type 2 diabetes is a heterogeneous disorder characterized by peripheral insulin resistance , defects in insulin secretion , and -cell apoptosis .
pancreatic -cell dysfunction is central to the pathogenesis of type 2 diabetes , and the loss of functional -cell mass in type 2 diabetes is at least in part secondary to increased -cell apoptosis .
obesity is a well - known risk factor for diabetes and is characterized by elevated levels of circulating free fatty acids ( ffas ) .
prolonged exposure to elevated levels of ffas has been shown to cause defective -cell proliferation and increased -cell apoptosis .
therefore , lipotoxicity plays an important role in underlying the mechanism of type 2 diabetes .
glucagon - like peptide-1 ( glp-1 ) , an incretin hormone secreted by intestinal l - cells , is a recent modality for the treatment of type 2 diabetes . glp-1 and its receptor agonists exhibit a variety of benefits to diabetic patients .
glp-1 can be used against hyperglycemia and obesity in most t2 dm diabetic patients by stimulating insulin secretion , inhibiting glucagon secretion , slowing gastric emptying , and promoting satiety . among all these effects ,
furthermore , recent data have shown that glp-1 and its receptor agonists promote the survival of -cell lines when challenged with various apoptotic stimulators , including hyperglycemia , inflammatory cytokines , oxidative stress , and endoplasmic reticulum stress [ 810 ] . however , until now whether exendin-4 , a glp-1 receptor agonist , exerts cytoprotective effects in pancreatic -cells under lipotoxic condition had not been determined completely .
therefore , the aim of the present study was to investigate the potential antiapoptotic and prosurvival actions of exendin-4 in pancreatic -cells under a chronic lipotoxic condition and the underlying signaling pathways involved .
fatty - acid - free bovine serum albumin ( bsa , fraction v ) , palmitate , hoechst33258 , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromides ( mtt ) , and pd98059 were purchased from sigma - aldrich ( st . louis , mo , usa ) .
the anti - phospho - erk1/2 and anti - total erk1/2 antibodies were obtained from cell signaling technology ( danvers , ma , usa ) .
the anti - bax and anti - bcl-2 antibodies were obtained from santa cruz biotechnology ( heidelberg , germany ) .
an enzyme - linked immunosorbent assay- ( elisa- ) based bromodeoxyuridine ( brdu ) incorporation kit was from roche ( basel , switzerland ) . the caspase-3 activity assay kit was from r&d systems ( minneapolis , mn , usa ) .
mouse pancreatic -cell line min6 was a kind gift from biochemistry lab of china medical university with passage number of 10 to 20 .
they were maintained in dulbecco modified eagle medium ( dmem ) containing 25  mm glucose , 15% fetal bovine serum ( fbs ) , 100  u / ml penicillin , 100  mg / ml streptomycin , 100  mg / ml l - glutamine , and 5  g / l -mercaptoethanol in a 37c humidified atmosphere with 5% co2 and 95% air .
cells at 80% confluence were washed with phosphate - buffered saline ( pbs ) and preincubated overnight in serum - free dmem before treatment .
min6 cells were cultured in a high glucose ( 25  mm ) serum - free dmem with 0.5% bsa or 0.4  mm palmitate added 0.5% bsa , with or without erk1/2 inhibitor , in the presence or absence of exendin-4 .
briefly , a 20  mm solution of palmitate in 0.01  m naoh was incubated at 70c for 30  min .
then 330  l 30% bsa and 400  l palmitate / naoh mixture were mixed together , filter - sterilized , and added into 20  ml dmem .
we selected a 25  mm glucose concentration for all tests because an increased glucose concentration was essential for min6 proliferation , and 25  mm glucose protected min6 cells from apoptosis . graded doses of exendin-4 ( 1 , 10 , 100 , and 500  nm ) were prepared freshly before tests .
, cells were incubated with 1  mg / ml mtt for 1  h. the medium was removed and the formazan product was solubilized with 150  l dimethyl sulfoxide .
viability was assessed by spectrophotometry at 570  nm absorbance using a 96-well plate reader .
briefly , min6 cells were set up onto 96-well plates at 80% confluence and incubated overnight in serum - free dmem before experiments , and brdu was added to the culture medium for 1  h before cells harvest .
cells were then fixed and incubated with a peroxidase - conjugated anti - brdu antibody , and immune complexes were quantified by spectrophotometry .
the hoechst positive cells represent the apoptotic cells , which were characterized with chromatin condensation or fragmented nuclear membrane .
briefly , hoechst staining was performed by exposing the cell slides to 10  g / ml hoechst33258 for 10  min at room temperature .
caspase-3 activity was measured in triplicate with a caspase-3 colorimetric assay kit , according to the manufacturer 's manual . in brief , after two time washes with ice - cold pbs , cells were harvested and centrifuged at 10,000  rpm for 10  min , followed by the addition of 1  l dithiothreitol ( dtt ) and 100  l lysis buffer .
cell lysates were placed in a 96-well plate and incubated for 2  h at 37c with 10  l caspase-3 colorimetric substrate ( devd - pna ) .
50  g protein samples ( in 20  l buffer ) were separated by sds - electrophoresis on 12% gradient polyacrylamide gels and transferred onto pvdf membranes .
the membranes were incubated with the primary antibodies with appropriate concentration , including anti - phospho - erk1/2 antibody ( 1  :  1,000 ) , anti - total erk1/2 antibody ( 1  :  1,000 ) , anti - bax antibody ( 1  :  200 ) , and anti - bcl-2 antibody ( 1  :  200 ) . immunodetection was performed with ecl advance , and resulting images were analyzed by scion image software version 4.0.3.2 ( scion corporation , frederick , md ) .
differences between groups were determined by one - way analysis of variance ( anova ) followed by post hoc testing and correction by least significant difference / dunnett 's t - test . if the f ratio was statistically significant , a turkey 's post hoc test was considered .
to determine the effects of exendin-4 on -cell survival and proliferation , min6 cells were incubated in the 0.5% bsa ( bsa ) and 0.4  mm palmitate added 0.5% bsa ( pa ) , with or without increasing concentrations of exendin-4 ( 1500  nm ) ( ex ) for 24  h. cell viability and proliferation were assessed by the mtt assay and an elisa - based brdu incorporation kit , respectively .
cell survival ( figure 1(a ) ) was significantly reduced by palmitate treatment ( 28% , p < 0.01 versus bsa ) .
exendin-4 treatment reversed palmitate - induced reduction of cell viability , and 100  nm exendin-4 displayed a major effect of restoring the cell viability by 27% ( p < 0.01 versus pa ) .
exendin-4 treatment alone showed a nonsignificant promotion to cell survival , and 100  nm exendin-4 provided the best potentiation ( 112% , p = 0.21 versus bsa ) .
cell proliferation ( figure 1(b ) ) was decreased under palmitate exposure ( 38% , p < 0.01 versus bsa ) .
this decrease was inhibited by exendin-4 treatment , most obviously at 100  nm ( 29% , p < 0.01 versus pa ) .
exendin-4 treatment alone displayed a nonsignificant increase in cell proliferation , and 100  nm exendin-4 provided the greatest tendency ( 120% , p = 0.11 versus bsa ) . in the presence of pa ,
100  nm exendin-4 achieved a significant proliferative effect ( 91% , p = 0.03 versus pa ) .
we next assessed cell apoptosis by hoechst33258 assay and caspase-3 activity assay . for hoechst33258 assay
, min6 cells were incubated with or without 0.4  mm palmitate , in the presence or absence of 100  nm exendin-4 ( figure 1(c ) ) .
pa exposure for 24  h induced apoptosis ( 34.3% , p < 0.01 versus bsa ) , which was reversed by 100  nm exendin-4 treatment by decreasing the apoptosis to 11.9% ( p < 0.01 , pa + ex versus pa ) .
apoptosis was significantly increased in cells treated with pa alone ( 133% , p < 0.01 , pa versus bsa ) .
100  nm exendin-4 treatment with pa presence resulted in a significant decrease of apoptosis ( 87% , p < 0.01 , pa + ex versus pa ) .
we investigated the effect of exendin-4 on erk1/2 phosphorylation under palmitate treatment , by detecting the ratio of phosphorylated erk1/2 expression to total erk1/2 expression ( figure 2(a ) ) .
the erk1/2 phosphorylation was blocked by palmitate exposure ( 0.624  0.048 versus 0.496  0.062 , p < 0.05 bsa versus pa + ex at 0  min ) . at the end of the preincubation period , 100  nm exendin-4 was added .
the maximal effect was observed at 5  min ( 0.721  0.135 versus 0.496  0.062 , p < 0.01 pa + ex at 5  min versus pa + ex at 0  min ) .
exendin-4 also induced the phosphorylation of erk1/2 in a concentration - dependent manner ( figure 2(b ) ) and 100  nm exendin-4 treatment produced the most effective potentiation ( 0.744  0.083 versus 0.494  0.117 , p < 0.01 pa + ex at 100  nm versus pa ) . to establish the induction of phosphorylation of erk1/2 by exendin-4 , we did further treatment using pd98059 , a specific erk1/2 inhibitor ( figure 2(c ) ) .
the exendin-4-induced phosphorylation of erk1/2 was obviously suppressed by pd98059 ( 0.707  0.096 versus 0.556  0.050 , p < 0.05 ex + pa versus ex + pd + pa ) , whereas the effect of pd98059 on erk1/2 phosphorylation without exendin-4 was similar to that of pa alone ( 0.459  0.057 versus 0.519  0.071 , p = 0.217 pa + pd versus pa ) .
we also determined the role of the erk1/2 inhibitor on the cytoprotective effect of exendin-4 by mtt assay and hoechst33258 assay ( figures 2(d ) and 2(e ) ) .
consistent with the aforementioned results , exendin-4 treatment promoted cell survival ( 95.3  3.7% versus 68.4  6.9% , p < 0.01 ex + pa versus pa ) and prevented apoptosis of min6 cells ( 21.2  2.1% versus 33.5  3.7% , p < 0.01 ex + pa versus pa ) under lipotoxic condition , whereas pd98059 suppressed this promotion of cell survival ( 71.0  4.6% versus 95.3  3.7% , p < 0.05 ex + pd + pa versus ex + pa ) and attenuated the restore of apoptosis ( 29.2  3.2% versus 21.2  2.1% , p < 0.05 ex + pd + pa versus ex + pa ) under lipotoxic condition .
all these results strongly suggested that exendin-4 protected min6 cells against lipotoxicity , at least in part , via activation of erk1/2 signaling pathway .
western blot analysis of bcl-2 and bax were conducted after 24  h culture under lipotoxic condition ( figure 3 ) .
we found a significant decreased expression of the antiapoptotic protein bcl-2 ( figure 3(a ) , p < 0.01 versus bsa ) and enhanced expression of the proapoptotic protein bax ( figure 3(b ) , p < 0.01 versus bsa ) in min6 cells under palmitate treatment . while the exendin-4 treatment showed a significant increased expression of bcl-2 ( figure 3(a ) , p < 0.05 versus pa ) and a nonsignificant decreased expression of bax levels ( figure 3(b ) , p = 0.22 versus pa ) , compared with the parallel lipotoxic condition cultured without exendin-4 .
the incretin glucagon - like peptide-1 ( glp-1 ) , the major gastrointestinal product of proglucagon processing , has been shown to exert trophic effects on -cells .
although roles of exendin-4 on cell proliferation and antiapoptosis have been demonstrated in -cells , its mechanisms are not fully understood . moreover , whether exendin-4 protects -cells against lipotoxicity remains to be determined . in the present study
, we found that exendin-4 inhibited palmitate - induced apoptosis via activating erk1/2 pathway and also through inhibiting mitochondrial apoptosis pathway in the min6 cells .
physiologic levels of lipids and glucose are not toxic but essential to -cell function , while prolonged exposure of pancreatic -cells to high levels of fatty acid impairs insulin gene expression , inhibits insulin synthesis and secretion , and induces -cell apoptosis [ 11 , 21 , 22 ] . in vivo ,
the normal plasma concentration of ffa is less than 0.6  mmol / l but could exceed 1  mmol / l in type 2 diabetic patients [ 23 , 24 ] .
palmitate and oleate are the two most represented ffas in plasma ( 20 to 25% each ) , linked to albumin in the circulation [ 25 , 26 ] . to mimic what is observed in vivo
, we used a mixture of palmitate with 5% fatty - acid - free bsa .
this high albumin concentration was used to obtain an appropriate albumin to nonesterified fatty acid ratio . in our study ,
the direct effects of long - term elevated palmitate on the survival of pancreatic -cells were studied .
consistent with previous observations [ 27 , 28 ] , theoretically , antilipotoxicity through blockade of apoptosis and enhancement of proliferation may be a novel approach to protect pancreatic -cells .
accumulating evidence supports the role of glp-1 mimetics and enhancers in regulating proliferation and protecting from cellular apoptosis .
these agents constitute a novel class of antidiabetes medications , which might have a major impact on the treatment of type 2 diabetes [ 2931 ] . in this study
, we demonstrated that treatment with exendin-4 , a glp-1 agonist , protected pancreatic -cell from palmitate - induced toxicity by enhancing proliferation and inhibiting apoptosis , in a dose - dependent manner ( figure 1 ) . in addition , exendin-4 treatment activated the erk1/2 signaling pathway .
erk1/2 , a well - known downstream target of raf-1 , has been shown to participate in pathways controlling -cell survival .
it has been reported in several studies that acute glucose and protein kinase a signaling may regulate -cell growth and survival through erk1/2 [ 3234 ] . on the other hand , others have shown that activation of erk1/2 is required for human islet apoptosis in response to chronic exposure to high glucose concentrations or interleukin-1 .
the outcome may depend on the timing and duration of erk1/2 activation . in the present study
, our findings showed that exposure of min6 cells to chronic lipotoxic conditions reduced the phosphorylation of erk1/2 .
in addition , this reduction was reversed by exendin-4 treatment through a rapid activation of erk1/2 .
further treatment with the erk1/2 inhibitor pd98059 blocked this protective effect of exendin-4 ( figure 2 ) .
these data indicate that exendin-4 exerted cytoprotective effect on pancreatic -cells against lipotoxicity , at least in part via the erk1/2 pathway .
the mechanisms underlying apoptosis are regulated by defined biochemical pathways , involving members of the bcl-2 family . in mammalian cells ,
the bcl-2 family of proteins plays a central role in modulating mitochondrial - dependent apoptosis .
both prosurvival and proapoptotic bcl-2 family members are critical death regulators via mitochondrial apoptosis pathway . among bcl-2 family proteins ,
prosurvival bcl-2 stabilizes the mitochondrial membrane and prevents the cytochrome c release from the mitochondria and the followed activation of caspases . in the present study
, bcl-2 was found to be downregulated and the proapoptotic bax was upregulated , which was in accordance with the increased activity of caspase-3 under lipotoxic condition .
after exendin-4 treatment , we observed that these alterations were partially counteracted ( figure 3 ) .
so , our results indicate that exendin-4 conducts its protective effect through inhibiting mitochondrial apoptosis pathway , by promoting bcl-2 protein expression .
interestingly , some bcl-2 family proteins were found to be involved in erk1/2 signaling pathway . as reviewed by cagnol and chambard ,
in contrast , antiapoptotic proteins bcl-2 and bcl - xl were downregulated by erk1/2 activation . on the other hand , phosphorylation of proapoptotic protein bad
was assumed to be involved in erk1/2 promoted cell survival . in human pancreatic cancer cells ,
inhibition of erk1/2 activities caused downregulation of antiapoptotic proteins bcl-2 , mcl-1 , and bcl - xl without affecting the proapoptotic proteins bax and bak .
these various reports might be interpreted depending on different cell types or conditions . in our study
, we found both erk1/2 activation and enhanced bcl-2 expression induced by exendin-4 treatment under lipotoxic condition .
further study on the relation between erk1/2 activation and inhibition of mitochondrial apoptosis pathway may reveal more detailed molecular mechanism of glp-1 mediated cytoprotective role on pancreatic -cells .
compared to other insulin - secreting cell lines , min6 cells express glut-2 and glucokinase and display a similar glucose stimulating insulin secretion to normal primary -cells .
the insulin secretion of min6 cells reaches the maximal level approximately sevenfold above the basal level at 25  mmol / l , which is consistent with the performance of our min6 cells ( data not shown ) .
but as an immortalized cell line , some of its characters , especially the proliferative specific , are not exact same with primary -cells . this could be a confounding factor within any study on this aspect .
in conclusion , it is likely that exendin-4 induced erk1/2 activation is an important pathway , by which exendin-4 exerts antiapoptotic actions against palmitate - induced lipotoxicity .
besides , the potential cytoprotective effect of exendin-4 on -cells involves the mitochondrial apoptosis pathway , by upregulating bcl-2 .
because lipotoxicity is supposed to be responsible for -cell loss in the pathological process of obesity - associated type 2 diabetes , the antilipotoxic effects on residual -cells of exendin-4 may arouse interest , considering its therapeutic utilization in type 2 diabetes treatment .
 type 2 diabetes is a heterogeneous disorder that develops as a result of relatively inappropriate insulin secretion and insulin resistance . increased levels of free fatty acids ( ffas ) are one of the important factors for the pathogenesis of type 2 diabetes and contribute to defective -cell proliferation and increased -cell apoptosis . 
 recently , glucagon - like peptide-1 ( glp-1 ) receptor agonists have been shown to possess an antiapoptotic effect , by increasing -cell mass and improving -cell function . 
 however , their effects on -cells in vitro against lipotoxicity have not been elucidated completely . in this study 
 , we investigated whether the glp-1 receptor agonist exendin-4 displays prosurvival effects in pancreatic -cells exposed to chronic elevated ffas . 
 results showed that exendin-4 inhibited apoptosis induced by palmitate in min6 cells . 
 after 24  h of incubation , exendin-4 caused rapid activation of extracellular signal - related kinase 1/2 ( erk1/2 ) under lipotoxic conditions . 
 the erk1/2 inhibitor pd98059 blocked the antilipotoxic effect of exendin-4 on min6 cells . 
 exendin-4 also inhibited the mitochondrial pathway of apoptosis . 
 this inhibition is associated with upregulation of bcl-2 . 
 our findings suggested that exendin-4 may exert cytoprotective effects through activation of erk1/2 and inhibition of the mitochondrial apoptosis pathway . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion