diabetes mellitus is clinically classified into three stages based on the degree of insulin dependence . the stages are non - insulin requiring ( nir ) , insulin requiring for control of blood glucose ( irc ) and insulin requiring for survival ( irs ) . a patients pharmacologic treatment is selected based on these stages because combinations of different oral agents may be useful for controlling hyperglycemia before insulin therapy becomes necessary . at the nir stage , adequate glycemic control can be achieved through weight reduction , exercise and/or oral glucose - lowering agents , and so individuals at this stage do not require insulin . individuals at the irc stage have some residual insulin secretion , but require exogenous insulin for adequate glycemic control , and can also survive without taking insulin . individuals at the irs stage with extensive -cell destruction and therefore no residual insulin secretion require insulin for survival . the c57bl / ksl db / db mouse ( db / db mouse ) is a diabetes mellitus animal model that is a spontaneous mutant strain of the c57bl / ksj db / db mouse resulting from a point mutation of the downstream intron of the leptin receptor gene rendering it unresponsive to leptin . leptin is a peptide hormone secreted by adipocytes and is involved in eating behavior and energy homeostasis . so , this animal models expresses unrepressed eating behavior , becomes obese and develops severe insulin resistance associated with hyperinsulinemia and hypertriglyceridemia , followed by hyperglycemia peaking at 34 months of age . pancreatic islet -cell mass is reduced as disease progresses , resulting in severe insufficiency of insulin secretion . it has also been well shown by immunohistochemistry that a decrease in insulin levels of islets of db / db mice occurs at 18 weeks of age , without referreing to the blood insulin levels , which is one of the most important biomarkers . in spite of the extensive use of the db / db mouse in this field , there are no reports on the three clinical stages in the db / db mouse . however , if pathophysiological staging were possible in the db / db mouse , drug candidates for diabetes mellitus could be better evaluated in preclinical studies to selectively target a specific pathophysiological stage . in this study , time course blood insulin and glycosylated hemoglobin ( hba1c ) levels , the clinical parameters for evaluation of the pathophysiological stages of diabetes mellitus in humans , and morphological features of pancreatic islets in db / db mice were examined in order to determine the pathophysiological stage of the disease in the diabetic mouse model . both the american diabetes association ( ada ) and the american association of clinical endocrinologists ( aace ) recommend monitoring glycemic control using hba1c as the parameter . the major advantage of measuring hba1c is that a specimen can be collected without regard to when the patient last ate . twenty five male db / db mice were purchased from charles river laboratories ( japan ) and subjected to experimentation at 5 weeks of age . the animals were housed in cages in an animal room maintained at a temperature of 23  2c and a humidity of 55  10% , with 14 to 16 air changes per hour and a 14-hour light and 10-hour dark cycle . all animal procedures were conducted in accordance with chugai pharmaceutical s guide for the care and use of laboratory animals , and all experimental protocols were approved by the institutional animal care and use committee . the db / db mice were divided into 5 groups ( n=5 per group ) , and the animals of each group were sacrificed by exsanguination under ether anesthesia at the age of 6 , 9 , 12 , 15 or 22 weeks after their body weights were measured and blood samples were collected . blood samples were obtained from the caudal vena cava for measurement of plasma insulin and hba1c . plasma insulin levels were measured using elisa ( institute of biological science , inc . , yokohama , japan ) , and the percentage of hba1c was measured using an auto analyzer ( type 7170 , hitachi high - technologies corporation , tokyo , japan ) . the pancreas was removed from all necropsied animals , fixed in 20% neutral buffer formalin solution , embedded in paraffin wholly , sectioned longitudinally and stained with hematoxylin and eosin . twenty five male db / db mice were purchased from charles river laboratories ( japan ) and subjected to experimentation at 5 weeks of age . the animals were housed in cages in an animal room maintained at a temperature of 23  2c and a humidity of 55  10% , with 14 to 16 air changes per hour and a 14-hour light and 10-hour dark cycle . all animal procedures were conducted in accordance with chugai pharmaceutical s guide for the care and use of laboratory animals , and all experimental protocols were approved by the institutional animal care and use committee . the db / db mice were divided into 5 groups ( n=5 per group ) , and the animals of each group were sacrificed by exsanguination under ether anesthesia at the age of 6 , 9 , 12 , 15 or 22 weeks after their body weights were measured and blood samples were collected . blood samples were obtained from the caudal vena cava for measurement of plasma insulin and hba1c . plasma insulin levels were measured using elisa ( institute of biological science , inc . , yokohama , japan ) , and the percentage of hba1c was measured using an auto analyzer ( type 7170 , hitachi high - technologies corporation , tokyo , japan ) . the pancreas was removed from all necropsied animals , fixed in 20% neutral buffer formalin solution , embedded in paraffin wholly , sectioned longitudinally and stained with hematoxylin and eosin . time course of changes of body weight ( a ) , plasma insulin level ( b ) and hba1c level ( c ) in the db / db mice . enlarged , circular islet at 6 weeks of age and islet cells with vacuoles ( insert ,  600 ) ( a ) , enlarged islet at 12 weeks of age ( b ) , small irregular - shaped islet at 12 weeks of age ( c ) and a smaller and irregular - shaped islet at 22 weeks of age ( d ) . the mean body weight was 33.67  0.39 g , the level of the plasma insulin was at its highest ( 16.82  1.03 ng / ml ) and the level of hba1c was at its lowest ( 2.06  0.14% ) during the observation period ( fig . histopathological examination revealed enlarged , circular - shaped islets , which were consistent with hypertrophic islet cells having abundant cytoplasm and a large vacuole with a curved lucent region considered to be the golgi apparatus ( fig . the mean body weights were 45.13 g  1.50 and 47.93 g  4.14 , respectively . the level of plasma insulin had decreased to mid levels ( 8.92  2.14 ng/ ml and 7.73  2.61 ng / ml , respectively ) , and the level of hba1c had increased to middle to high mean levels ( 5.79  0.54% and 10.11  0.84% ; fig . , two types of islets were coexisting : enlarged islets similar to those observed in the 6-week - old animals ( fig . 