worldwide prevalence of diabetes has been recently increased over two - fold and is estimated to affect 439 million people by the year 2030 ( 1 , 2 ) .
diabetes is a complex metabolic disease characterized by high blood glucose level resulting from defects in insulin secretion or action ( 2 ) . 1
, 25-dihydroxyvitamin d3 ( vit d ) , the most potent metabolite of vitamin d , plays an important role in calcium and phosphate homeostasis , also vital for a number of other biological activities including immunomodulation and normal release of insulin from  cells ( 3 - 5 ) .
pancreatic islets have both vitamin d receptors and vitamin d - dependent calcium - binding proteins , suggesting a role for vitamin d in insulin secretion ( 6 , 7 ) .
it has been reported that vitamin d deficiency impairs insulin synthesis and secretion in both human and animal models and its replenishment improves  cell function and glucose tolerance ( 4 , 8 , 9 ) .
have shown improvement in glucose metabolism and insulin secretion in vitamin d - deficient rats following a single subcutaneous injection of vitamin d ( 10 ) .
in addition , it has been reported that de novo insulin synthesis is reduced in isolated islets in vitamin d - deficient rats and insulin biosynthetic capacity can be restored in vitro by injection of vitamin d ( 9 ) .
vitamin d deficiency may be related to type 2 diabetes , cardiovascular disease and cancer ( 11 , 12 ) .
subjects with type 2 diabetes have lower circulating vitamin d levels compared to healthy controls ( 3 , 13 ) .
furthermore , epidemiological studies have shown that vitamin d receptor restriction site polymorphisms are associated with genetic susceptibility to type 1 diabetes in different populations , while vitamin d supplementation in early childhood is associated with a reduced risk of type 1 diabetes ( 14 ) .
nikooyeh et al . have reported that daily consumption of vitamin d improves glycemic status in patients with type 2 diabetes ( 15 ) .
interventional studies investigating vitamin d for treatment of type 2 diabetes have shown negligible effects , and the results available in humans have been contradictory ( 15 ) .
previous studies have reported that high glucose concentrations significantly decreased total insulin secretion from  cells ( 16 ) .
the effects of coincubation and preincubation of vitamin d with high glucose concentration on insulin secretion from islets have not been fully understood yet .
therefore , the aim of this study was to determine the effects of vitamin d on insulin release from isolated islets of rats .
adult male wistar rats ( two months old , weighing 200 - 250 grams ) were obtained from the laboratory animal house of the research institute for endocrine sciences , shahid beheshti university of medical sciences and housed in an animal room at 22  3c , relative humidity of 50  6% , an inverse 12 : 12 hour light / dark cycle .
rats had free access to standard rat chow ( pars co. , tehran ) and tap water during the study .
all experimental procedures and rat care and handling were performed in accordance with guidelines provided by the local ethics committee of the research institute for endocrine sciences , shahid beheshti university of medical sciences .
animals were anesthetized by intraperitoneal injection of ketamine / xylazine ( 50 mg / kg and 10 mg / kg ) and the abdomen was exposed , and 0.5 mg / ml of collagenase p ( roche , cat .
# 1213 , germany ) in 10 ml cold hanks balanced salt solution ( hbss ) [ ph = 7.4 ; containing kcl , 5.36 ; cacl2 , 1.26 ; nacl , 136 ; mgso4 7 h2o , 0.8 ; na2hpo4 2 h2o , 0.33 ; nahco3 , 4.16 ; kh2 po4 , 0.44 ) all in mm ( merck , germany ) and gassed with 95% o2 , 5% co2 for five minutes at the beginning ] was injected into the common bile duct ; the expanded pancreas was detached , transmitted to a 50 ml falcon container and placed in a water bath ( 37c ) for 15 minutes . at the end of digestion , 30 ml cold hbss was added and the tube was shaken for one minute .
tube contents were filtered through a 500 m plastic mesh to discard any undigested tissue .
after three washes with cold hbss , islets were selected under a stereomicroscope ( kyowa , sdz - tr - pl japan ) ; a group of 10 islets were incubated in a cell strainer containing 4 ml of rpmi 1640 medium containing 10% fetal bovine serum and 1% of penicillin / streptomycin ( 17 , 18 ) .
insulin release was assessed in response to vitamin d ( d1530 , sigma ) following 24 and 48 hours coincubation of islets with vitamin d ( 0.1 , 1 , and 10 nm ) and glucose ( 5.6 , 11.1 and 16.7 mm ) .
