postprandial glucose homeostasis is controlled by insulin release in response to the absorbed nutrients and to gastric inhibitory polypeptide ( gip ) and glucagon - like peptide 1 ( glp-1 ) [ 14 ] , which are responsible for the so - called incretin effect , that is , the enhanced insulin secretion after oral versus intravenous administration of glucose . in healthy , nondiabetic subjects ,
the quantitative contribution of this incretin effect to the overall postprandial insulin secretion has been estimated to be 5070% [ 6 , 7 ] , depending on meal size and composition . on the contrary , a marked reduction of the incretin effect
was demonstrated in type 2 diabetes patients , thereby contributing to their excess in postprandial glucose excursions .
although the exact mechanisms underlying the loss of incretin activity in type 2 diabetes patients are unclear , it is evident that while the effects of glp-1 are largely preserved [ 911 ] , the insulinotropic effect of gip is almost lost in type 2 diabetes , potentially due to a defective expression of gip receptors , a downregulation of gip signaling , or a general reduction of beta cell function and mass [ 1118 ] .
it is well known that hyperglycemia enhances the endogenous nonenzymatic glycosylation of proteins , lipids , and nucleic acids .
this process might result in the accumulation of heterogeneous molecules , such as the advanced glycation end products ( ages ) .
several studies showed a positive correlation between the accelerated formation of ages and the complications of diabetes . in the last decade , our research group demonstrated a direct role of ages on pancreatic beta cell dysfunction , showing that exposure of pancreatic beta cells to ages was able to increase oxidative stress and decrease their antioxidant activity [ 2123 ] .
furthermore , evaluating the effects of ages in glutag , an enteroendocrine cell line that produces and secretes glp-1 , we found that the exposure of glutag cells to ages results in impaired glp-1 secretion and induction of insulin resistance .
the aim of this study is to investigate whether ages impair pancreatic beta cell responsiveness to gip , thus altering gip - induced insulin secretion .
glycated serum ( gs ) was prepared by adding 50  mmol / l ribose to heat - inactivated ( 56c for one hour ) fbs , as described previously .
aliquots of fbs were processed the same way but without ribose ( nonglycated serum ( ngs ) ) and used for standard medium preparation .
the concentration of pentosidine in the experimental media containing ngs was 70  pmol / ml , whereas the concentration of pentosidine in the experimental media containing gs was 400  pmol / ml which corresponds to the levels within the pathophysiological range detected in the plasma of diabetic patients [ 2527 ] .
hit - t15 cells were grown in rpmi 1640 supplemented with 10% fetal bovine serum ( fbs ) , 2  mmol / l l - glutamine , 100  iu / ml penicillin , and 100  g / ml streptomycin . before each experiment ,
the cells were split into 6-well plates and cultured for 5 days in the following experimental conditions : rpmi 5.6  mmol / l ( ctr ) or 11.1  mmol / l glucose ( hg ) supplemented with gs . at the end of the culture we evaluated the expression of gip receptor , the secretion of insulin and glp-1 in response to gip , and the intracellular gip signaling .
insulin release was evaluated under static conditions . to study secretion of insulin in response to gip
, hit - t15 cells were preincubated 1  h in kreb 's ringer bicarbonate buffer ( 118.5  mmol / l nacl , 2.54  mmol / l cacl2 , 1.19  mmol / l kh2po4 , 4.75  mmol / l kcl , 25  mmol / l nahco3 , 1.19  mmol / l mgso4 , 10  mmol / l hepes , 0.1% bsa , ph 7.4 ) , then cells were challenged for 60  min either with 4  mmol / l glucose alone or in presence of 10  nmol / l gip [ 18 , 28 ] .
results were shown as percentage change from ctr level ( 100% ) . in order to evaluate gipr activation ,
hit - t15 cells were incubated in serum - free medium supplemented with 1% bsa and then exposed for 5  min to 100  nmol / l gip [ 2931 ] .
