type 1 diabetes mellitus ( t1 dm ) is characterized by a loss of beta cell mass due to an autoimmune process . in a phase prior to the onset , immune cells infiltrate the islets creating an inflammatory microenvironment responsible for the -cell - specific toxicity .
proinflammatory cytokines , such as interleukin-1 beta ( il-1 ) , tumor necrosis factor alpha ( tnf- ) , and interferon gamma ( ifn- ) , secreted by activated lymphocytes and macrophages during insulitis , induce nfkb mediated inos expression which has a key role in -cell apoptosis in the early t1 dm stage .
it is produced in tissues by the enzyme nitric oxide synthase ( nos ) by oxidation of the amino acid l - arginine .
there are three different tissue - specific nos isoforms : ca / calmodulin - independent and inducible nos ( inos ) , neuronal nos ( nnos ) , and endothelial nos ( enos ) .
these last two isoforms also exhibit constitutive expression and ca / calmodulin - dependent activity .  
expression of nos isoforms has been studied in pancreatic islets , and the nnos form has been observed in -cell cells , located , mainly , in insulin secretory granules .
it exhibits cytochrome c reductase activity in addition to no production activity , and a balance between both functions is essential for a normal insulin secretion in response to glucose stimulation . in contrast , enos expression has not been found in rat islets under basal conditions [ 2 , 3 ] .  
prooxidant agents formed from no induce damages in dna which activate p53 which induces proapoptotic expression of genes such as bax , fas , noxa , and puma .
a p53-independent mechanism has been described in which no acts by inhibiting the sarcoendoplasmic reticulum pump ca atpase 2b ( serca2b ) protein and , subsequently , by inducing the endoplasmic reticulum stress mechanisms [ 4 , 5 ] .
the importance of the mitochondrial pathway has also been highlighted in no - mediated -cell cell apoptosis [ 6 , 7 ] .
paradoxically , an antiapoptotic effect of low concentration of no has been reported in several systems , including  cells [ 812 ] .  
in addition to apoptosis , cell proliferation has been described as a no - regulated process . although certain activating effects have been reported in physiological systems [ 13 , 14 ] , the main role of no in cellular proliferation is inhibitory . in the subventricular zone , no induces inhibition of stem cell proliferation by a nitrosylation process .
other proposed mechanism for no antiproliferative action is a g1-s inhibition mediated by an induction of the cell cycle inhibitor p21   or cyclins inhibition .
recently , no - mediated neogenesis stimulation has been observed in an alloxan - induced murine model of diabetes . a proinflammatory cytokine - mediated inhibition of cultured -cell proliferation in addition to the apoptotic effect has been observed by our group . as described previously , no production is one of the most important events mediated by proinflammatory cytokines .
therefore , the aim of this study was to assess the importance of no in the cytokine - mediated antiproliferative effect on cultured  cells and to determine the role(s ) of different no synthase isoforms present in pancreatic islets .
all animal procedures were performed with the approval of the cdiz university school of medicine ( cdiz , spain ) committee for the ethical use and care of experimental animals .
bio - breeding ( bb ) and wistar rats were kept under conventional conditions in an environment - controlled room ( 20 - 21c , 12  h light - dark cycle ) with water and standard laboratory rat chow available ad libitum and their weight was daily recovered .
blood extracted from the tail vein was used in bb rats for weekly random glucose measurements using an automatic glucose monitor ( accu - chek optimum , roche diagnostic , basel , switzerland ) .
pancreatic islets were isolated from adult male wistar rats as described previously by mcdaniel et al . .
louis , mo , usa ) supplemented with 2  mm l - glutamine ( gibco invitrogen , carlsbad , ca , usa ) , 100  u / ml penicillin , 100  g / ml streptomycin ( pen - strep , bio - whittaker europe , verviers , belgium ) , and 10% fetal bovine serum ( fbs , gibco invitrogen , carlsbad , ca , usa ) .
proinflammatory cytokines ( peprotech ec ltd , london , uk ) used in the experiments were recombinant human il-1 ( 50  u / ml ) , recombinant rat ifn- ( 1000  u / ml ) , and recombinant rat tnf- ( 1000  u / ml ) .
these concentrations were selected as being appropriate based on the results of previous published studies [ 20 , 21 ] .
no donors , s - nitroso - n - acetyl - dl - penicillamine ( snap ) , and diethylenetriamine / nitric oxide adduct ( deta - no ) , obtained from sigma - aldrich ( st . louis , mo , usa ) show different no release rates .
deta - no is a member of the nonoates family and has a half - life ( t1/2 ) of 20  h at 37c , ph 7.4 .
the t1/2 of snap is approximately 6  h at 37c and ph 68 although its biological activity is highly influenced by the molecular environment of the parent thiol .
inhibitors of nos 7-nitroindazole ( 7-ni ) , n-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide , dihydrochloride ( 1400  w ) , and n5-(1-iminoethyl)-l - ornithine , dihydrochloride ( l - nio ) , purchased from sigma - aldrich ( st . louis , mo , usa ) were used to inhibit the activity of neuronal , inducible , and endothelial isoforms of nos , respectively .
proliferating  cells were detected using 5-bromo-2-deoxyuridine ( brdu ) 5  mol / l label ( sigma - aldrich , st .
louis , mo , usa ) which was added to the cultures from the start of the assay together with the drugs being tested , when appropriate . following the appropriate culture duration ,
the islets were recovered and incubated for 15  min with trypsin - edta ( 0.25% trypsin , 1  mm edta ) in hanks ' balanced salt solution without ca and mg ( gibco invitrogen , carlsbad , ca , usa ) at 37c , and the islets were gently dispersed . after washing with pbs , cells were cytospin on poly - l - lysine - coated slides and fixed in 4% methanol - free formaldehyde
. slides were immuno - stained using monoclonal mouse anti - brdu ( dako cytomation , denmark ) and polyclonal guinea pig anti - insulin ( sigma - aldrich , st .
cells were permeabilized by incubation for 30  min with 0.1% triton - x100 in pbs and washed twice with 100  mm glycine buffer containing 0.1% triton - x100 and 3% bovine serum albumin .
cells were then treated with hcl ( 2n ) in pbs for 30  min , neutralized with borax / borate buffer ( 0.1  m , ph 8.9 ) for 30  min , washed , and incubated overnight at 4c with anti - brdu and anti - insulin antibodies .
stained cells were revealed using anti - mouse igg antibody ( alexa-546 conjugated ) and anti - guinea pig igg ( alexa-488 conjugated ) antibody ( molecular probes inc , eugene , or , usa ) .
cell nuclei were stained with 4-6-diamidino-2-phenylindole ( dapi ) . to determine the proliferating fraction ,
total and insulin - positive / brdu - positive cells were analyzed using a fluorescence microscope in randomized conditions , by a single investigator . for statistical purposes ,
48  h cultured islets in presence and absence of cytokines were processed for histology study by histogel and paraffin inclusion .
10  m sections were stained using a polyclonal rabbit anti - inos ( ab15323 , abcam , cambridge , uk ) and a polyclonal guinea pig anti - insulin ( sigma - aldrich , st .
louis , mo ) and examined using a microscope equipped with a digital camera and the image analysis celldsoftware ( olympus , hamburg , germany ) . randomly grouped bb rats daily received an intraperitoneal injection of vehicle ( 0.1% dmso diluted in injection water ) alone or containing l - nmma at a dose of 30  mg / kg , from 4 to 9 weeks of age .
animals were sacrificed at 4 ( before treatment ) , 7 , and 9 weeks of age .
wistar , bb , and thymectomized bb rats were fasted overnight ( 1618  h ) and a blood sample was collected from the tail vein ( fasting or 0  min sample ) .
then , an intraperitoneal injection of 40% solution of glucose was administered ( 2  g / kg ) , followed by blood sampling at 15 , 30 , 60 , and 120  min after the glucose administration .
glycemia was measured with an automatic glucose monitor ( accu - chek optimum , roche diagnostic , basel , switzerland ) .
blood samples obtained from fasting and 15  min after glucose administration were also used to plasma extraction and insulin quantification by elisa technique ( insulin rat ultrasensitive kit .
weeks of age bb rat pancreas were resected , weighed , fixed in bouin 's solution for 6  h , and postfixed in formalin for 12  h. then , they were dehydrated , paraffin embedded , and longitudinal 10  m microtome sections were obtained .
insulin was stained in obtained pancreas by immunohistochemical techniques using a mouse anti - rat insulin monoclonal antibody and a peroxidase conjugated goat anti - mouse igg antibody and revealed with dab kit ( sigma aldrich , st .
insulin - positive areas were quantified in two complete sections of each pancreas using a microscope equipped with a digital camera and the image analysis cell d software ( olympus , hamburg , germany ) .
-cell mass values were calculated by multiplying the total insulin - positive area / total pancreatic area ratio by the total pancreas weight .
equivalent numbers of islets under the different experimental conditions described previously were lysed in lysis buffer ( 125  mm tris - hcl buffer ph 68 , 2% sds , 1  mm dtt and containing protease and phosphatase inhibitors ) .
protein ( 40  g ) was loaded and electrophoresed on 8% sds - page .
proteins were transfer - blotted on to polyvinylidene fluoride ( pdfv ) membranes and then incubated in blocking buffer ( 5% nonfat milk in 10  mm tris - hcl , 1.15  m nacl and 0.1% tween-20 ) for 1  h at room temperature .
the blots were then incubated with rabbit polyclonal antibody against enos ( santa cruz biotechnology , santa cruz ca , usa ) and inos ( transduction laboratories bd , nj , usa ) overnight according to the manufacturer 's instructions , followed by incubation with peroxidase conjugated anti - rabbit igg for 1  h at room temperature .
stained bands were revealed using immun - star western c kit ( bio - rad , hercules ca , usa ) and quantified by quantity one software version upgrade ( bio - rad , hercules , ca , usa ) .
-tubulin expression was used as loading control and enos expression was calculated as ratio of enos : -tubulin densities .
histological examination of 7 and 9 weeks of age rat pancreatic islets was performed in harris ' h&e stained pancreas sections using 20 objective lens .
the severity of insulitis was graded as a function of the mononuclear cell infiltration of the pancreatic islets : 0 = no infiltrate ; 1 = periductular infiltrate ; 2 = periislet infiltrate ; 3 = intraislet infiltrate ; 4 = intraislet infiltrate associated with -beta cell destruction .
thirty islets were examined in each pancreas and the mean score was calculated by dividing the total score by the number of islets examined .  
results are presented as means  sem of measurements performed in at least 3 animals .
to determine the role of no donors on -cell proliferation , pancreatic islets were treated with proinflammatory cytokines or different concentrations of no donors snap and deta , alone or in combination with the caspase-3 inhibitor z - vad - fmk .
-cell proliferation measured by brdu incorporation showed that the no donors , deta - no ( figure 1(a ) ) and snap ( figure 1(b ) ) exert an antiproliferative action on  cells in a dose - dependent manner .
this effect of no donors was not modified by addition of z - vad - fmk to the cultures . to determine the contribution of no to the antiproliferative effect of proinflammatory cytokines on pancreatic  cells
, pancreatic islets were cultured over a 48  h period and treated with proinflammatory cytokines , alone or in the presence of l - nmma ( an inhibitor of nitric oxide synthase ) .
inhibition of -cell proliferation induced by proinflammatory cytokines was completely abolished by l - nmma treatment ( figure 2 ) .
guanylate cyclase is the enzyme through which no exerts the greater part of its effect . to assess the implication of the guanylate cyclase pathway in the no effect on -cell proliferation
, cultures of pancreatic islet were treated for 48  h with 8br - cgmp ( a guanylate cyclase analogue ) and its inhibitor odq , alone or in combination with proinflammatory cytokines . as shown in figure 3 ,
the cgmp analogue exerts no effect on -cell proliferation , and the antiproliferative effect of cytokines is not altered by the cgmp inhibitor odq .   to study the role of constitutive and inducible nos isoforms in the antiproliferative action of cytokines , cultured pancreatic islets were treated with proinflammatory cytokines in addition to 1400  w and 7-ni , inhibitors of inducible and constitutive isoforms of nitric oxide synthase , respectively ; the resultant -cell proliferation was quantified .
the antiproliferative effect of proinflammatory cytokines underwent no modification when islet cultures were incubated with the constitutive nos inhibitor ( figure 4(e ) ) .
related to inos , cytokine - dependent induction of protein was observed from 24  h of culture ( figure 4(a ) ) .
inos expression study by immunohistochemical techniques in control cultured pancreatic islets showed a scarce stain with peripheral profile .
however , cultured islet in presence of cytokines showed an increased inos expression localized internally in the islet ( figure 4(b ) ) . the specific inhibitor 1400  w partially reverted cytokine - induced decreased proliferation in a dose - dependent manner ( figure 4(d ) ) .
endothelial isoform of nos expression was initially examined in pancreatic islets cultured under control condition , or after stimulation with proinflammatory cytokines . as shown in figure 4(c ) , enos isoform was expressed in pancreatic islets and was not altered by exposure to proinflammatory cytokines . to study the role of enos on the antiproliferative effect of cytokines , l - nio ( an inhibitor with higher affinity for enos than the other isoforms )
a partial inhibition of the antiproliferative effect of proinflammatory cytokines was observed in treated cultures ( figure 4(f ) ) mediated by l - nio .
weight and random glycemia monitoring along treatment period show no differences in those parameters between control and l - nmma - treated animals .
glucose homeostasis was studied by ipgtt and insulin determination before and 15  min after intraperitoneal glucose administration . as shown in figure 5
, ipgtt displays a normal curve at studied times in l - nmma treated as in control animals ( figure 6(a ) ) .
in addition , insulin quantification also shows no changes between control and treated animals at none of studied times ( figure 6(b ) ) .
bb rats treated with l - nmma from 4 to 7 or 9 weeks of age were sacrificed and pancreas was removed from each rat to quantify beta cell mass by immunohistological techniques . in figure 7 , a beta cell mass loss undergone by bb rats between 7 and 9 weeks of age can be observed .
l - nmma reverts this effect at both studied times . to test the effect of l - nmma treatment on infiltration levels ,
h&e - stained sections obtained from treated and control bb rats were evaluated and infiltration scores were calculated .
no changes were observed in infiltration level in response to l - nmma at none of studied times ( figure 8) .
currently , no is considered an important mediator of cell signaling via its classical guanylate cyclase pathway or by exerting posttranslational modifications on an increasing number of proteins and hence regulating their activity . in the present study
, we analyzed the role of no in the previously described antiproliferative action of proinflammatory cytokines on pancreatic  cells .
loss of membrane integrity and cytochrome c release induced by deta - no and snap in insulin producing cell lines has been reported [ 22 , 23 ] . in our system , no donors snap and deta - no induced a decrease in cultured -cell proliferation at concentrations up to 50  m for deta - no and up to 400  m for snap .
this effect is dose dependent and similar to that obtained with proinflammatory cytokines ( il-1  +  tnf-  +  ifn- ) treatment ( figure 1 ) . the antiproliferative effect observed in response to no donors
is not dependent on the biological t1/2 and suggests that no exerts its biological action in the first hours after exposure . to discard the possibility that the decrease in proliferation was due to a cytokine - induced apoptosis in proliferating  cell , we treated pancreatic -cell cultures with the caspase-3 inhibitor zvadfmk , in addition to the no donors .
-cell apoptosis inhibition induced by zvadfmk exerts no effect on the antiproliferative effect of no donors suggesting that the two processes are unrelated .
although studies in neonatal rat islets indicated that cytokine - induced no production regulates the expression of up to 42 proteins , a considerable number of proteins are regulated in a no - independent manner .
thus , the cytokine - induced apoptosis in mouse  cells is not caused by the inducible isoform of nos alone .
endoplasmic reticulum stress has been described as a possible mechanism by which no induces apoptosis .
its presence regulates unfolded protein response ( upr ) signaling pathway that determines apoptotic response to reticulum stress .
in addition to cytokines , there is evidence of no participation in mitochondrial dysfunction and cell death occurring in pc12 cells in response to amyloid  .  
results from our study suggest that , in addition to apoptosis , no can be implicated in the antiproliferative effect of proinflammatory cytokines on pancreatic  cell since treatment with nos inhibitor l - nmma is capable of reverting this cytokine - induced effect ( figure 2 ) .
very few studies have investigated the possible mechanisms underlying the effect of no on cellular proliferation . in pulmonary microvascular
smooth muscle cells , exogenous no upregulates p21 , a protein which mediates cell cycle arrest .
further , no is capable of inhibiting erk 1/2 and akt activation in breast cancer cell lines and pancreatic  cells [ 29 , 30 ] .
these results are in accord with those observed by our group ; that is , proinflammatory cytokines induced an inhibition of erk 1/2 in islet cultures   and in early stages of insulitis .
however , no has also been described as an inducer of proliferation , when acting at low concentrations .
for example , in mesangial cells , the no donor snap increases proliferation at concentrations of up to 200  m and inhibits proliferation at higher concentrations , in a process implying akt signaling and cox-2 protein .
activation of no - sensitive guanylyl cyclase by no binding to the enzyme 's prosthetic heme group has been described as one of the most important no - mediated cellular effects .
nevertheless , results observed in our system suggest that this pathway is not involved in the no - mediated antiproliferative effect observed in  cells from islets treated with proinflammatory cytokines ( figure 4 ) .
these results are in accord with those previously reported in tumor cell lines in which the proliferative effect of low no concentration is mediated by the cgmp pathway , but the antiproliferative effect induced by high concentrations of no is independent of this pathway .
a proposed cgmp - independent mechanism of no action is its direct interaction with protein and , as such , performing posttranslational modifications such as nitration or s - nitrosylation .
for example , s - nitrosylation of egf receptor has been reported in human neuroblastoma nb69 as a mechanism of no - mediated antiproliferative effect .
the role of nos isoforms in no production in response to proinflammatory cytokines has not been well defined .
our results show that inos , the expression of which is induced in our system after 24  h culture in the presence of proinflammatory cytokines , is implicated while constitutive isoform is not implicated in cytokine - induced antiproliferative response in  cells ( figure 4 ) . of note
is that although inos inhibitor 1400  w inhibited the cytokine - mediated antiproliferative effect , its effect was only partial , and was unlike the total reversion by l - nmma ( a general nos inhibitor ) .
it is also interesting the effect of 1400  w increasing beta cell proliferation in absence of cytokines which could be due to a certain level of islet cells inos activation induced by islet isolation process .
this partial effect of 1400  w suggests that other nos isoforms are participating in antiproliferative response to cytokines .
endothelial nos determination by western blotting was performed in cultured rat islets in the presence of proinflammatory cytokines and , contrary to the results reported by lajoix et al . , enos expression was observed .
this expression is maintained in culture and was not affected by the presence of cytokines ( figure 4(b ) ) and could be attributed to endothelial cells in the profuse vascular network present in pancreatic islets .
once the presence of enos in islets is confirmed , its implication in -cell antiproliferative response to cytokines was tested in islet cultures treated with l - nio   ( an inhibitor whose primary target is enos ) alone or in combination with cytokines .
l - nio induced a partial reversion of cytokine - induced -cell antiproliferation in a similar manner to that induced by the inos inhibitor 1400  w. these results suggest participation of both no isoforms in the antiproliferative response induced by proinflammatory cytokines , possibly by enos producing no in a paracrine manner . in this sense , it has recently reported that inflammatory environment induces changes in enos which may adopt functional features of inos in a ca - independent way . to assess no involvement in the pathophysiology of t1 dm
, we treated biobreeding rats , an animal model of autoimmune diabetes , with the general nos inhibitor l - nmma at early stages of the insulitis process .
previous observations from our group indicated a halting of -cell proliferation between 4 and 7 weeks of age and which can be reversed by administering neutralizing antibody against ifn- .
treatment with l - nmma of bb rats from 5 weeks of age induced a reversal of -cell proliferation loss observed in untreated animals ( figure 5 ) .
in addition , a relative recovery of -cell area with a regional pattern in treated animals at 7 weeks of age was observed .
the effects , either on proliferation of  cell or on -cell area , were very much more evident in the head than in the tail of the pancreas .
a similar regional pattern of response was reported in earlier studies using a malnutrition model . a plausible explanation for this regional effect could be a different vascularization in the head area compared to that in the tail which precluded equivalent drug availability in all areas of the pancreas volume .
further , a different response to environmental factors due to a different islet composition could underlie this observed effect .
t1 dm prevention strategies focus , essentially , on early stages of the disease before clinical onset when there is enough -cell mass in the pancreas to maintain its metabolic function .
our present study highlights the importance of no as a mediator of the observed impaired -cell proliferative response from the initial stages of insulitis .
this impairment in proliferative capacity precludes an appropriate regenerative response of  cells to autoimmune damage .
our data also highlight the property of l - nmma to maintain -cell proliferative capacity in early insulitis stage and , as such , confirms that nitric oxide might be an important therapeutic target in the strategies of t1 dm prevention .
 nitric oxide ( no ) is involved in several biological processes . in type 1 diabetes mellitus ( t1 dm ) 
 , proinflammatory cytokines activate an inducible isoform of nos ( inos ) in  cells , thus increasing no levels and inducing apoptosis . 
 the aim of the current study is to determine the role of no ( 1 ) in the antiproliferative effect of proinflammatory cytokines il-1 , ifn- , and tnf- on cultured islet  cells and ( 2 ) during the insulitis stage prior to diabetes onset using the biobreeding ( bb ) rat strain as t1 dm model . 
 our results indicate that no donors exert an antiproliferative effect on  cell obtained from cultured pancreatic islets , similar to that induced by proinflammatory cytokines . 
 this cytokine - induced antiproliferative effect can be reversed by l - nmma , a general nos inhibitor , and is independent of guanylate cyclase pathway . 
 assays using nos isoform specific inhibitors suggest that the no implicated in the antiproliferative effect of proinflammatory cytokines is produced by inducible nos , although not in an exclusive way . in bb rats , early treatment with l - nmma improves the initial stage of insulitis . 
 we conclude that no is an important mediator of antiproliferative effect induced by proinflammatory cytokines on cultured  cell and is implicated in -cell proliferation impairment observed early from initial stage of insulitis . 
1. Introduction
2. Methods
3. Results
4. Discussion