in addition , islets were preincubated with vitamin d for 24 and 48 hours and glucose - stimulated insulin secretion ( gsis ) was assessed for one hour in the presence of 5.6 and 16.7 mm glucose . for in vitro assessment of insulin secretion , batches of eight islets were transferred into 1.5 ml plastic cups , and 1 ml of krebs ringer solution [ ( ph = 7.4 ) ; nacl , 115 ; kcl , 5 ; mgcl2 6 h2o , 1 ; cacl2 , 2.5 ; nahco3 , 24 ( merck , germany ) ; hepes , 16 ( sigma , usa ) all in mm ] and 5 g / dl bsa ( fluka , usa ) with different glucose concentrations ( 5.6 and 16.7 mm ) were added into the cups , incubated for 60 minutes in 37c water bath and gassed with 95% o2 , 5% co2 for five minutes at the beginning .
intra and inter - assay coefficients of variations for insulin measurements were 6.1% and 9.7% , respectively .
all data were expressed as mean  sem and analyzed using graphpad prism software ( version 5.00 for windows , graphpad software , san diego california usa ) .
two - way analysis of variance ( anova ) and bonferroni post - hoc test were used to compare serum insulin concentrations in different levels of glucose and vitamin d. p value below 0.05 was considered statistically significant .
all data were expressed as mean  sem and analyzed using graphpad prism software ( version 5.00 for windows , graphpad software , san diego california usa ) .
two - way analysis of variance ( anova ) and bonferroni post - hoc test were used to compare serum insulin concentrations in different levels of glucose and vitamin d. p value below 0.05 was considered statistically significant .
effects of coincubation of vitamin d with glucose on insulin release from islets were shown in figure 1 . as shown ,
coincubation of islets with 10 nm of vitamin d and 11.1 mm glucose increased insulin release , while 1 and 10 nm vitamin d with 16.7 mm glucose decreased insulin release .
vitamin d had no effect on insulin release from islets during 24 hours coincubation with 5.6 mm glucose or 48 hours coincubation with 5.6 , 11.1 , and 16.7 mm glucose .
effects of 5.6 and 16.7 mm glucose on gsis in islets , following 24 and 48 hours preincubation with vitamin d were shown in figure 2 . as seen , 24 and 48 hours preincubation with 10 nm of vitamin d and 48 hours preincubation with 1 and 10 nm of vitamin d increased gsis in the presence of 16.7 mm glucose .
insulin release in response to 24-hours ( a ) and 48-hours ( b ) incubation with different concentrations of vitamin d in presence of 5.6 , 11.1 , and 16.7 mm glucose . * : p < 0.05 compared to control ( vitamin d= 0 ) .
insulin release in response to 5.6 and 16.7 mm glucose for a duration of one hour in islets from normal rats following 24-houes ( a ) and 48-hours ( b ) preincubation with vitamin d. * : p < 0.05 compared to control ( vitamin d = 0 ) ;  : p < 0.05 compared to vitamin d 1 nm .
preincubation of vitamin d with glucose for 24 or 48 hours increased gsis from islets only at high doses of vitamin d and glucose .
in addition , when vitamin d and glucose were coincubated for 24 hours , insulin release was increased with 10 nm vitamin d and 11.1 mm glucose ; whereas , insulin release decreased with 1 and 10 nm vitamin d and 16.7 glucose .
these results were consistent with those studies reporting an association between vitamin d and insulin release from  cells ( 19 - 21 ) .
preincubation with vitamin d increased gsis in response to 16.7 mm glucose , findings in agreement with the results of billaudel et al . who reported that gsis is eliminated by vitamin d deficiency in rats ; however , six hours preincubation with 0.001 - 1000 nm vitamin d had a stimulatory effect on gsis in response to 8.3 mm glucose ( 8) .
furthermore , it has been reported that 24 hours preincubation with 10 nm vitamin d increases gsis in the presence of 8.3 mm glucose in vitamin d - deficient rats ( 22 ) .
tanaka et al . have also reported that insulin secretion induced by 16.7 mm glucose in vitamin d deficient rats was only 35% of that of control rats , but was restored to that of the controls in vitamin d - treated rats ( 23 ) .
have also reported that insulin secretion is destroyed in vitamin d deficient rats and restored by 1 , 25-(oh)2d3 ( 24 ) .
results of this study showed that vitamin d affects insulin secretion only in the presence of high glucose level , whereas it does not affect insulin secretion in presence of 5.6 mm glucose .
vitamin d enhances sensitivity of  cells to glucose , and has been suggested that the effect of vitamin d on insulin secretion may be atp - dependent ( 4 , 22 ) .
have shown that coincubation of 0.1 nm and 1 nm vitamin d with 10 mm glucose did not increase insulin release in medium for up to 180 hours ( 25 ) .
faure et al . have confirmed that coincubation of 0.001 nm vitamin d with 8.3 mm glucose did not affect insulin secretion ( 22 ) .
chan et al . have reported that in vitro addition of vitamin d had no effect on insulin secretion from normal or vitamin d deficient rats ( 26 ) .
in addition , two human studies found no change in insulin release following vitamin d supplementation in adults with either obesity or insulin resistance ( 19 - 21 ) .
contradictory to our results , billaudel et al . have reported that coincubation of 0.001 - 1000 nm of vitamin d with 8.3 mm glucose had an incremental dose - dependent effect on insulin secretion ( 8) .
moreover , demden et al . have demonstrated that coincubation of 10 nm vitamin d with 10 mm glucose increased insulin secretion 2.5 fold in a  cell medium ( 25 ) . to date , no study has investigated the effect of vitamin d on insulin secretion in the presence of 16.7 mm glucose in short duration ( up to 24 hours ) . lower insulin secretion has been reported in the presence of high glucose concentrations compared to low glucose concentrations in  cell medium .
in addition , high glucose concentrations significantly decrease insulin secretion in cultured  cells ; these data indicates that exposure to high glucose levels induces a glucotoxic situation , which could impair insulin secretion in response to glucose ( 16 ) .
contrary to the finding of our study , demden et al . have demonstrated that coincubation of 10 nm vitamin d with 20 mm glucose increased insulin secretion in a  cell medium ( 25 ) , but suggested a significantly increased insulin release in islets following 96 hours coincubation or longer
. the exact mechanisms of vitamin d effect on insulin secretion in the presence of glucose are not yet fully understood and need further studies , but it has been suggested that vitamin d directly increases insulin secretion in  cells ( 25 ) .
faure et al . have reported that vitamin d enhances ca entry or ca mobilization in  cells , which could amplify insulin secretion in the presence of glucose ( 22 ) .
have also shown that vitamin d requires a delay to increase insulin secretion from  cells ; this delay varies from 6 hours pre - incubation in vitro , 3 hours to 20 hours in vivo and 48 hours in culture , which can indicates the genomic effects of vitamin d ( 9 ) .
in conclusion , effects of coincubation of islets with vitamin d and glucose depend on glucose concentration .
vitamin d significantly decreased insulin secretion in the presence of 16.7 mm glucose , but increased insulin secretion in the presence of 11.1 mm glucose .
in addition , this study showed that preincubation of islets with vitamin d increased gsis at high levels of glucose ( 16.7 mm ) , while no effect was observed at low levels .
 background : vitamin d ( vit d ) affects glucose metabolism . receptors of vitamin d have been identified in  cells and studies show that vitamin d deficiency reduces glucose - stimulated insulin secretion ( gsis).objectives : the aim of this study was to examine the effect of vitamin d on insulin release from isolated islets of rats.materials and methods : islets were isolated from male wistar rats , weighing 200 - 250 grams , using the collagenase digestion method . 
 insulin release was assessed following 24 and 48 hours coincubation of islets with vitamin d ( 0.1 , 1 and 10 nm ) and glucose ( 5.6 , 11.1 and 16.7 mm ) . 
 in addition , islets were preincubated with vitamin d for 24 and 48 hours and gsis was assessed for one hour in the presence of 5.6 and 16.7 mm glucose.results:coincubation of islets with vitamin d ( 10 nm ) and 11.1 mm glucose increased islet insulin release ( 37.27  3.75 vs. 24.64  2.83 ng / islet/24 hours ; p < 0.05 ) , while vitamin d ( 1 and 10 nm ) decreased insulin release in the presence of 16.7 mm glucose ( 21.14  3.58 and 18.65  3.84 vs. 37.71  4.63 ng/ islet/24 hours ; p < 0.05 ) . 
 islets preincubation with vitamin d ( 1 and 10 nm ) increased gsis in the presence of 16.7 mm glucose ( 4.39  0.73 and 4.39  0.63 vs. 2.07  0.43 ng / islet/1 hour ; p < 0.05).conclusions : preincubation of islets with vitamin d increased gsis but decreased insulin release in coincubation with high levels of glucose . 
 insulin secretion from  cells in the presence of glucose seems to be related to the dosage of vitamin d and duration of preincubation . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Statistical Analysis
4. Results
5. Discussion