2b ) and small , irregular - shaped islets consisting of atrophic islet cells with acinar cells and spindle cells thought to be myofibroblasts ( fig . 2c ) . at 15 and 22 weeks of age , the mean body weights were 52.35  4.75 g and 52.37  5.24 g , respectively , the level of plasma insulin had decreased further to low mean levels ( 2.38  0.99 ng / ml and 5.15  0.78 ng / ml , respectively ) and the level of hba1c had increased to high mean levels ( 10.47  2.06% and 11.21  0.97% ; fig . islet cells were even more atrophic than in the 12-week - old animals , and the presence of acinar cells and spindle cells was obvious ( fig . staging of diabetes mellitus is clinically based on insulin dependence , namely non - insulin requiring ( nir ) , insulin requiring for control ( irc ) of blood glucose and insulin requiring for survival ( irs ) . the purpose of this study was to determine the pathophysiological stage of diabetes mellitus in db / db mouse at various ages and discuss the relevance of the stages in the animal model to those in humans . in patients at the nir stage of diabetes mellitus , adequate glycemic control can be achieved with weight reduction , exercise and/or oral glucose - lowering agents . the patients in this stage have insulin secretion ability , and the glucose level is occasionally high but controllable . in this study , the 6-week - old db / db mice can be considered equivalent to being in the nir stage of human diabetes mellitus patients according to the following results : the animals showed obesity , unlike the nonobese heterozygote ( db/+ ) mouse , hypertrophic islet cells were observed in the histopathological examination and the findings well reflected blood parameters . these findings are considered to be an insulin secretion reaction of islet cells caused by hyperphagia , a characteristic of the db / db mouse . based on the blood parameters of the animals at this stage , sufficient insulin secreting ability ( the highest insulin level ) was preserved to counteract high blood glucose levels . at the irc stage in human diabetes mellitus , patients have some residual insulin secretion but require exogenous insulin for adequate glycemic control . the findings in the db / db models at 9 and 12 weeks of age were equivalent to the irc stage . the animals showed mid - level residual insulin secretion and mildly increased blood glucose level ( mid levels of hba1c ) . the histopathological findings showed some irregular - shaped islets , which correlates to the existence of acinar cells and spindle cells . it is known that the fibrotic distraction of islets is commonly observed in humans and several animal models of type 2 diabetes . pathophysiological stages of dm in humans and as represented in the db / db mouse . , insulin secretion in the mice was very low ( low levels of insulin ) , because islets were destructed ( irregular - shaped and small ) , and the blood glucose levels had increased ( high levels of hba1c ) . in addition , the decrease of insulin in the islets of db / db mice at 18 weeks of age was shown well in a previous report , and the morphology of islet is similar to that in this study . our results indicate that the sustained secretion of insulin induced -cell destruction and that the ability to secrete insulin had almost disappeared . we consider these findings to be equivalent to the irs stage in humans . in the db / db mouse used in this study , three distinctive pathophysiological stages of diabetic change the results elucidate a time course relationship between the blood parameters and the morphology of pancreatic islets in the db / db mouse , where pathophysiological stages based on plasma insulin levels , hba1c levels and histopathological findings are clearly distinguishable and characteristically reflect the stages of human diabetes mellitus . to our knowledge , this is the first report of a db / db mouse exhibiting pathophysiological stages similar to those in human diabetes mellitus . in the evaluation of new drug candidates for diabetes mellitus , indicating the time point in the life cycle of the disease would be very important , as the pharmacologic effects of a drug must be selected to match patient status . in addition , there is evidence that environmental factors contribute to the development of obesity and type 2 diabetes . being able to understand the pathophysiological stage of an animal model based on blood glucose and insulin would be beneficial .
the db / db mouse is one of the diabetes mellitus animal models and if the pathophysiological stages of diabetic changes in the mouse model could simulate the stages in human diabetes , the db / db mouse could be used to better evaluate drug candidates . 
 blood insulin , hba1c levels and morphological features of pancreatic islets in db / db mice were evaluated to determine the pathophysiological stage . at 6 weeks of age , 
 db / db mice showed the highest level of plasma insulin and lowest level of hba1c , and histopathological examination revealed enlarged islets with a circular shape and hypertrophic islet cells . by 9 and 12 weeks of age , 
 the plasma insulin levels had decreased to mid levels and hba1c had increased to mid to high levels ; histopathological examination at this time revealed two types of islets coexisting , enlarged circular islets and small irregular - shaped islets . by 15 and 22 weeks of age , 
 plasma insulin had decreased further to low levels and hba1c was at its highest level ; the histopathological examination at this time revealed an increase in irregular - shaped and small islets . based on blood insulin levels , hba1c levels and histopathology findings in the db / db mice in this study , the clinical staging of diabetic changes were recognized . 
 the pathophysiological stages of diabetes mellitus in this animal model were similar to the stages in humans .