and zf strains were similar at earlier ages , those of fa / fa rats in the zfdm strain were significantly lower at 11 weeks of age and later , most likely due to the onset of diabetes ( supplementary figure 1(a ) available online at http://dx.doi.org/10.1155/2013/103731 ) .
there was no difference in nonfasting blood glucose level between fa / fa and + /+ rats in the zf strain during the experimental period , and none of the fa / fa and + /+ rats in the zf strain exhibited diabetes by 21 weeks of age ( figure 5(b ) ) ; the character is different from that of the zfdm strain ( supplementary figure 1(b ) ) . nonfasting plasma insulin level was significantly higher in fa / fa rats than that in + /+ rats at 6 weeks of age ( fa / fa 1.49  0.27  ng / ml versus + /+   0.83  0.09  ng / ml , p = 0.045 ) and that in fa / fa rats was increased until 20 weeks of age ( fa / fa 2.00  0.26  ng / ml versus + /+  0.62  0.06  ng / ml , p = 0.0007 ) ( figure 5(c ) ) .
while there were no obvious pathological changes in endocrine and exocrine pancreas of fa/+ rats in the zfdm strain ( figures 6(a ) , 6(b ) , 6(e ) , 6(f ) , 6(i ) , and 6(j ) ) , various degrees of pathological change were observed in the pancreas of the fa / fa rats at 16 and 24 weeks of age , including loss of islet architecture and fibrosis ( figures 6(g ) , 6(h ) , 6(k ) , and 6(l ) ) . to determine whether these pathological changes are specific to the fa / fa rats in the zfdm strain , we performed histological characterization of the pancreas in the normoglycemic zf strain .
although the pancreatic islets of fa / fa rats in the zf strain were enlarged , islet architecture was maintained even at 24 weeks of age ( figures 6(o ) and 6(p ) ) .
similarly to the fa/+ rats in the zfdm strain , + /+ rats in the zf strain exhibited no obvious pathological changes in the pancreas ( figures 6(m ) and 6(n ) ) .
in this study , we describe the establishment and initial characterization of a novel type 2 diabetes model derived from normoglycemic zf rats .
we first established an outbred zf rat colony in which fa / fa males have reproductive ability .
we then incidentally identified an fa / fa male rat exhibiting diabetes and performed selective breeding using fa / fa male rats showing relatively high blood glucose levels at 10 weeks of age .
we successfully maintained this colony by mating between fa / fa male and fa/+ female rats , which resulted in establishment of an outbred diabetic rat colony that we designated hos : zfdm - lepr .
the male fa / fa rats in the zfdm strain are fertile , exhibit obesity , and develop diabetes as early as 10 weeks of age , reaching 100% incidence at around 20 weeks of age .
the zdf strain was established in 1990   and has been widely used to study t2d associated with obesity .
the zdf strain is an inbred rat model of early - onset diabetes in which all of the fa / fa male rats develop diabetes at 10 to 12 weeks of age when fed a special diet of purina 5008 ( charles river laboratories international , inc . , wilmington , ma , usa ) .
the phenotype is homogeneous , mainly due to the facts that the strain is genetically inbred and the special diet is provided .
in contrast , the zfdm strain is an outbred rat model of young- to middle - aged adult - onset diabetes in which the fa / fa male rats develop diabetes as early as 10 weeks of age and most develop diabetes at 12 to 20 weeks of age ( table 1 ) .
after the onset of diabetes , high blood glucose levels are maintained , a feature is similar to that of the zdf strain .
since the prediabetic stage of the zfdm strain is much longer than that of the zdf strain , the zfdm strain is more suitable for pathophysiological studies of diabetes and therapeutic intervention studies . in the zdf strain ,
the fa / fa male rats exhibit diabetic complications including nephropathy   and neuropathy . given that the zfdm strain harbors a similar genetic background to that of the zdf strain and exhibits severe diabetes , diabetic complications found in the zdf strain may well occur in the zfdm strain .
usually , humans , mice , and rats lacking normal lepr are infertile due to hypogonadotropic hypogonadism
. however , there is some evidence for spontaneous pubertal development in humans homozygous for a splicing mutation resulting in lepr lacking both the transmembrane and intracellular domains , among whom one woman was able to give birth to a healthy son [ 18 , 19 ] .
in addition , human subjects with lepr missense mutations have been reported to show less severe clinical features than those with the splicing mutation described above . in rat species , l. m. zucker and
t. f. zucker reported that fa / fa female rats are always infertile , while chelich and edmonds found that a small portion of young fa / fa female rats are fertile .
furthermore , young fa / fa male rats are more likely to be fertile ; about 70% of fa / fa male rats were reported to be fertile in a colony at vassar college , but their current status is unknown . in our zf rat colony ,
more than 10 generations of selective breeding for fertility of fa / fa male rats enabled us to obtain fertile fa / fa male rats stably .
we have established a novel diabetic rat strain , zfdm , in which fa / fa male rats are fertile . in this strain ,
fa / fa male rats develop diabetes as early as 10 weeks of age , which reaches 100% incidence at around 20 weeks of age , while no fa/+ rats develop diabetes .
the zfdm rat strain possesses high reproductive efficiency and therefore should serve as a useful model of young- to middle - aged adult - onset t2d in studies of the pathophysiology , therapeutic interventions , and complications of the disease .
 the zucker fatty ( zf ) rat harboring a missense mutation ( fatty , fa ) in the leptin receptor gene ( lepr ) develops obesity without diabetes ; zucker diabetic fatty ( zdf ) rats derived from the zf strain exhibit obesity with diabetes and are widely used for research on type 2 diabetes ( t2d ) . 
 here we establish a novel diabetic strain derived from normoglycemic zf rats . in our zf rat colony , 
 we incidentally found fa / fa homozygous male rats having reproductive ability , which is generally absent in these animals . during maintenance of this strain by mating fa / fa males and fa/+ heterozygous females , we further identified fa / fa male rats exhibiting diabetes . 
 we then performed selective breeding using the fa / fa male rats that exhibited relatively high blood glucose levels at 10 weeks of age , resulting in establishment of a diabetic strain that we designated hos : zfdm - leprfa ( zfdm ) . 
 these fa / fa male rats developed diabetes as early as 10 weeks of age , reaching 100% incidence by 21 weeks of age , while none of the fa/+ male rats developed diabetes . 
 the phenotypic characteristics of this diabetic strain are distinct from those of normoglycemic zf rats . 
 zfdm rat strain having high reproductive efficiency should serve as a more useful animal model of t2d . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions