procyanidins are the second most abundant natural phenolic after lignin , and they are widely distributed in fruits , berries , beans , nuts , cocoa , and wine .
they participate in glucose homeostasis   and modulate insulin synthesis , secretion , and degradation .
moreover , changes in -cell insulin production may also be due to variations in the number of insulin - producing cells .
-cell mass adapts to increased metabolic demands caused , for example , by obesity , pregnancy , or insulin resistance .
however , when -cells are unable to compensate for increased insulin demand , there is a decrease in -cell mass characteristic of the onset of type 2 diabetes mellitus ( t2 dm ) .
moreover , they protect cells from diverse drug- or chemical - induced toxic assaults by decreasing apoptosis and inducing cell growth . however , there is little information available regarding their effects on -cells
. other studies by our research group have reported that procyanidins modulate proliferation and apoptosis of the pancreatic -cell line ins-1e under altered conditions .
procyanidins also alter the protein and/or gene expression of factors involved in apoptosis in zucker fatty rats .
obesity has become a worldwide problem , leading to an explosion of obesity - related health issues .
obese individuals develop resistance to the cellular actions of insulin , a key etiological factor for t2 dm , which is also becoming an epidemic .
t2 dm is characterised by peripheral insulin resistance as well as pancreatic -cell dysfunction and decreased -cell mass .
these lifestyle changes , involving high - energy diets and reduced physical activity , are linked to the pandemics of obesity and t2 dm . given the high prevalence of the disease , obtaining knowledge about natural compounds with potential beneficial effects on glucose homeostasis is of great interest .  
several animal models have been used to study obesity , including both genetic and diet - induced obesity models . however , the cafeteria diet is a more robust model to reproduce the diet in western society . in a previous study , we analysed the effects of procyanidins in an insulin resistance model induced by cafeteria diet administration in female wistar rats .
we also considered the effects of the cafeteria diet on insulin production and apoptosis in the pancreas .
the study showed that the cafeteria diet increased insulin production as well as activated apoptosis biomarkers .
furthermore , procyanidin administration caused a reduction in the homeostasis model assessment for insulin resistance ( homa - ir ) index , suggesting improved insulin resistance .
moreover , in the pancreas , procyanidins caused a decrease in insulin production   and modulated pro- and antiapoptosis markers .
the pharmacokinetics and pharmacodynamics in females are different from the same parameters in males because of the female 's unique anatomy and physiology [ 16 , 17 ] .
thus , the aim of the present study was to evaluate the effects of gspe in male wistar cafeteria - induced obese rats and to compare the results on insulin synthesis , apoptosis , and proliferation in the pancreas with those observed in the previous study of female rats .
the procyanidin extract was derived from grape seed and contained the following : catechin ( 58  mol / g ) , epicatechin ( 52  mol / g ) , epigallocatechin ( 5.50  mol / g ) , epicatechin gallate ( 89  mol / g ) , epigallocatechin gallate ( 1.40  mol / g ) , dimeric procyanidins ( 250  mol / g ) , trimeric procyanidins ( 15.68  mol / g ) , tetrameric procyanidins ( 8.8  mol / g ) , pentameric procyanidins ( 0.73  mol / g ) , and hexameric procyanidins ( 0.38  mol / g ) .
wistar male rats weighting between 250330  g were purchased from charles river laboratories ( barcelona , spain ) and housed in animal quarters at 22c with a 12  h light / dark cycle .
after 1 week in quarantine , the animals were divided in two groups , a diet - control group ( 7 animals ) fed a standard diet ( panlab a03 ) and a cafeteria group ( 21 animals ) fed a cafeteria diet ( bacon , biscuits with pt , biscuits with cheese , muffins , carrots , and milk with sugar ) in addition to standard chow and water . every day at 9 am , food was withdrawn , and it was replaced at 6 pm .
afterwards , the diet - control group and 7 animals from the cafeteria - fed group were sacrificed , as a reference for the state of the animals before the beginning of treatment .
the rest of the cafeteria - fed rats were divided in two subgroups ( 7 animals / group ) .
these two groups were the ( i ) cafeteria group : rats treated with a vehicle ( gum arabic 5% w / v ) and the ( ii ) gspe - treated group : rats treated with 25  mg of gspe / kg of body weight ( bw ) per day .
the treatment was administrated every evening for 21 days before the replacement of the food .
three days before the end of the treatment and after 8  h of fasting , blood was collected from the tails of the rats to measure glucose and insulin levels . at the end of the treatment regimen and after 3  h of fasting , the animals were anesthetised using sodium pentobarbital ( 50  mg / kg of bw , sigma - aldrich , st .
the pancreas was isolated from all of the animals , frozen immediately in liquid nitrogen , and stored at 80c until analysis .
all of the procedures were approved by the experimental animals ethics committee of the universitat rovira i virgili .  
insulin plasma levels were assayed using an elisa method following the manufacturer 's instructions ( mercodia , uppsala , sweden ) .
glucose plasma levels were determined using an enzymatic colorimetric kit ( qca , amposta , spain ) .  
the homa - ir and homa- were calculated using the fasting values of glucose and insulin and the following formulas : 
 ( 1)homa - ir = insulin  ( u / ml )    glucose  ( mm ) 
22.5,homa-=20insulin  ( u / ml )  glucose  ( mm)3.5 . triglycerides ( tag ) and nonesterified fatty acids ( nefas ) from the pancreas were extracted by homogenising the tissue with pbs containing 0.1% triton x-100 ( sigma - aldrich , st .
louis , mo ) , and their concentrations were determined using enzymatic colorimetric kits ( qca , amposta , spain for tag and wako chemicals gmbh , neuss , germany for nefas ) .
the pancreas was homogenised with six volumes of pbs containing 50  mm edta at ph 7.4 and centrifuged at 3000  g for 5  min at 4c .
reactive oxygen species ( ros ) in the supernatants were quantified using 20  m dcfh - da ( 2,7-dichlorofluorescin diacetate ) ( sigma - aldrich , st .
louis , mo ) , and the fluorescence was measured after 50 minutes at 37c at ex = 485  nm and em = 530  nm .
the values were normalised to the protein content , which was analysed by the bradford method .
total rna from the pancreas was extracted using the rneasy mini kit ( qiagen , barcelona , spain ) .
cdna was generated with the high - capacity cdna reverse transcription kit ( applied biosystems , madrid , spain ) and was subjected to quantitative real - time pcr amplification using the taqman master mix ( applied biosystems , madrid , spain ) .
specific taqman probes ( applied biosystems , madrid , spain ) were used for each gene : rn99999125_m1 for bcl2 , rn01480160_g1 for bax , rn01492401_m1 for ccnd2 ( cyclin d2 ) , rn01774648_g1 for ins , and rn00565544_m1 for cpe .
reactions were run on a quantitative rt - pcr 7300 system ( applied biosystems , madrid , spain ) according to the manufacturer 's instructions .
the protein levels of bax and bcl-2 were quantified by western blot as previously described .
25  g of protein was loaded onto the gel , and the antibody dilution was 1  :  1500 for bax and bcl-2 .
after incubation with peroxidase - conjugated monoclonal anti - rabbit secondary antibody ( sigma - aldrich , madrid , spain ) at a 1  :  10000 dilution , immunoreactive proteins were visualised with the ecl plus western blotting detection system ( ge healthcare , buckinghamshire , uk ) .
chemiluminescence and densitometric analysis of the immunoblots was performed using imagej 1.44p software , and all proteins were quantified relative to the loading control .
we first examined the effects of the cafeteria diet on pancreatic insulin production after 52 days of diet administration .
insulin and glucose plasma levels were quantified at day 49 , and the cafeteria diet - fed rats showed significantly higher plasma insulin levels and no changes in glucose levels ( table 1 ) .
the homa - ir index indicated that the cafeteria - fed animals had peripheral insulin resistance , and their homa- index tended to increase ( table 1 ) .
therefore , there was a tendency to increase pancreatic functionality response to glucose in order to counteract peripheral insulin resistance .
the increased plasma insulin levels agree with an increase in the insulin gene expression , as well as with increased gene expression of carboxypeptidase e ( cpe ) ( table 2 ) .
after induction of obesity via the cafeteria diet , rats were treated with 25  mg / kg of bw gspe for 21 days , concomitantly with cafeteria diet administration .
the animals treated with the procyanidin extract had lower insulinemia and decreased homa - ir and homa- indexes ( table 1 ) , counteracting the effects observed in the cafeteria - fed rats .
moreover , insulin gene expression tended to decrease in these rats , and decreased expression of cpe was also observed ( table 3 ) . to examine the effects of the cafeteria diet and gspe on apoptosis and proliferation in the pancreas
the cafeteria - fed rats showed a decrease in the antiapoptotic marker bcl-2 at both the gene ( table 2 ) and protein levels ( figure 1(a ) ) .
for the pro - apoptotic marker bax , the mrna levels of this gene were not significantly altered ( table 2 ) , but we did observe an increase in the protein levels of bax in the cafeteria - diet - fed group ( figure 1(a ) ) .
therefore , the ratio of bcl-2/bax was reduced both at the gene and protein levels , suggesting an increase in apoptosis in the pancreas ( table 2 and figure 1(a ) ) .  
the administration of gspe had no effect on bcl-2 and bax at gene expression compared to the cafeteria diet ( table 3 ) and on bcl-2 at protein expression ( figure 1(b ) ) .
in contrast , bax protein levels were increased by the gspe treatment , enhancing the effects observed in the cafeteria - fed rats ( figure 1(b ) ) .
the ratio of bcl-2/bax was significantly reduced at protein level ( figure 1(b ) ) compared to the cafeteria - fed animals . finally , we also analysed cyclin d2 , a proliferation marker , but no changes were observed in the cafeteria - fed animals or in the gspe - treated rats ( table 2 ) .
pancreas malfunction is in part due to the accrual of tag in its cells . to measure it , we examined the tag content in this tissue and found that tag triplicated its levels in the pancreas of cafeteria - fed rats compared to the standard - diet - fed rats ( 33.15  2.7 versus 11.42  0.3  g tag / mg tissue , p  0.01 ) .
after 21 days of treatment , the tag contents in the pancreas increased , due to cafeteria diet , but gspe avoided this ( figure 2 ) .  
in contrast , the content of nefas was not modified neither in the cafeteria group compared to the standard - diet - fed rats ( 3.37  0.9 versus 4.40  0.4  g nefa / mg tissue ) nor in the gspe - treated rats compared to the vehicle - treated rats ( 2.43  0.3 versus 10.29  4.6  g nefa / mg tissue ) .  
ros content in the pancreas was also analysed , and no significant differences were observed in either the cafeteria - fed rats or in the gspe - treated animals ( figure 3 ) .
this study was designed to examine the effects of the cafeteria diet on insulin production in male wistar rats by evaluating pancreas functionality , apoptosis , and proliferation .
we have also evaluated the effects of procyanidins on these processes , since procyanidins were shown to have positive effects on glucose metabolism under conditions of slightly disrupted homeostasis .
we had previously shown that 17 weeks of a cafeteria diet led to insulin resistance , high plasma insulin levels , and increased insulin synthesis and secretion in female wistar rats .
it has been reported that female rats are more sensitive to cafeteria - induced obesity than males [ 20 , 21 ] . however , with respect to insulin resistance , we now show that 52 days of cafeteria diet ( nearly 7 and a half weeks ) administrated to male wistar rats confirms the effects observed in the previous study of female rats . in the males ,
we have observed high insulin plasma levels and elevated homa - ir index , indicating peripheral insulin resistance .
additionally , we have seen an elevated homa- index , which indicates an increase in pancreatic functionality in terms of glucose response to counteract peripheral insulin resistance . we have also found an increase in the expression of the insulin gene in the cafeteria - fed rats and an increase in the gene expression of cpe .
cpe is the enzyme thought to be involved in the cleavage of proinsulin , which results in insulin and c - peptide molecules .
therefore , the pancreas of cafeteria diet - fed rats is still functional , and it tries to counteract peripheral insulin resistance despite the increased lipid accumulation in the pancreas .  
increased deposits of fat are associated with obesity and lead to an increase in free fatty acids ( ffas ) .
the induction of apoptosis in in vivo high - fat diet models and in vitro models of ffa - induced apoptosis is important evidence for -cell lipotoxicity . in mice ,
administration of a high - fat diet for 12 weeks led to increased -cell mass , despite showing an increase in -cell apoptosis . in our study , we have found a decrease in the bcl-2/bax ratio both at the gene and protein levels in the pancreas of the cafeteria - fed rats compared to the standard chow - fed rats which also suggest an increase in apoptosis in the cafeteria - fed animals .
palmitate was reported to induce -cell endoplasmic reticulum stress and death mediated by cpe degradation [ 26 , 27 ] .
we have found that cpe is not decreased but increased by the cafeteria diet , suggesting that the apoptosis observed in the cafeteria - fed rats is not mediated by lipotoxicity .
in fact , the levels of nefa in the pancreas are not altered in the cafeteria - fed rats when compared to the standard - diet - fed rats .
the results from the apoptosis markers are in accordance with the data obtained in the previous study which evaluated the cafeteria diet in wistar female rats .
we have also analyzed the expression of the proliferation marker cyclin d2 and found no changes due to the cafeteria treatment suggesting that at the time of the analysis , -cell mass had likely already increased .
this also agrees with the pervious results in females , as well as with those found in rats fed high fat diets , in which no changes in ki67 ( a proliferation marker ) expression were observed .  
taken together , the data suggests that the effects of the cafeteria diet on insulin synthesis , secretion , and apoptosis are not influenced by gender or treatment duration .  
once the effects of the cafeteria have been established , we have analyzed the effects of a gspe treatment on the cafeteria - fed animals .
after 52 days of cafeteria diet administration , male wistar rats have been treated with 25  mg / kg of gspe for 21 days concomitant with the cafeteria diet .
the gspe treatment has decreased insulin production , plasma insulin levels , and pancreatic insulin cpe gene expression compared to the cafeteria - vehicle - fed rats . moreover , the decreased insulin production could be at least in part explained through gspe 's lipid - lowering effect , since the triglyceride content has also been reduced in the pancreas of gspe - treated rats .
previously , we showed that 25  mg of gspe / kg of bw administered to female wistar rats for 30 days resulted in decreased insulin production and reduced triglyceride content in the pancreas , likely via decreased fatty acid synthesis and increased -oxidation . despite this lower insulinemia , gspe improved glycemia in female wistar cafeteria - fed rats acting peripherally on adipose tissue .
present results reinforce the effects of gspe decreasing insulin production and ameliorating lipid accumulation in cafeteria - fed rats and support that these effects are not gender dependent .  
gspe has counteracted the effects of the cafeteria diet reducing the accumulation of triglycerides in the pancreas but has not counteracted the cafeteria - diet effects on the apoptosis markers . instead , gspe - treated rats have shown an increase in bax protein levels and a decreased ratio of bcl-2/bax , suggesting an enhancement in the apoptosis .
therefore , the lipid - lowering effects of gspe do not involve a reduction in apoptosis in the pancreas of cafeteria - fed male rats .
in fact , gspe increases glucose uptake in -cells under high - glucose conditions and impairs mitochondrial and cellular membrane potentials .
moreover , gspe is reported to enhance the pro - apoptotic effects of high glucose in vitro .
therefore , the enhanced apoptosis observed in the gspe - treated rats could be due to increased glucose uptake in the -cells that potentiate glucotoxicity .
ros is one of the players in glucose - induced apoptosis in -cells ; ros is increased as a consequence of chronically increased glucose metabolism .
-cells have relatively low expression of antioxidant enzymes and are more sensitive to ros attack when they are exposed to oxidative stress .
however , the levels of ros in the pancreas have not been modified by the cafeteria diet or by gspe treatment , suggesting that glucose toxicity could be mediated by another mechanism .   the apoptosis marker results conflict with those in the previous study of female wistar rats , which showed that 25 and 50  mg / kg of gspe seemed to counteract the deleterious effects of the cafeteria diet by inhibiting the down - regulation of bcl-2 protein expression after 10 and 30 days of treatment .
in addition , 50  mg / kg bw of gspe also counteracted the decrease in the bcl-2/bax ratio at the protein level after 10 days of administration .
however , no gspe effects were observed with respect to the ratio of bcl-2/bax gene expression at any dose or treatment duration .
therefore , the modulation of apoptosis biomarkers by gspe in cafeteria - fed rats is clearly dependent on the dose and treatment period ; these effects may also be dependent on gender . in conclusion
, the present study has confirmed that the cafeteria model is a suitable reproduction of the prediabetic state .
this model induces an insulin resistance state , shows increased insulin synthesis and secretion , and exhibits increased apoptosis in the pancreas .
moreover , gspe treatment in male rats treated with 25  mg / kg of gspe for 21 days improves the insulin resistance state and counteracts the cafeteria - induced effects on insulin synthesis .
however , procyanidins enhance the elevated levels of bax , a pro - apoptotic protein observed in the cafeteria - fed rats , potentially suggesting an increase in apoptosis .
this result indicates that the effects of gspe on apoptosis markers are dose , time , and/or gender dependent .
 in a previous study , the administration of a grape seed procyanidin extract ( gspe ) in female wistar rats improved insulin resistance , reduced insulin production , and modulated apoptosis biomarkers in the pancreas . considering that pharmacokinetic and pharmacodynamic parameters in females are different from these parameters in males , the aim of the present study was to evaluate the effects of gspe on male wistar cafeteria - induced obese rats . 
 the results have confirmed that the cafeteria model is a robust model mimicking a prediabetic state , as these rats display insulin resistance , increased insulin synthesis and secretion , and increased apoptosis in the pancreas . 
 in addition , gspe treatment ( 25  mg / kg of gspe for 21 days ) in male rats improves insulin resistance and counteracts the cafeteria - induced effects on insulin synthesis . 
 however , the administration of the extract enhances the cafeteria - induced increase in bax protein levels , suggesting increased apoptosis . 
 this result contradicts previous results from cafeteria - fed female rats , in which gspe seemed to counteract the increased apoptosis induced by the cafeteria diet . 
 
1. Introduction 
2. Materials and Methods
3. Results
4. Discussion