  diabetes mellitus is a heterogeneous group of metabolic diseases that is characterised by hyperglycaemia .
it can result in blindness , kidney and heart disease , stroke , loss of limbs , and reduced life expectancy if left untreated and is recognized as a primary threat to human health in the 21st century .
type 2 diabetes mellitus is the most common form of diabetes and accounts for approximately 90% of cases ; its development is controlled by interactions between multiple genetic and environmental factors [ 24 ] .
the genetics and pathophysiology of type 2 diabetes remain poorly understood because detailed investigations , such as genetic dissection , have been restricted in humans for practical and ethical reasons .
animal models of type 2 diabetes mellitus have provided important information , and many rat strains of spontaneous diabetes have been reported [ 511 ] . among these strains
, goto - kakizaki ( gk ) , otsuka long - evans tokushima fatty ( oletf ) , and spontaneously diabetic torii ( sdt ) rats have been widely used for elucidating the genes responsible for the development of diabetes , the physiological course of the disease , and the complications related to diabetes [ 1214 ] .
the different diabetes model strains exhibit different aspects of the disease , and thus additional animal models are needed to elucidate the complete pathogenesis of diabetes .
we have developed a novel , nonobese rat strain with spontaneous diabetes , long - evans agouti ( lea ) rat , which was established from a long - evans closed colony together with the long - evans cinnamon ( lec ) rat .
they do not exhibit any signs of obesity throughout their lives but experience late onset of the disease and exhibit histological changes ( macrophage infiltration and fibrosis ) of the pancreatic islets .
thus , lea rats may serve as a new model of nonobese type 2 diabetes mellitus caused by impairing insulin secretion . in the present study , we examined the pathophysiological characteristics of the strain and demonstrated that the lea rat is a useful model of human nonobese diabetes .
  lea rats , also known as lea / sendai or sendai rats , were maintained at the institute for animal experimentation , tohoku university graduate school of medicine , japan .
wistar rats were purchased from japan slc ( hamamatsu , japan ) and were used as controls .
all rats were housed in air - conditioned animal rooms at an ambient temperature of 23  3c and relative humidity of 50  10% , under specific pathogen - free conditions with a 12  h light / dark cycle .
food , consisting of a labo mr standard diet ( nosan , yokohama , japan ) , and water were available ad libitum .
all animal care procedures were approved by the animal care and use committee of tohoku university graduate school of medicine , and complied with the procedures of the guide for the care and use of laboratory animals of tohoku university .  
urinary glucose and protein were monitored at 1-month intervals using uro - paper ag2 ( eiken , tokyo , japan ) .
the detection limits of the uro - paper were 1020  mg / dl for urine protein and 4060  mg / dl for urine glucose .
the body weight ( bw ) and body length ( bl ) , that is , the distance from the nose to the anus , of five male rats ( 6 months old ) were measured . the body mass index ( bmi )
after 16  h fast , a dose of 2  g / kg bw of glucose was given orally , and blood samples were collected from the tail vein at 0 , 30 , 60 , 90 , and 120  min after loading .
the blood glucose levels were measured with a glutest eii blood glucose monitoring meter with a monitoring range of 40500  mg / dl ( sanwa kagaku , nagoya , japan ) .
the rats were classified according to the three - grade system of diabetes mellitus ( dm ) , impaired glucose tolerance ( igt ) , and normal .
dm was defined as 120  min blood glucose levels of 200  mg / dl .
igt was defined as 120  min blood glucose levels between 140 and 199  mg / dl .
the plasma insulin concentration was determined as immunoreactive insulin ( iri ) by elisa with a rat insulin assay kit ( morinaga milk industry , yokohama , japan ) using separated plasma from the blood collected from the tail vein at 0 , 30 , 60 , 90 , and 120  min after glucose loading . to study early - phase insulin secretion ,
an ogtt was performed on male rats ( 2 and 12 months of age ) as described previously , and blood glucose levels ( bg ) and insulin concentrations ( iri ) at 0 and 30  min after glucose loading were measured . the insulinogenic index ( i.i . )
= iri/bg , where iri and bg are the differences between their respective values at 0 and 30  min .
the animals were intraperitoneally challenged with a dose of 0.75  u / kg bw of human insulin ( novolin r ; novo nordisk , denmark ) .
blood samples were drawn from the tail vein at different time points , and glucose levels were determined as described previously .   
the tissues from rats were fixed overnight at 4c in phosphate - buffered saline ( pbs ) that contained 4% paraformaldehyde .
they were rinsed with pbs , dehydrated , embedded in paraffin , cut into 5-m - thick sections , and stained with haematoxylin and eosin ( h&e ) . for insulin detection ,
the pancreatic sections were processed for immunostaining by an indirect method using guinea pig anti - insulin polyclonal antibody ( 1  :  100 , daco , carpinteria , ca ) as the primary antibody and peroxidase - labelled goat anti - guinea pig igg antibody ( 1  :  200 , chemicon international , temecula , ca ) as the secondary antibody .
the specific reactions were visualised with a dab substrate kit ( vector , burlingame , ca ) . to distinguish the inflammatory cells infiltrated in the islets ,
additional deparaffinised sections of pancreas were processed for immunostaining using mouse monoclonal antibodies against rat cd4 clone w3/25 ( mca 55r ; abd serotec , oxford , uk ) , cd8 clone ox-8 ( mca48r ; abd serotec ) , cd45ra clone ox-33 ( mca340 g , abd serotec ) , and macrophage antigen clone ed1 ( mca341 , abd serotec ) .
specific reactions were visualised with a sab - po kit ( nichirei , tokyo , japan ) .
the volume of -cells relative to the pancreas volume was calculated as the proportion of the total area of -cells to the total area of pancreatic tissue , according to the method of bouwens et al .
three serial paraffin sections ( 5-m - thick ) of pancreatic tissue from three animals of each strain were obtained at intervals of 100  m .
the sections were immunostained with guinea pig anti - insulin antibodies ( 1  :  100 ) and analysed under an olympus bx51 microscope ( olympus , tokyo , japan ) connected to a computer running the winroof software ( mitani corp . , tokyo , japan ) .
the image analysis quantified the total pancreatic tissue area and the insulin - positive area , permitting the calculation of the ratio of islet -cell area to total pancreatic area . to ensure that any change in the relative -cell volume was not attributable to a change in the size of individual -cells ,
the density of nuclei per insulin - positive area was also measured in 20 islets of the five rats of each strain .  
  the incidence of diabetes in lea rats as determined by ogtt is shown in figure 1(a ) .
diabetes mellitus was observed only in male rats , and its incidence increased with age : 10% , 61% , and 86% at 6 , 12 , and 14 months of age , respectively .
the onset of diabetes was not observed in females , although 33% of the females showed only igt at 12 months of age . as the onset of diabetes differed according to sex
glucosuria appeared at 5 months of age , before the onset of diabetes in male rats , and was present in 100% of the males at 8 months of age . in female rats
, glucosuria appeared at 7 months of age and was present in 100% of the females at 9 months ( table 1 ) .
proteinuria appeared at 6 months of age , concomitant with the onset of diabetes , in male rats and was present in 57% of the males at 9 months of age , whereas proteinuria appeared in 20% of female rats at 9 months of age and did not exceed 30% of the females thereafter .  
the average bws of male and female lea rat increased gradually throughout the experimental period and were 506  37.8  g ( n = 5 ) and 312  27.7  g ( n = 5 ) at the 12 months of age , respectively ( figure 1(b ) ) . a significant decrease in bw
the bmi of the lea rats at 6 months of age ( 0.57  0.02  g / cm , n = 5 ) was not significantly different from that of the control wistar rats ( 0.59  0.02  g / cm , n = 5 ) , confirming that the lea rats were nonobese .  
the survival rate of lea rats was examined(supplementary figure 1 available online at http://dx.doi.org/10.1155/2013/986462 ) .
we found that 95% of the male rats survived to 12 months of age , and 50% survived to 22 months of age .
the survival rate of male lea rats was not significantly different from that of normal control wistar rats , which indicates that diabetes does not influence the survival of lea rats .  
the results of the ogtt in male rats at different ages are shown in figure 2 .
two - month - old male lea rats showed impaired glucose tolerance compared with age - matched male wistar rats ( figure 2(a ) ) . at 12 and 14 months of age ,
the lea rats presented with typical diabetic glucose levels of 200  mg / dl at 120  min after glucose loading ( figures 2(c ) and 2(d ) ) .
the wistar rats did not show any change in blood glucose level in relation to age .  
the plasma insulin concentrations in male rats at 2 months of age showed that the pre - ogtt values did not differ among the rats , whereas the values at 30  min after glucose loading were significantly lower in lea rats ( figure 2(e ) ) .
the plasma insulin level was significantly lower in lea rats at 12 months of age after glucose loading ( figure 2(f ) ) .
these results indicate that lea rats have impairment of insulin secretion in response to glucose stimulation .
the low insulin levels measured at 30  min after glucose loading suggest that lea rats have a decreased ability to secrete insulin at an early phase of the disease ( figures 2(e ) and 2(f ) ) .
the insulinogenic index ( i.i . ) of male lea rats was significantly lower at both 2 and 12 months of age compared with that of wistar rats ( table 2 ) , which indicates that early - phase insulin secretion is significantly impaired at an early age .   
we performed an insulin tolerance test on nonfasting , 12-month - old male rats by intraperitoneal injection of insulin , to examine insulin response ( figure 3 ) . before insulin injection , the blood glucose levels were significantly higher in lea rats than in wistar rats .
after insulin injection , the blood glucose levels in lea rats significantly decreased by maximum 51.1% over 120  min to the same levels observed in the wistar rats .
these results indicate that lea rats have a normal glycaemic response to exogenous insulin and are not insulin - resistant .  
the age - dependent histological changes in the pancreas were examined ( figure 4 ) .
male lea rats at 2 months of age had inflammatory reaction in fraction of the pancreatic islets ( figure 4(a ) ) , although the most of islets were intact .
immunostaining for insulin revealed that insulin - positive cells were irregularly distributed within inflammatory foci ( figure 4(b ) ) .
the inflamed islets were infiltrated by cells positive for anti - rat macrophage antibody ( figure 4(c ) ) but not for antibodies against cd4 + , cd8 + , or cd45ra ( data not shown ) .
however , inflammatory reactions disappeared by 6 months of age , and the islets had been replaced by fibrotic remnants in rats over 12 months of age ( figure 4(d ) ) .
the number of -cells was reduced , although -cells were present at 12 months of age ( figure 4(e ) ) . a few tiny islets without fibrosis , which appeared to be regenerative islets ,
were intermingled in the affected pancreas in rats from 12 months of age ( figures 4(f ) and 4(g ) ) .
the pancreatic islets from female lea rats had no pathological changes , including fibrosis and inflammatory reaction .  
the volume of -cells relative to the gross volume of the pancreas was determined using the winroof software to analyse sections that were immunostained for insulin ( figure 4(h ) ) . in male lea rats ,
the volume of -cells significantly decreased from 0.74  0.23% at 2 months to 0.52  0.08% at 6 months .
the control wistar rat strain showed a rising , albeit not statistically significant , trend from 0.79  0.05% at 2 months to 0.96  0.24% at 6 months .
these results reveal that the significant decrease in the insulin - positive area with age appears in lea rats owing to severe fibrosis of the islets .  
to identify whether the diminished -cell mass in lea rats was caused by a reduction in cell number or atrophy of the cells , we counted the number of nuclei in the insulin - positive areas of 20 islets in five of each of 6-month - old lea and wistar rats .
the number of nuclei did not differ significantly between the two strains ( 117.97  4.13  m / nuclei versus 128.82  13.35  m / nuclei for lea versus wistar rats , resp . ) .
these results indicate that a reduction in the cell number ( not atrophy of -cells ) is responsible for the reduction of -cell volume in lea rats .   in lea rats , glucosuria and proteinuria
were present in 100% at 8 months of age and in 66% of males at 12 months of age , respectively .
the histopathological analysis was performed to examine the renal lesions in 12-month - old male lea rats ( figure 5 ) .
the large dilatation of tubular lumen was present , mostly in superficial cortex regions ( figure 5(a ) ) .
atrophy of tubular epithelium and flattend / detached renal tubules were also observed ( figure 5(b ) ) .
the intracytoplasmic hyaline droplet accumulation and the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in proximal tubules ( figure 5(c ) ) .
there were no obvious pathological changes in the glomeruli at 12 months of age ( figure 5(d ) ) .
  two inbred strains , long - evans agouti ( lea ) and long - evans cinnamon ( lec ) , which were selected for coat color , were established from a closed colony of long - evans rats at the center for experimental plants and animals , hokkaido university ( japan ) .
the lea rat has been known as the control strain for the lec rat , an animal model of wilson disease . however , the large amount of urine and the strong odor of urine , a possible indicator of diabetes , are often observed in lea rats during long - term breeding . in 1996 , we found three male rats that were positive for glucosuria and hyperglycaemia among littermates from an inbred colony of lea / hkm .
the lea rats exhibit several distinctive diabetes - related characteristics : ( 1 ) onset of diabetes is observed only in male rats , not in female , at over 6 months of age ; ( 2 ) the early - phase insulin secretion is impaired at 2 months of age ; ( 3 ) the progressive fibrosis in islet in an age - dependent manner ; ( 4 ) a normal glycaemic response to exogenous insulin ; ( 5 ) nonobese . from these results , we conclude that the lea rat is a new rat model for nonobese type 2 diabetes mellitus .  
several rat model strains of spontaneous type 2 diabetes mellitus have been identified to date , and they are classified into two types , obesity and nonobesity models .
the obesity models of type 2 diabetes , such as sand , wistar fatty , and oletf rats , are characterised by hyperglycaemia , hyperinsulinemia , and insulin resistance .
in contrast , the nonobesity models , such as gk , wbn / kob , and spontaneously diabetic torii ( sdt ) rats [ 11 , 19 ] , are characterised by hyperglycaemia , hypoinsulinemia , and noninsulin resistance .
we classify the lea rat as a nonobesity model because bmi of lea rats is not different from that of control rats .
however , there are several differences between the gk and lea rats . in gk rats , there is no sex difference with respect to the occurrence of diabetes and no age - dependent deterioration of impaired glucose tolerance ; in addition , hyperglycaemia occurs 8 days after birth [ 8 , 20 ] .
the sdt rat is a new model of nonobese , severe type 2 diabetes mellitus with hyperglycaemia , hemorrhage in and around the islets , and hyposecretion of insulin ( hypoinsulinemia ) resulting from a significantly decreased number and size of islets .
although lea rats displayed no hemorrhage in and around the islets , macrophage infiltration was present around the islets , leading to the progressive fibrosis of islet ( figure 4 ) .  
the glucose intolerance in correspondence with the impairment of the insulin secretion was observed in male lea ( figure 2 and table 2 ) , suggesting that the main cause of diabetes in lea rats is hypoinsulinemia attributable to a decreased number of -cells in the islets ( figure 4 ) .
it is also likely that significant decreased capability of early - phase insulin secretion is caused by the hypofunction of -cells .
although there was inflammatory reaction in fraction of the pancreatic islet in male lea rats at 2 months of age , the volume of -cells in lea rats was comparable to that of control rats ( figure 4(h ) ) , suggesting that lea rats have congenital defect of insulin secretion in addition to the progressive reduction of -cell mass in age - dependent manner .
the ability to secrete insulin in the early phase reflects the first phase of insulin secretion from pancreatic -cells and contributes to the suppression of gluconeogenesis in the liver .
therefore , we suggest that the lea rat is unable to suppress the increasing blood glucose concentration that occurs after feeding because of a decreased ability to secrete insulin in the early phase , which eventually leads to the deterioration of -cell function by glucose toxicity and causes the rats to experience chronic hyperglycaemia .
the reduction in the number of -cells is thought to be the main cause of type 2 diabetes in lea rats , and this is supported by previous observations in human type 2 diabetes patients and rat models of type 2 diabetes mellitus [ 2226 ] .   
both congenital and acquired factors are involved in the mechanism of -cell reduction . in regard to congenital factors , mutations in transcription factor genes , such as insulin promoter factor-1 ( ipf-1 )   and hepatocyte nuclear factor-1 ( hnf-1 ) , have been verified .
have revealed that apoptosis reduces -cells in human diabetes and that it progresses by amyloid deposition and hyperglycaemia .
zhu et al .   have reported that the impairment of -cell proliferation causes a decrease in -cells under hyperglycaemic conditions in oletf rats , and movassat et al .  
free - fatty - acid- ( ffa- ) induced -cell apoptosis has been proposed by shimabukuro et al .   as the underlying cause in zucker diabetic fatty ( zdf ) rats .
although it is speculated that the impairment of -cell proliferation , progression of apoptosis by hyperglycaemia , and impairment by cytokines produced by macrophages cause the reduction in -cells in lea rats , further analyses are required to clarify the pathogenesis of diabetes in lea rats .  
the abnormality of the renal tubules at 12 months of age was observed in the lea rats with glycosuria and proteinuria ( figure 5 and table 1 ) .
onset of diabetes as determined by ogtt was observed in only male lea rats ( figure 1(a ) ) , and its incidence increased with age : 10% and 61% at 6 and 12 months of age , respectively . however , glucosuria appeared at 5 months of age before the onset of diabetes in male rats , and was present in 100% of the females at 9 months ( table 1 ) , which did not develop diabetes ( figure 1(a ) ) .
based on these findings , it is unlikely that the onset of diabetes and impairment of glucose intolerance are not associated with glucosuria and proteinuria .
although the lea rat is used as the control strain of the lec rat since they do not harbor atp7b mutation , the several phenotypes such as hypersensitivity to x - rays   and the lack of d - amino acid oxidase ( dao ) activity , which is involved in the degradation of d - serine , a key coagonist for n - methyl - d - aspartate ( nmda ) receptor , have been reported .
we are now performing quantitative trait locus ( qtl ) analyses for impaired glucose tolerance and urinary glucose to lead us to identification of genes for glucose intolerance , renal glucose excretion , and the development of diabetes in lea rat .   in conclusion
, the lea rat has distinctive characteristics that are different from the previously described model rats .
the lea rats develop late onset diabetes in correspondence with the impairment of the insulin secretion , which is caused by progressive fibrosis in pancreatic islets in age - dependent manner . in japan ,
the prevalence of type 2 diabetes mellitus is increasing rapidly , and more than 10% of individuals over 40 years of age are affected .
relatively few diabetic individuals in japan are obese , and impairment of insulin secretion often develops before onset of diabetes .
the unique characteristics of lea rat are a great advantage to analyze the progression of diabetes mellitus with age .
the additional studies are expected to disclose the genes involved in type 2 diabetes mellitus .
 animal models have provided important information for the genetics and pathophysiology of diabetes . 
 here we have established a novel , nonobese rat strain with spontaneous diabetes , long - evans agouti ( lea ) rat derived from long - evans ( le ) strain . 
 the incidence of diabetes in the males was 10% at 6  months of age and 86% at 14  months , while none of the females developed diabetes . 
 the blood glucose level in lea male rats was between 200 and 300  mg / dl at 120  min according to ogtt . 
 the glucose intolerance in correspondence with the impairment of insulin secretion was observed in male rats , which was the main cause of diabetes in lea rats . 
 histological examination revealed that the reduction of -cell mass was caused by progressive fibrosis in pancreatic islets in age - dependent manner . 
 the intracytoplasmic hyaline droplet accumulation and the disappearance of tubular epithelial cell layer associated with thickening of basement membrane were evident in renal proximal tubules . 
 the body mass index and glycaemic response to exogenous insulin were comparable to those of control rats . 
 the unique characteristics of lea rat are a great advantage not only to analyze the progression of diabetes , but also to disclose the genes involved in type 2 diabetes mellitus . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion