studies have shown that statins can inhibit the action of hmg - coa reductase in the liver , thereby slowing down the cholesterol production process . clinical studies have also shown that statins can reduce the risk of heart attack , stroke , and death in patients with heart disease such as coronary artery disease and cardiovascular disease [ 2 , 3 ] . in addition , researchers have reported that statins may reduce the risk of developing colon cancer , non - small lung cancer , pancreatic cancer , and esophageal cancer [ 47 ] . although these drugs have a satisfactory safety record , the increased risk for developing new - onset diabetes mellitus during extended statin use has recently generated attention ; this includes simvastatin ( 10% ) , atorvastatin ( as , 22% ) , and rosuvastatin ( 18% ) [ 811 ] . have recently found that these statins reduced insulin sensitivity and pancreatic -cell function , possibly because of the effect on ca channels in -cells , glucose transporter 4 translocation , insulin receptor substrate-1 and insulin receptor expression or phosphorylation , adipocyte maturation or differentiation , isoprenoid and coenzyme q10 biosynthesis , and adiponectin and leptin levels [ 12 , 13 ] . however , studies on pravastatin showed significant improvements in insulin sensitivity and -cell function [ 14 , 15 ] . therefore , demonstrating the proposed mechanisms of statins therapy during the development of new - onset diabetes may be valuable for designing a new generation of statins without the aforementioned side effects . poly(adp - ribosyl ) polymerase-1 ( parp1 ) is a group of nuclear enzymes that has been associated with three different modes of cell death induced by dna damage , namely , apoptosis , necrosis , and parthanatos . in addition , its overactivation results in nad / atp energy depletion and eventually causes necrotic cell death [ 1619 ] . however , studies have identified a novel function of parp1 in mediating autophagy ; thus , parp1 exhibits a dual role in modulating autophagy and necrosis under oxidative stress and dna damage [ 2022 ] . autophagy is an intracellular bulk degradation system for the removal of long - lived proteins and damaged organelles such as lysosomes . studies have indicated that autophagy may prevent neurodegeneration , aging , and tumorigenesis [ 2327 ] . by contrast , several studies have suggested that autophagy may trigger and mediate type ii programmed cell death , which has been referred to as autophagic cell death [ 28 , 29 ] . autophagy has also been demonstrated as a prosurvival mechanism against cell death , especially under stress conditions such as starvation , metabolic stress , oxidative stress , and dna damage [ 30 , 31 ] . thus , this study examined the signaling pathway linking parp1 activation to autophagy under pharmaceutical stress , as well as the functional role of autophagy in pharmaceutical stress - mediated cell death . mouse pancreatic nit-1 cells were obtained from bcrc ( bioresource collection and research center , taiwan ) and cultured in f-12k medium ( sigma - aldrich , usa ) supplemented with 10% fbs ( gibco , usa ) at 37c in a humidified 5% co2 . cells were seeded into a 96-well plate ( 6  10/well ) on f-12k medium and treated with statin ( 1020  m , merck millipore , germany ) for 48  h , beginning 24  h after seeding . wst-1 solution ( 10  l , roche , germany ) was added to each well and the cells were incubated for 1  h. the absorbance at 440  nm was measured using a microplate reader ( fluostar galaxy , germany ) . in some experiments , the cells were pretreated with various inhibitors such as necrostatin-1 ( nec-1 , an inhibitor of rip1 , 10  m , enzo life sciences , usa ) , necrosulfonamide ( nsa , an inhibitor of mlkl , 1.0  m , millipore ) , bafilomycin a1 ( baf - a1 , 2  nm , sigma - aldrich ) , 3,4-dihydro-5[4-(1-piperidinyl)butoxyl]-1(2h)-isoquinoline ( dpq , 60  m , calbiochem ) , z - val - ala - asp - fluoromethylketone ( zvad - fmk , 2  m , biovision , usa ) , and mtor inhibitors ( rapamycin , 20  nm , adooq bioscience , usa ) before statin treatment . one - day cultured nit-1 cells were treated with statin for 48  h. the supernatants were collected and the insulin concentrations were measured using rat / mouse insulin 96-well plate assay ( millipore ) according to the manufacturer 's instruction ; the absorbance at 450  nm and 590  nm was measured by an elisa reader ( fluostar galaxy ) . il-6 was determined using the mouse il-6 elisa kit ( bd biosciences , usa ) according to the manufacturer 's instruction ; the absorbance at 450  nm was measured by an elisa reader ( fluostar galaxy ) . the lower limit of detection for this elisa was 3.8  pg / ml il-6 . nit-1 cells were seeded into a 96-well plate ( 6  10/well ) and cultured in statin - containing medium supplemented with 1% fbs for 48  h. after applying 100  l of the lactate dehydrogenase reaction mixture using the cytotoxicity detection kit ( roche ) , the absorbance at 490  nm was measured by an elisa reader ( fluostar galaxy ) : cytotoxicity  ( % ) = ( exp . nit-1 cells were seeded in 12-well plates at 4  10 cells / well and treated with statin for 48  h. subsequently , the cells were collected , fixed with cold 70% absolute ethanol , and stored at 20c overnight . before detection subsequently , 1  ml of staining solution was added into each tube ( 20  g / ml of pi ) , followed by incubation in darkness for 30  min . finally , the stained cells were analyzed using a flow cytometer ( cytomics fc 500 , beckman coulter ) . cells were plated in a 6 cm culture dish at a seeding density of 5  10 cells / dish . the cells were trypsinized , collected by centrifugation , resuspended in 500  l of 1x annexin v binding buffer mixed with 1  l of annexin v ( biovision ) and 1  l of propidium iodide ( biovision ) , and incubated at room temperature for 15  min in darkness . the cells were analyzed using a flow cytometer ( cytomics fc 500 ) with a single laser emitting excitation light at 488  nm . the intracellular reactive oxygen species were assessed using the cellrox oxidative stress reagent ( life technologies , usa ) according to the manufacturer 's instructions . cells were plated in 12-well plates with a cell density of 8  10 cells / well and treated with statin for 48  h. after statin treatment , the cells were stained with 5  m of cellrox green reagent and incubated in darkness for 30  min . after incubation , the stained cells were analyzed using the flow cytometer ( cytomics fc 500 ) . the 2.5  10 cells were seeded in a 3.5 cm confocal dish and pretreated with various inhibitors before statin treatment . following a 48 h treatment with statin , the cells were washed with pbs to remove dead cells and serum proteins . immediately , treated or untreated nit-1 cells were stained with an antibody against rabbit monoclonal aif ( 1  :  400 , cell signaling ) or cyto - id autophagy detection kit ( enzo life sciences , farmingdale , new york , usa ) and imaged via confocal microscopy using an olympus fv1200 microscope ( tokyo , japan ) . the images were converted to 8-bit grayscale and analyzed by the imagej software . the integrated density ( intden ) provided a measure of intensity proportional to total volume and was calculated using area . after statin treatment , the cells were stained with mitotracker red and incubated at 37c for 30  min in darkness . the cells were washed three times with pbs to remove unbound dye and recultured in f-12k medium . the fluorescence of the bound dyes was analyzed using an olympus ix81 fluorescence microscope ( tokyo , japan ) . cellular atp synthesis was determined using the phosphoworks luminometric atp assay kit ( aat bioquest , usa ) according to the manufacturer 's instructions . in brief , cells were seeded into 96-well plates and pretreated with inhibitors . after statin treatment , the cells were added with 100  l of atp assay solution and incubated for 20  min at room temperature . nuclear and mitochondrial fractions were prepared from renal tissue using nuclear protein isolation - translocation assay kit ( fivephoton biochemicals , san diego , ca , usa ) and allpure mammalian mitochondria isolation kit ( allbio science inc . , whole - cell lysates were prepared using ripa lysis buffer ( millipore , 20188 ) and protein concentration was detected using a bca protein assay kit ( thermo scientific ) . a total of 150  g of cell lysates was mixed with anti - rip3 ( 2  g , genetex , usa ) or magsi - protein a / g beads ( 50  l , magnamedics , netherlands ) at 4c overnight . the beads were then collected using a magnet for 2  min , washed with pbst washing buffer three times , and subjected to elution with 40  l of 1x sds loading buffer ; the samples were incubated at 95c for 10  min . protein was separated using sds - page and transferred to 0.2  m pvdf membranes ( bio - rad , usa ) . blots were then probed with anti - rip1 , anti - rip 3 , anti - mlkl ( genetex ) , monoclonal anti - aif ( cell signaling ) , polyclonal anti - cox4 ( genetex ) , polyclonal anti - pcna ( genetex ) , monoclonal anti - parp1 , monoclonal anti - par ( cell signaling ) , anti - phospho - ampk ( thr172 ) , anti - ampk , anti - phospho - p70 s6 kinase ( thr389 ) , anti - p70 s6 kinase ( cell signaling , usa ) , mitoprofile total oxphos rodent antibody cocktail ( mitosciences , or ) , and mouse anti - gapdh ( abcam , usa ) . signals were obtained using an enhanced chemiluminescence kit ( millipore ) and densitometry was performed using fusion - capt software ( vilber lourmat , fusion fx7 , france ) . to determine the effects of statin on the cell viability of pancreatic  cells , nit-1 cells were treated with various concentrations of as or ps for 48  h by using the wst-1 assay . after treatment with as ( 10  m and 20  m ) , the cell viabilities were determined to be approximately 52.3% and 41.0% , respectively , compared with the vehicle control . moreover , after treatment with ps ( 10  m and 20  m ) , the cell viabilities were determined to be approximately 104.9% and 98.6% , respectively , compared with the vehicle control . accordingly , treatment with as results in the dose - dependent inhibition of cell viability in nit-1 cells , but not treatment with ps ( figure 1(a ) ) . similarly , the reduction of cell viabilities under the 10 m and 20 m as treatment could be associated with the decreased nit-1 cell insulin secretion and increased lactate dehydrogenase ( ldh ) activity release , which is a cytosolic marker . although the 20 m ps treatment resulted in less insulin secretion in the nit-1 cells , it was insufficient to change the amount of ldh activity release ( figures 1(b ) and 1(c ) ) . to further examine the inhibitory effects of as or ps on cell viability the treatment of nit-1 cells with as resulted in the significantly increased accumulation of cells in the sub - g1 phase ( necrotic / apoptotic cells ) in a dose - dependent manner , and the treatment with 20  m as resulted in significantly fewer cells in the g0/g1 phase ( p < 0.05 ) . the ps treatment did not change cell cycle parameters ( figure 2(a ) ) . significantly increased necrosis phase ( pi annexin v ) percentages were found in proportion to the as treatments ( 15.95% and 24.08% in the presence of 10 and 20  m as , resp . , p < 0.05 ) , and the apoptotic phase ( pi annexin v , data not shown ) percentages were not significantly different among the groups . in addition , no significant difference was identified in the necrosis phase and apoptotic phase among the groups during the experimental period with ps treatment , compared to the vehicle control ( figure 2(b ) ) . to further confirm whether the nit-1 cell death was caused by necrosis after as treatment the il-6 expression level was significantly increased in the as - treated groups 1.3- and 1.6-fold . however , no significant differences were found in the ps - treated groups compared with the vehicle control ( figure 2(c ) ) . in addition , the as treatment of nit-1 cells induced a dose - dependent increase of intracellular ros production ( p < 0.05 ) , whereas ps treatment did not result in significant differences among the groups ( figure 2(d ) ) . the results indicate that as treatment can diminish nit-1 cell viability primarily by triggering necrosis and possibly increase production of ldh , il-6 , and ros . next , we examined the inhibitor cell death response in the nit-1 cells treated with as and ps . nec-1 , nsa , and zvad - fmk are typically used as necrosis and pan - caspase inhibitors in vitro , respectively [ 30 , 31 ] . after 48  h , nsa and nec-1 treatment increased the numbers of viable nit-1 cells compared with the 20 m as - treated group ( 61% versus 58% versus 41% , p < 0.05 ) ; zvad - fmk treatment exhibited no effect on nit-1 cell viability after the 20 m as treatment . in addition , nsa , nec-1 , and zvad - fmk treatments showed no significant differences when compared to the 20 m ps - treated group ( 98% versus 101% versus 98% , figure 3(a ) ) . by contrast , the necrosis induced by the as treatment specifically caused the formation of the rip1-rip3-mlkl complex . the interactions between mlkl and rip1 and rip3 were significantly eliminated by nsa and nec-1 ; thus , the rip1 and mlkl signals appeared significantly reduced , and the rip3 signal was significantly augmented in the presence of nsa and nec-1 . the ps treatment did not induce the formation of the rip1-rip3-mlkl complex ( figure 3(b ) ) . here , we investigated the upstream signaling pathways controlling the as- and ps - mediated cell death . parp1 activation has been suggested to be associated with the necrotic and parthanatos cell death progress [ 17 , 19 , 32 , 33 ] . dpq markedly reduced the statin - associated necrotic cell death in nit-1 cells and significantly increased nit-1 cell viability in the 20 m as - treated group ( 58% versus 41% , p < 0.05 , figure 3(a ) ) . confocal microscopy and subcellular fractionation revealed that as - treated or ps - treated cells did not induce apoptosis - inducing factor ( aif ) translocation from mitochondria into the nucleus ( figures 3(c)3(e ) , figure s1a in supplementary material available online at http://dx.doi.org/10.1155/2016/1828071 ) , suggesting that parp1 activation plays crucial roles in as - induced necrotic cell death , not via parthanatos . several studies have demonstrated that parp1 exhibits a dual role in modulating autophagy and necrosis under oxidative stress and dna damage , whereas parp1 activation is associated with autophagy induction in as and ps treatment . rapamycin ( rapa ) is an mtor inhibitor and has been reported to promote autophagy processing , whereas bafilomycin a1 ( baf - a1 ) is an autophagy inhibitor . the nit-1 cell viability was significantly increased when the cells were treated with 20  m as with rapa , and the cell viability decreased mildly with baf - a1 pretreatment , compared with the 20 m as - treated group ( 56% versus 43% versus 45% , p < 0.05 ) . similarly , the nit-1 cells with rapa pretreatment showed considerably increased cell viability compared with the 20 m ps - treated group ( 125% versus 97% , p < 0.05 , figure 4(a ) , figure s1b ) . ldh activity release also was measured to determine the extent of nit-1 cell death after exposure to inhibitor agents such as dpq , baf - a1 , and rapa . the treatment with dpq and rapa markedly reduced ldh release after the 20 m as treatment ( 1.3- versus 1.2- versus 1.5-fold , p < 0.05 ) , whereas the baf - a1 treatment mildly increased ldh compared to the 20 m as - treated group ( figure 4(b ) , figure s1c ) . similarly , the baf - a1 treatment enhanced the ldh release content considerably , and the dpq treatment only slightly reduced ldh activity release . rapa treatment can attenuate baf - a1-associated increase in the ldh activity release of nit-1 cells after 20 m ps treatment ( 1.3- versus 1.4- versus 1.1-fold , p < 0.05 ) . these results showed that autophagy acts as a cell survival mechanism in as - induced necrotic cell death and that ps may be able to immediately induce autophagy to prevent nit-1 cell death under pharmaceutical stress . to evaluate whether parp1 expression plays a crucial role in autophagic induction , nit-1 cells were incubated in medium containing vehicle ( control ) , statin , statin+dpq ( parp1 inhibitor ) , statin+baf - a1 ( negative autophagy ) , and statin+rapa ( positive autophagy ) for 48  h. the cells were then stained with cyto - id green dye autophagy detection kit . both as - treated groups showed significantly stimulated further microtubule - associated protein 1a/1b - light chain 3- ( lc3- ) ii accumulation , as autophagosome formation in nit-1 cells compared with the vehicle control . as+baf - a1-treated group showed significantly reduced lc3-ii expression compared with as - treated group . no significant difference was found in the number of lc3-ii between control and ps - treated or ps+baf - a1-treated nit-1 cells . both dpq- and rapa - treated nit-1 cells showed significantly bright green fluorescence compared with only as - treated cells but only showed light green fluorescence when compared with ps - treated cells , respectively ( figures 5(a)5(c ) , figure s2a ) . these findings indicated that as - treated cells significantly induced the autophagic activity ; both dpq- and rapa - treated cells further enhanced the as - treated increase of autophagy flux in nit-1 cells . parp1 is known as an energetically expensive process that leads to cellular atp depletion and contributes to necrotic cell death . thus , atp depletion was determined in as - treated and ps - treated nit-1 cells , and the results showed that the cellular atp levels of as - treated , as+baf - a1-treated , as+dpq - treated , and as+rapa - treated nit-1 cells were significantly reduced compared with the untreated control ( p < 0.05 ) . however , dpq- and rapa - treated cells showed significantly increased as - stimulated cellular atp consumption , decreasing 0.86- and 0.66-fold ( p < 0.05 , figure 5(d ) , figure s2b ) . in addition , the rapa - treated cells similarly exhibited significantly increased ps - stimulated cellular atp consumption , but no significant difference was found in the cellular atp levels among the ps - treated groups . the results clearly indicated that as - induced parp1 activation , mtor suppression , and autophagy induction are mediated by inhibited parp1 activation and enhance cellular atp depletion . after demonstrating the prosurvival role of autophagy and the role of parp1 activation in as - induced necrotic cell death , we investigated whether the signaling pathway of parp1-ampk - mtor autophagy controls as- and ps - mediated cell survival . treatment with as+dpq markedly blocked as - enhanced parp1 cleavage and par expression , with a simultaneous decrease in the p - ampk level and increase in p - p70s6k level ( p < 0.05 , figure 6(a ) , figure s3a ) . as+baf - a1 was expected to inhibit parp1 cleavage and par formation ; it reduced the p - ampk level , restored p - p70s6k activation , and blocked autophagy induction . moreover , cells that were treated with rapa markedly enhanced as - induced parp1 cleavage and par and ampk activation and eliminated the phosphorylation of p70s6k to induce autophagy . similar effects were also found in the nit-1 cells with ps+dpq , ps+baf - a1 , and ps+rapa ( p < 0.05 , figure 6(b ) ) . the results show that parp1 exhibits dual roles in changing the outcome of nit-1 cells in response to as . parp1 activation is the cause of necrotic cell death through atp depletion , and parp1 activation can elicit a self - protective mechanism through the induction of autophagy via the ampk - mtor signaling pathway ; however , necrotic cell death still eventually occurs . parp1-ampk activation is an important prosurvival mechanism in ps - treated cells through the suppression of mtor and activation of autophagy . we examined the mitochondrial status of the as- and ps - treated cells conjugated with mitotracker red ; the accumulation of the cells is dependent on the membrane potential , and the cells are fusible into actively respiring cells . the as- and ps - treated cells showed reduced red fluorescence intensity with fewer mitochondria ; for the as - treated cells , the reduction was approximately 0.5-fold relative to the control , and they contained round , discrete mitochondria and widely diffused weak cytoplasmic fluorescence ( figure 7 , figure s3b ) . similar effects were also found in nit-1 cells treated with as+baf - a1 and ps+baf - a1 . dpq and rapa pretreatment to some extent protected against as- and ps - induced leakage of mitotracker staining and substantially enhanced the mitochondrial morphology and staining intensity in nit-1 cells . these results indicated that as treatment reduced mitotracker staining , thereby reducing mitochondrial activity ; this explains the close association between parp1 activation and mtor suppression . the detailed mechanisms of the increased risk for developing diabetes during statin therapy remain unclear . we used the mouse nit-1 insulinoma cell line as a pancreatic -cell model , which is an effective tool for analyzing -cell function and apoptosis [ 35 , 36 ] . to examine whether statins mediate the outcome of nit-1 cells , we used chemical inhibitor reagents to inhibit the expression of the target gene in nit-1 cells . our study results showed that atorvastatin treatment markedly reduced cell viability compared to untreated nit-1 cells through increasing parp1 activation and subsequently inducing necrosis and autophagy induction . this finding confirms previous findings that atorvastatin induced autophagic activity in vivo and in vitro [ 3740 ] . in 2012 , lim et al . demonstrated that pravastatin can improve renal function in csa - induced autophagic cell death through reducing lc3-ii and p62 expression . , we find that pravastatin treatment may increase parp1 activation and immediately elicit a basal self - protective autophagy mechanism in conservative nit-1 cells under pharmaceutical stress . in contrast , other studies showed that pravastatin did not result in induced autophagy activation in ovarian cancer cell , smooth muscle cell , and human rhabdomyosarcoma cell [ 4244 ] . the reason for the differences observed is that the role of pravastatin in autophagy activation and function are cell type - specific . in this study , parp1 exhibited dual roles in regulating the cell outcome in response to as or ps treatment . the findings provide a reasonable basis for the future improvement of statin - based cardiovascular disease therapies . apoptosis has been considered to be the predominant mechanism induced by free fatty acids , cytokines , and glucose in nit-1 cells [ 36 , 45 , 46 ] . in addition , several studies have shown that as and ps could trigger different cells to mediate apoptosis in vitro and regulate cell growth in vivo [ 4750 ] . we found that cell death was not inhibited by the pan - caspase inhibitor zvad - fmk in nit-1 cells treated with as . the lack of apoptosis was further indicated by the absence of the apoptotic phase ( annexin v / pi ) . given that as killed 50%60% of nit-1 cells , these observations suggest that the effect of as - induced nit-1 cell death may not depend on apoptosis . according to the aforementioned studies , more than 50% of bladder cancer cells , t24 , died 2 - 3 days after as treatment ( 30  m ) via activated caspase 3 and cleaved parp . in another study , increased annexin v - positive cells and accumulated sub - g1 cell fractions were observed on day 3 after as treatment with an increased dose ( 100  m ) , whereas as treatment at 10  m showed a protective trend against apoptosis . thus , mechanisms other than apoptosis might exist in as - mediated cell death , such as necrosis . in recent years , the crosstalk among autophagy , apoptosis , and necrosis has been intensively studied . most studies showed that autophagy is cytoprotective in cells under stress and inhibits apoptosis and necrosis [ 21 , 5356 ] . moreover , necrosis is found to be typically accompanied by autophagy , but how autophagy counteracts necrosis and why autophagy can not protect against as - induced cell death remain unclear . we found that as induces autophagy and necrotic cell death mediated by the activation of parp1 and ros . a major finding in the present study is the crucial role of ampk in as- and ps - induced autophagy and cell death , as well as the downstream of parp1 activation . ampk is a highly conserved cellular energy sensor , and it is activated under stress conditions such as heat shock , hypoxia , ischemia , and glucose starvation [ 39 , 57 , 58 ] . some evidence suggests that autophagy induction is regulated through ampk - dependent phosphorylation , which leads to the inactivation of mtor for homeostatic mitochondria and promotes cell survival [ 40 , 59 , 60 ] . consistent with these findings , our results revealed that the activation of ampk leads to the suppression of mtor and induction of autophagy in nit-1 cells exposed to as and ps . moreover , this study clearly suggested that autophagy is a cell survival mechanism in as- and ps - mediated cell death , based on the suppression of autophagy caused by baf - a1 . by contrast , the activation of autophagy caused by rapamycin protected the as- and ps - mediated cell death . therefore , our findings suggest that targeting autophagy or parp1-ampk - mtor pathways should be considered in the development of effective statin therapies for diabetes . in conclusion , based on a literature review , this study was the first to demonstrate a novel function of parp1 in the regulation of as- and ps - induced autophagy via the parp1-ampk - mtor signaling pathway and that such autophagy serves as a cell survival mechanism against as - mediated necrosis , although it is insufficient to prevent necrosis - induced cell death ( figure 8) . these findings contribute to the understanding of the complex relationship among statin - related diabetes , autophagy , and cell death .
statins have been widely used drugs for lowering low - density lipoprotein and for preventing heart attack and stroke . however , the increased risk for developing diabetes during extended stain use and the molecular mechanisms remain unclear . 
 the objective of this study was to elucidate the signaling pathway and biological function between necrosis and autophagy induced by atorvastatin ( as ) and pravastatin ( ps ) . 
 here we observed that atorvastatin ( as ) can increase intracellular reactive oxygen species ( ros ) and induce necrotic cell death and autophagy in nit-1 cells , whereas pravastatin ( ps ) does not cause ros and cell death but also induces autophagy . 
 parp1 exhibited a dual role in modulating necrosis and autophagy in as- and ps - treated nit-1 cells through rip1-rip3-mlkl pathway and parp1-ampk - mtor pathway . 
 lastly , as treatment induced mitochondrial morphology injury significantly more than ps treatment did . 
 thus , the parp1 activation should be considered in the development of effective statin therapies for diabetes . 
 future studies may examine specific mechanisms and pathways in mitochondria , autophagy , and oxidative stress in vivo .