then , cells were lysed and phosphorylation of members of the gipr signaling cascade was evaluated in western blot using specific antibodies . at the end of the experiments
, hit - t15 cells were lysed in ripa buffer ( 50  mm tris hcl ph 7.5 , 150  mm nacl , 1% np40 , 0.1% sds , supplemented with protease and phosphatase inhibitors ) , and protein concentrations were determined using the bca protein assay kit .
thirty micrograms of total cell lysate was separated on a sds - page and transferred onto nitrocellulose .
filters were blocked in 5% bsa and incubated overnight at 4c with primary specific antibodies ( anti - gip receptor ( h-70 ) and anti - gapdh ( fl-335 ) from santa cruz biotechnology , inc .
santa cruz , ca , usa ; anti - phosphoakt ( ser473 ) , -pi3 kinase ( 19h8 ) , and -phospho - p44/42 mapk ( erk1/2 ) ( thr202/tyr204 ) from cell signaling technology , beverly , ma , usa ) .
secondary specific horseradish - peroxidase linked antibodies were added for 1 hour at room temperature .
bound antibodies were detected using an enhanced chemiluminescence lighting system ( luminata classico , millipore , billerica , ma , usa ) , according to manufacturer 's instruction . to verify equal loading of the proteins , membranes were stripped , reblocked , and reprobed to detect gapdh .
all analyses were carried out with the graphpad prism 5.0 software ( graphpad software , san diego , ca , usa ) .
data were expressed as the mean  se . comparison between control and single treatments was done using unpaired t - test .
firstly , we confirmed the protein expression of gipr in the pancreatic beta cell lines hit - t15 ( figures 1(a ) and 1(b ) ) .
then , we investigated whether treatments with hg or gs would affect gipr protein expression .
incubation with gs or hg alone did not affect gipr expression , while combined treatment with gs and hg significantly decreased gipr protein expression ( figures 1(a ) and 1(b ) ) .
activation of gipr signaling is coupled to increased phosphorylation of several substrates including p44/42 erk and akt . in order to investigate whether decreased gipr expression is associated with altered intracellular signaling , we stimulated hit - t15 cells with 100  nmol / l gip and then analyzed the phosphorylation of p42/44 erk and akt , two validated intracellular kinases downstream gipr . treatment with gs and
hg ( alone or in combination ) did not affect gip - induced p42/44 erk phosphorylation ( figures 2(a)2(c ) ) .
on the contrary , gs and hg ( alone and in combination ) abrogated gip - induced akt activation ( figures 3(a ) and 3(b ) ) .
since pi3k is known to be the upstream activator of akt , we determined whether hg or gs regulates pi3k protein expression .
treatment with hg alone or in combination with gs , but not gs alone , significantly reduced the expression of pi3k in hit - t15 cells ( figures 4(a ) and 4(b ) ) . to verify whether decreased expression of gipr and altered intracellular signaling induced by gip were associated with loss of beta cell function , we evaluated gip - induced insulin secretion in hit - t15 cells grown at low or high glucose levels in presence of gs . under static incubation conditions in krb buffer containing 4  mmol / l glucose , the rate of insulin secretion
was significantly decreased in cells cultured at low glucose concentration with gs as compared to cells cultured with low glucose alone ( ctr ) ( figure 5(a ) ) .
intracellular insulin content in hit - t15 cells grown with gs alone was comparable to control cells but significantly decreased in cells cultured under hyperglycemic condition ( figure 5(b ) ) .
the rate of insulin secretion in response to 10  nmol / l gip stimulation was significantly reduced in cells grown with hg in combination with gs ( figure 5(c ) ) .
in this study , we demonstrated that stimulation with ages might be associated with the loss of gip responsiveness in pancreatic beta cells .
indeed , our results showed that high glucose levels might modulate pancreatic beta cell function , reducing intracellular insulin content and affecting activation of gip substrates .
however , these alterations did not result in loss of gip - induced insulin secretion .
in fact , the secretory response to gip was lost , only when cells were cultured under hyperglycemic conditions in combination with gs .
the direct and deleterious role of ages on microvascular diabetic complications and on pancreatic beta cell function is well validated .
evidence also from our group previously identified the adverse effects of ages in hit - t15 cell pathophysiology [ 2123 ] . as expected , treatment with gs alone was able to alter gip - triggered intracellular signaling and reduce glucose - induced insulin secretion .
however , cells grown under this condition were found to maintain a gip - induced insulin secretion that was comparable to control cells .
the binding of gip to its cognate receptor gipr triggers several intracellular signaling pathways , including pi3k , akt , and mapk , which potentiate glucose - stimulated exocytosis of insulin - containing granules . in this study
, we showed that treatment with gs and hg was able to alter gip - triggered intracellular signaling by selectively abrogating akt activation .
although both gs and hg were able to abrogate gip - induced akt phosphorylation , their inhibition did not affect gip - induced insulin secretion .
indeed the loss of increment in insulin secretion in response to gip was associated with significant reduction in gipr expression , suggesting that the loss of gip - stimulated insulin secretion was mainly related to the levels of gipr expression rather than to gip - triggered intracellular signaling .
these findings are consistent with the hypothesis that the unresponsiveness to gip could be due to a decreased expression of its receptor [ 16 , 18 , 35 ] .
ages have been suggested as key mediators in the  metabolic memory .  this hypothesis explains how diabetic complications are evolving even after glucose control is achieved [ 3638 ] . since molecular alteration of ages is  permanent ,  culture in the presence of gs alone may represent the milieu of improved glucose control reached after chronic hyperglycemia .
it has been reported that an improved control of hyperglycemia may reverse gipr expression downregulation and resistance to gip in zucker rats .
our finding suggests that improved glucose control was associated with recovery of gip- but not glucose - induced insulin secretion .
therefore , the  memory  of hyperglycemia may compromise the complete recovery of pancreatic beta cells even when glucose control is improved .
since chronic hyperglycemia leads to formation of ages , an hg combined with gs might be more representative of a 
our findings suggest that pancreatic beta cells are capable of counteracting detrimental action of chronic hyperglycemia . however , when hyperglycemia is combined with ages , beta cells are not able to react to the insults and become insensitive to gip .
our results are in agreement with findings reported by meier and nauck , showing that at hyperglycemic clamp conditions the insulin response to gip was relatively normal in the individuals with fasting glucose concentrations of less than ~100  mg / dl , but , when glucose concentrations exceeded this level , there was a progressive decline in gip activity on insulin secretion , with an almost complete loss of response in patients with 150250  mg / dl serum glucose .
these results suggest that the loss of gip responsiveness might be due to the downregulation of its receptor .
this condition was related to the deterioration of beta cell function due to the combined stimulation in the presence of hg and gs .
these findings may contribute to explain the loss of gip responsiveness in type 2 diabetes patients ( figure 6 ) .
 glucose - dependent insulinotropic peptide ( gip ) is an incretin hormone produced in the gastrointestinal tract that stimulates glucose dependent insulin secretion . 
 impaired incretin response has been documented in diabetic patients and was mainly related to the inability of the pancreatic beta cells to secrete insulin in response to gip . 
 advanced glycation end products ( ages ) have been shown to play an important role in pancreatic beta cell dysfunction . 
 the aim of this study is to investigate whether the exposure to ages can induce gip resistance in the pancreatic beta cell line hit - t15 . 
 cells were cultured for 5 days in low ( ctr ) or high glucose ( hg ) concentration in the presence of ages ( gs ) to evaluate the expression of gip receptor ( gipr ) , the intracellular signaling activated by gip , and secretion of insulin in response to gip . 
 the results showed that incubation with gs alone altered intracellular gip signaling and decreased insulin secretion as compared to ctr . 
 gs in combination with hg reduced the expression of gipr and pi3k and abrogated gip - induced akt phosphorylation and gip - stimulated insulin secretion . in conclusion , we showed that treatment with gs is associated with the loss of the insulinotropic effect of gip in hyperglycemic conditions . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions