gestational diabetes mellitus ( gdm ) is defined as  impaired glucose tolerance appearing specifically during pregnancy  ( 1 ) .
gdm develops by excessive insulin resistance , inadequate -cell compensation , reduced -cell function , or any combination of these ( 2 ) .
a large number of epidemiological studies demonstrated that diabetes in pregnant women is associated with an increased risk of maternal and neonatal morbidity ( 3 ) .
infants of a diabetic mother ( idms ) have been shown to be prone to the development of complex diseases , including obesity , and metabolic and cardiovascular complications , during childhood and adulthood ( 4 , 5 ) . in animal models of gdm , it has been shown that idms overtly develop diabetes throughout life ( 6 , 7 ) .
furthermore , population - based studies have also demonstrated that idms have an increased risk for type 2 diabetes in later childhood and as adults ( 8) .
insulin - producing -cells in the endocrine pancreas play a pivotal role in maintaining glucose homeostasis .
adult -cells can dynamically respond to systemic increases in insulin demand by expan - ding their functional mass .
compensatory changes in -cells mass are controlled by increases in cell size ( hypertrophy ) or increase in number of cells ( hyperplasia ) ( 9 , 10 ) .
recent findings have shown that defect in these mechanisms is a key feature of the pathogenesis of diabetes ( 11 , 12 ) . similarly , idm mature animals show -cells dysfunction and decreased insulin secretion in response to glucose ( 7 ) . however , the mechanisms responsible for -cells malfunction in idms are unknown .
therefore , understanding how -cells proliferate and function is important and may lead to development of new therapeutic strategies for diabetes .
strikingly , recent studies have shown a link between the cell cycle regulators and the risk of type 2 diabetes ( 13 , 14 ) .
cyclin - dependent kinases ( cdk )  a family of serine / threonine protein kinases  phosphorylate a number of substrates like retino - blastoma protein ( prb ) mainly implicated in cell cycle progression .
subsequent phosphorylation of prb by the cdk results in the release of e2f . upon activation , e2f1 is able to turn on genes required for progression through g1 into the s phase of cell cycle ( 15 ) .
in addition to activation of cell cycle , e2f directly contributes to insulin secretion through the regulation of kir6.2 ( also referred as kcnj11 ) expression .
it has been proven that kir6.2 channels play a pivotal role in the regulation of insulin secretion .
so , cdk4-prb - e2f1 pathway directly contributes to both proliferation and regulation of insulin secretory capacity of -cells ( 16 ) .
indeed , no study has investigated the gene expre - ssion of kir 6.2 and cdk4-prb - e2f1 factors in adult offspring of diabetic rats .
thus , the purpose of the present investigation was to evaluate the expression changes of these genes in pancreatic islands extracted from offspring of streptozotocin - induced mildly hyperglycemic rats .
this experimental study was performed to evaluate the effect of gestational diabetes on expression of cdk4-prb - e2f1 pathway genes in pancreatic islets of rat offspring .
all animal procedures followed the guidelines set by the institutional animal care and use committee at the golestan university of medical sciences , gorgan , iran .
the rats were kept in a temperature - controlled environment ( 212 c ) on 12-hr light / dark cycles and allowed free access to standard rat chow and water .
vaginal plaque was mentioned daily as a positive sign of pregnancy and the day on which vaginal plaque was observed , considered as day 0 of pregnancy .
a total of 20 dams were made diabetic by single ip injection of freshly prepared streptozotocin ( stz ) solution ( 40 mg / kg body weight ) in sterile saline solution ( 0.85% ) on day zero of gestation ( 17 ) ; 7 dams were injected with equivalent volume of normal saline as control group .
four days later , blood glucose levels were checked using a glucometer ( accu - chek active glucometer , roche diagnostics , germany ) .
if glucose levels were between 120 - 250 mg / dl , the rats were selected and used as gdm .
mildly hyperglycemic dams in the current series of experiments were ~40% , or 8 out of 20 stz- injected rats .
totally six diabetic offspring from gdm mothers at the age of 12 and 15 weeks were selected .
islets were isolated from pancreases of diabetic and control group rats by a modification of the collagenase digestion technique ( 18 ) .
this involved cannulation of the common bile duct and the sequential administration of 2 ml digest solution containing 0.2 mg / ml liberase tl ( roche , cat # 05 401 020 001 ) and 10 pg / ml dnase ( takara ) in serum free rpmi 1640 medium .
the organ was then incubated at 37 c for 15 min and dispersed using pipetting action . at the end of the incubation
, the tubes moved to ice and 10 ml rpmi 1640 with 10% serum added .
the islets were separated by centrifugation on a ficoll gradient ( histopaque1077 , sigma - aldrich10771 ) and collected from the histopaque / media interface with a disposable 10 ml serological pipette and resuspended in serum containing rpmi .
the islets were then isolated by passage through a 100 m cell strainer ( bd falcon ) and handpicked with a pasteur pipette using a dissecting microscope .
rna was isolated from islets using the genabioscience rna extraction kit according to the manufacturer s instructions .
residual dna was digested with 10 u rnase - free dnase ( dnase i , takara ) in the presence of 20 units of rnase inhibitor at 37 c for 20 min . after heat inactivation for 10 min at 75 c in 2 mm edta ,
concentration and purity of the dnase i - treated samples was measur- ed using a nanodrop nd-1000 spectrophotometer ( a260/a280>1.8 and a260/a230>1.6 ) .
the integrity and stability of the rnas was confirmed by demonstrating the intact 28s and 18s bands on gel electrophoresis . for real - time rt - pcr ,
the cdna was synthesized from 1 g of dnasei - treated total rna using prime script rt reagent kit ( takara ) with random hexamer and oligo dt primers following the manufacturer s protocol .
the forward and reverse pcr primers for the 7 genes were designed in accordance to the real - time pcr conditions , using perlprimer software ( bio - rad , usa ) , and the sequences are listed in table 1 .
for each genes , the cdna amplified by specific primers using taq polymerase kit ( takara ) , and correct product was confirmed by running on gel electrophoresis
. real - time pcr primer name , sequences , size , genbank accession number and pcr condition real - time rt - pcr was performed using the sybr - green pcr master mix kit ( takara ) in the thermo cycler ( abi , 7300 ) .
the cycling conditions were 95 c for 30 sec followed by 40 cycles at 95 c for 5 sec , 55 c for 30 sec and 72 c for 1 min .
we used rat -actin as internal control and non- diabetic offspring islets cdna as calibrator .
amplification specificity was confirmed by gel electrophoresis . the relative expression level of mrna between the diabetic and non - diabetic samples was determined with the comparative ct ( cycle threshold ) method .
first , both ct values of target gene from control islets cdna ( n=3 ) and diabetic islets cdna ( n=3 ) were normalized by ct value of the internal control ( ct ) .
then , the former was subtracted by the latter , namely ct . the value of 2 , the fold change of gene expression , real - time serial data were statistically analyzed .
every real - time pcr experiment was repeated with three samples and each sample was run in duplicate .
relative target gene expression and blood glucose level was analyzed with one - way anova using spss 16.0 statistical analysis software .
the differences between groups were compared using unpaired t test and p<0.05 was chosen as the level of significance .
the rats were kept in a temperature - controlled environment ( 212 c ) on 12-hr light / dark cycles and allowed free access to standard rat chow and water .
vaginal plaque was mentioned daily as a positive sign of pregnancy and the day on which vaginal plaque was observed , considered as day 0 of pregnancy .
a total of 20 dams were made diabetic by single ip injection of freshly prepared streptozotocin ( stz ) solution ( 40 mg / kg body weight ) in sterile saline solution ( 0.85% ) on day zero of gestation ( 17 ) ; 7 dams were injected with equivalent volume of normal saline as control group .
four days later , blood glucose levels were checked using a glucometer ( accu - chek active glucometer , roche diagnostics , germany ) .
if glucose levels were between 120 - 250 mg / dl , the rats were selected and used as gdm .
mildly hyperglycemic dams in the current series of experiments were ~40% , or 8 out of 20 stz- injected rats .
totally six diabetic offspring from gdm mothers at the age of 12 and 15 weeks were selected .
islets were isolated from pancreases of diabetic and control group rats by a modification of the collagenase digestion technique ( 18 ) .
this involved cannulation of the common bile duct and the sequential administration of 2 ml digest solution containing 0.2 mg / ml liberase tl ( roche , cat # 05 401 020 001 ) and 10 pg / ml dnase ( takara ) in serum free rpmi 1640 medium .
the organ was then incubated at 37 c for 15 min and dispersed using pipetting action . at the end of the incubation
, the tubes moved to ice and 10 ml rpmi 1640 with 10% serum added .
the islets were separated by centrifugation on a ficoll gradient ( histopaque1077 , sigma - aldrich10771 ) and collected from the histopaque / media interface with a disposable 10 ml serological pipette and resuspended in serum containing rpmi .
the islets were then isolated by passage through a 100 m cell strainer ( bd falcon ) and handpicked with a pasteur pipette using a dissecting microscope .
rna was isolated from islets using the genabioscience rna extraction kit according to the manufacturer s instructions .
residual dna was digested with 10 u rnase - free dnase ( dnase i , takara ) in the presence of 20 units of rnase inhibitor at 37 c for 20 min . after heat inactivation for 10 min at 75 c in 2 mm edta ,
concentration and purity of the dnase i - treated samples was measur- ed using a nanodrop nd-1000 spectrophotometer ( a260/a280>1.8 and a260/a230>1.6 ) .
the integrity and stability of the rnas was confirmed by demonstrating the intact 28s and 18s bands on gel electrophoresis .
for real - time rt - pcr , the cdna was synthesized from 1 g of dnasei - treated total rna using prime script rt reagent kit ( takara ) with random hexamer and oligo dt primers following the manufacturer s protocol .
the forward and reverse pcr primers for the 7 genes were designed in accordance to the real - time pcr conditions , using perlprimer software ( bio - rad , usa ) , and the sequences are listed in table 1 . for each genes ,
the cdna amplified by specific primers using taq polymerase kit ( takara ) , and correct product was confirmed by running on gel electrophoresis .
real - time pcr primer name , sequences , size , genbank accession number and pcr condition real - time rt - pcr was performed using the sybr - green pcr master mix kit ( takara ) in the thermo cycler ( abi , 7300 ) .
the cycling conditions were 95 c for 30 sec followed by 40 cycles at 95 c for 5 sec , 55 c for 30 sec and 72 c for 1 min .
we used rat -actin as internal control and non- diabetic offspring islets cdna as calibrator .
amplification specificity was confirmed by gel electrophoresis . the relative expression level of mrna between the diabetic and non - diabetic samples was determined with the comparative ct ( cycle threshold ) method .
first , both ct values of target gene from control islets cdna ( n=3 ) and diabetic islets cdna ( n=3 ) were normalized by ct value of the internal control ( ct ) .
then , the former was subtracted by the latter , namely ct . the value of 2 , the fold change of gene expression , real - time serial data were statistically analyzed .
every real - time pcr experiment was repeated with three samples and each sample was run in duplicate .
data were presented as meanstandard deviation ( sd ) . relative target gene expression and blood glucose level was analyzed with one - way anova using spss 16.0 statistical analysis software .
the differences between groups were compared using unpaired t test and p<0.05 was chosen as the level of significance .
fasting blood glucose concentration was significantly increased in idm rats ( figure 1 ) . by 12 weeks of age ,
about 40% of the idms developed mild hyperglycemia and at 15 weeks of age , glucose levels were markedly elevated in idms compared to controls ( p<0.001 ) .
blood glucose concentrations in idms ( infants of a diabetic mother ) and control animals ( week 12 and week 15 )
. blood glucose level of offspring was obtained via tail vein and was measured with accu - chek glucometer .
values are means sem . * * * p<0.001 we assessed whether the expression levels of cell cycle regulator genes ( cdk4-prb - e2f1 ) were affected by gestational diabetes in adult offspring .
real - time pcr melting curve for e2f1 , cdk4 , kir6.2 , prb and -actin as internal control ( a ) .
agarose gel electrophoresis of pcr products following real - time sybr green amplification ( b ) the expression levels of prb in isolated islets were not significantly different between the two groups , indicating that the expression of prb was not disturbed by gdm . but
analysis of the mrna levels of cdk4 and e2f1 showed significant alterations in the diabetic group compared to controls ( figure 3 ) .
on the other hand , kir6.2 mrna expression severely reduced in idms , which followed the expected decrease in e2f1 protein ( figure 3 ) .
mrna levels were measured using gene specific primers ( table 1 ) and the values were normalized to -actin .
statistical significance was calculated using the t test ( * p<0.05 , * * p<0.01 , n=3 ) .
fasting blood glucose concentration was significantly increased in idm rats ( figure 1 ) . by 12 weeks of age ,
about 40% of the idms developed mild hyperglycemia and at 15 weeks of age , glucose levels were markedly elevated in idms compared to controls ( p<0.001 ) .
blood glucose concentrations in idms ( infants of a diabetic mother ) and control animals ( week 12 and week 15 )
. blood glucose level of offspring was obtained via tail vein and was measured with accu - chek glucometer .
we assessed whether the expression levels of cell cycle regulator genes ( cdk4-prb - e2f1 ) were affected by gestational diabetes in adult offspring .
real - time pcr melting curve for e2f1 , cdk4 , kir6.2 , prb and -actin as internal control ( a ) .
agarose gel electrophoresis of pcr products following real - time sybr green amplification ( b ) the expression levels of prb in isolated islets were not significantly different between the two groups , indicating that the expression of prb was not disturbed by gdm . but
analysis of the mrna levels of cdk4 and e2f1 showed significant alterations in the diabetic group compared to controls ( figure 3 ) . on the other hand ,
kir6.2 mrna expression severely reduced in idms , which followed the expected decrease in e2f1 protein ( figure 3 ) .
mrna levels were measured using gene specific primers ( table 1 ) and the values were normalized to -actin .
statistical significance was calculated using the t test ( * p<0.05 , * * p<0.01 , n=3 ) .
maintenance of appropriate insulin - producing -cells growth and mass is critical for metabolic balance ( 19 , 20 ) ; thus , the molecular mechanisms by which -cells proliferate are the focus of recent studies .
the first years of life in humans and the first postnatal months of life in rodents are crucial periods of islet -cells growth that result in establishment of appropriate -cells mass ( 21 - 24 ) .
fetal hyperglycemia is a consequence of maternal mild hyperglycemia that may disturb -cells growth during early postnatal period and causes diabetes in offspring later in life ( 25 ) .
previous studies have demonstrated that cdk4-prb - e2f1 pathway plays a crucial role in the control of glucose homeostasis .
as a cell cycle regulatory pathway , cdk4-prb - e2f1 genes not only regulate -cells proliferation , but also control the expression of genes implicated in insulin secretion , such as kir6.2 ( 15 , 16 ) . in spite of several studies regarding the effects of diabetes i and ii on pancreas structure and function , there is no investigation about the effect of gdm on cell cycle regulators expression in offspring s pancreatic islets .
thus , this study was designed to investigate the effects of hyperglycemic intrauterine environment on the expression of cell cycle regulator genes in langerhans islets of adult diabetic offspring .
consistent with some previous studies ( 6 , 7 ) , our offspring rats developed diabetes at 12 weeks of age . in this study , we observed a 51% decrease for cdk4 expression in langerhans islets of diabetic offspring ( p<0.05 ) .
many studies implicate cdk4 as a crucial factor for successful expansion of -cells mass in various conditions .
for instance , when cell cycle arrest is altered specifically in -cells by deletion of cdk4 postnatally , -cells mass decreases or fails to expand ( 13 ) .
also rieck et al showed that during the peak of -cells dna synthesis , cdk4 expression is induced in the islets of pregnant mice ( 26 ) .
also , it has been shown that pharmacological inhibition of cdk4 activity on glucose tolerance in mice drama - tically decreases the clearance of glucose in treated , compared to non - treated mice with idcx , which is a specific cdk4 inhibitor ( 15 ) .
correlated with previous studies , our observation links cdk to the risk of diabetes in offspring of gdm mothers .
furthermore , we showed that gdm also causes reduction of prb and e2f1 gene expression in pancreatic islets of diabetic offspring by 35% and 86% ( p<0.01 ) , respectively .
findings have shown that e2f1 mice have decreased pancreatic size and insulin secretion , as the result of impaired postnatal pancreatic growth .
the results have also demonstra - ted that e2f1 was highly expressed in non - proliferating pancreatic -cells , suggesting that e2f1 , besides the control of -cells number could have a role in pancreatic -cells function ( 27 ) .
these findings provided enough evidence to propose that cdk4-prb - e2f1 pathway genes are critical mediators of insulin secretion ( 16 ) . regarding the down - regulation of cdk4-prb - e2f1 pathway genes in pancreatic islets of rat ( idms )
, we can conclude that induction of diabetes in offspring of gdm is mediated by the reduction of cdk4 activity and subsequent e2f1 transcriptional activity .
in addition to evaluation of genes involved in -cells proliferation , we also wanted to evaluate whether gdm affects -cells function .
so , we analyzed the expression of kir6.2 , a key component of the atp - sensitive potassium channel involved in the regulation of glucose - induced insulin secretion in -cells .
our data indicated that gdm causes reduction in kir6.2 gene expression by 0.12 fold compare to control group .
recent in vitro and in vivo investigations have shown that e2f1 directly controls the expression of kir6.2 .
several studies on pancreas of e2f1-/- mice have shown that expression of kir6.2 is downregulated that causes insulin secretion defects in these animals ( 15 , 16 , 27 ) .
our data are in agreement with other reports and suggest that uncontrolled gdm may cause diabetes in offspring by repression of cdk4-prb - e2f1 pathway in their pancreases .
our data showed that down - regulation of cdk4-prb - e2f1 pathway genes is related with development of diabetes in offspring of gestational diabetic rats . furthermore , in this study , decreased kir6.2 mrna expression in diabetic offspring underscores a dual effect of the gdm on both proliferation , and function of -cells .
taken together , this study may open up new ways for understanding the molecular basis of gdm and type 2 diabetes in the offspring .
however , many advances must be made to fully appreciate the exact molecular mechanism of inducing diabetes in offspring by gestational diabetes .
 objective(s):the link between a hyperglycemic intrauterine environment and the development of diabetes later in life has been observed in offspring exposed to gestational diabetes mellitus ( gdm ) , but the underlying mechanisms for this phenomenon are still not clear . 
 reduced -cells mass is a determinant in the development of diabetes ( type 1 and type 2 diabetes ) . 
 some recent studies have provided evidence that the cdk4-prb - e2f1 regulatory pathway is involved in -cells proliferation . 
 therefore , we postulated that gdm exposure impacts the offspring s 
 -cells by disruption in the cdk4-prb - e2f1 pathway.materials and methods : adult wistar rats were randomly allocated in control and diabetic group . 
 the experimental group received 40 mg / kg / body weight of streptozotocin ( stz ) on day zero of gestation . 
 after delivery , diabetic offspring of gdm mothers and control dams at the age of 15 week were randomly scarified and pancreases were harvested . 
 langerhans islets of diabetic and control groups were digested by collagenase digestion technique . after rna extraction , we investigated the expressions of the kir 6.2 and cdk4-prb - e2f1 pathway genes by quantitative real - time pcr.results:gdm reduced the expression of cdk4-prb - e2f1 pathway genes in langerhans islets cells of offspring . 
 cdk4 , prb and e2f1 pathway genes were downregulated in diabetic islets by 51% , 35% and 84% , respectively . 
 also , the expression of kir 6.2 was significantly decreased in diabetic islets by 88%.conclusion : we suggest that the effect of gestational diabetes on offspring s -cells may be primarily caused by the suppression of cdk4-prb - e2f1 pathway . 
Introduction
Materials and Methods
Generation of the diabetic rat model
Isolation of langerhans islets
RNA extraction
Real-time RT-PCR analysis
Data analysis
Results
Glucose level
qRT-PCR results
Discussion
Conclusion
Conflict of Interest