functional foods containing vitamins and/or antioxidants in addition to other substances are used to treat metabolic disorders and have been shown to mitigate metabolic syndrome progression [ 1 , 2 ] . there is evidence supporting a role for foods containing essential polyunsaturated fatty acids ( pufas ) , especially omega-3 ( n-3 ) fatty acids , in preventing cardiometabolic diseases . the western diet is known to contain high amounts of omega-6 ( n-6 ) fatty acids and low levels of n-3 fatty acids . high doses of n-6 fatty acids in the diet can cause metabolic dysfunction , induce the onset of obesity , and potentially induce cardiometabolic diseases [ 4 , 5 ] . diets with ratio of n-6/n-3 fatty acids close to 1  :  1 offered during pregnancy and lactation reportedly have beneficial effects on early brain development in the mice offspring . interestingly , disruption of the brain hypothalamus areas controlling body mass and energy metabolism is strongly involved in inducing several metabolic diseases in animal model . considering that metabolic dysfunctions have one of its possible origins in early life [ 8 , 9 ] , in the current work , we focus on the role of maternal diet enriched with essential pufas as a preventive tool against the metabolic syndrome . among other obesity - models , the obesity induced by neonatal treatment with monosodium l - glutamate ( msg ) is an interesting tool to study the effects of obesity and diabetic condition on different metabolic parameters . this model is characterized by hyperinsulinemia and higher insulin secretion associated with early hyperglycemia in msg - treated mice [ 1012 ] . msg - induced obesity that is caused in neonatal rodents by partial destruction of the hypothalamus brain area which concentrates neuronal nuclei controlling body weight and energy metabolism has been long reported [ 13 , 14 ] . all these dysfunctions in this model are essential to make the msg - obese and prediabetic mice a good model to study metabolic diseases and its associated disturbances . using msg - induced obesity in mice , in the present study we tested the hypothesis that perinatal diet supplemented with a mixture of n-6/n-3 fatty acids in a ratio of 1.2  : 1.0 supplied to lean dams , throughout pregnancy and lactation , can attenuate the onset of obesity in offspring treated with msg during the first 5 days of life . in agreement with that , we assessed in the mice adult offspring the body composition and patterns regarding obesity and their related - complications such as impaired glucose homeostasis and pancreatic islets dysfunction . the ethical committee for animal experiments at the state university of maringa , which adheres to the brazilian federal law , approved this protocol ( protocol number 084/2009 ) . virgin female mice aged 70 days with regular cycles were fed a commercial diet ( nuvital , curitiba , pr , brazil ) and maintained under controlled lighting and temperature ( lights on from 07:00 to 19:00  h with temperature maintained at 23  2c ) for acclimation . after that , virgin female mice were mated with proven male breeders and then the vaginal smear washed with saline solution ( nacl , 0.9% , w / v ) was collected to evaluate the presence of spermatozoa to mark the first day of conception . after impregnation , females were kept in individual plastic cages and assigned to one of the two dietary treatments . a batch of mice ( n = 10 ) received a diet containing n-6 essential fatty acids from sunflower oil supplemented with flaxseed powder as source of n-3 fatty acids to reach an n-6/n-3 fatty acid ratio of 1.2  :  1.0 ( flax - diet , table 1 and table s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/9242319 ) throughout pregnancy and lactation . while control mice ( n = 10 ) were fed a commercial diet ( com - diet , nuvital , curitiba , pr , brazil ) throughout pregnancy and lactation . the composition of diets as well as the mixtures of salts and vitamins follows the recommendations of the ain93 g . upon delivery , newborn mice from both dam groups ( flax - diet , n = 5 litters , and com - diet , n = 5 litters ) were subcutaneously injected with msg ( 4  mg  g body weight once a day ) in the cervical region within the first 5 days of life , while the other half of litters ( n = 5 ) from each dam group was injected with an equimolar amount of saline solution . the male mice were kept , and all females were discarded at weaning ( 21st day ) . thus , the number of 24 male mice was used as a standard number for each experimental offspring group , where a number between four and five mice , from each litter , were used . indeed , as recommended , we used the number of litters ( n = 5 ) for the statistical calculation in the present study . msg - treated offspring were divided into two groups : mice from mothers that received the flaxseed powder supplemented diet ( msg - flax ; n = 5 ) and mice from mothers fed the commercial diet ( msg - com ; n = 5 ) . the control offspring were divided into two additional groups : mice from mothers fed the flaxseed powder diet ( control - flax ; n = 5 ) and mice from mothers fed the commercial diet ( control - com ; n = 5 ) . after weaning , the mice offspring were kept under controlled conditions with a temperature of 23  2c , a light cycle between 07:00 and 19:00  h , and water and commercial food ad libitum . the commercial diet contained n-6/n-3 fatty acids in a ratio of 10.7  :  1.0 , while the sunflower oil diet supplemented with golden flaxseed flour used in this study contained n-6/n-3 fatty acids in a ratio of 1.2  :  1.0 ( table 2 ) . total lipids extraction and fatty acids methyl esters preparation were performed as described previously [ 17 , 18 ] . the quantification of essential pufas in both com - diet and flax - diet was performed by gas chromatography ( thermo scientific 3300 , waltham , ma , usa ) fitted with a flame ionization detector and a fused - silica cp-7420 capillary column ( 100  m  0.25  mm i.d . the operating parameters were 220c temperature injection port , 240c temperature detector , and 165c column temperature for 12  min , which was programmed to increase at 40c per min up to 180c ( hold time of 15  min , being raised to 240c at 15c per min with final holding time of 18.6  min ) . the carrier gas was hydrogen at 1.4  ml  min ; the nitrogen was used as the makeup gas at 30  ml min , with split injection at 1  :  80 ratio . to evaluate the incorporation of pufas in the brain and pancreatic tissues from weaned mice offspring , a batch of 21-day - old mice from all experimental groups ( n = 5 per group ) were sacrificed and brain and pancreas removed for lipids extraction . lipid extraction was performed by the bligh and dyer method , as described above ; thus , a volume of 500  l chloroform and 1000  l methanol were added per each 500  mg of tissue and the homogenate was subjected to posterior sonication ( three times , 30  s pulses , using a sonic dismembrator model 100 ; fisher scientific ) . after the lipid extraction and fatty acids methyl esters preparation , the quantification of essential fatty acids incorporated in the brain and pancreas tissues was performed in three replicates by gas chromatography , as described above . the quantification of fatty acids incorporation in the tissues ( brain and pancreas ) is depicted as g of fatty acids per mg of tissue . it was made against a c23:0 as internal standard purchased from sigma - aldrich ( sigma - aldrich , st . the ipgtt was performed by intraperitoneally injecting glucose ( 2  g  kg ) into overnight - fasted mice . the number is 10 mice from 5 different litters ( n = 5 for each group ) . the blood glucose levels were determined prior ( 0 ) to injection and 30 , 60 , 90 , and 120  min after injection . all blood samples were obtained from the tail vein , and the plasma was used to measure the glucose concentration by the glucose oxidase method . the total area under the curve ( auc ) for the ipgtt was calculated as previously reported . in another batch of mice ( n = 5 for each group ) , the ipitt was performed by intraperitoneally injecting insulin ( 1  ui  kg body weight ) into the mice . the blood glucose levels were determined prior ( 0 ) to injection and 5 , 15 , 30 , and 45  min after injection . all blood samples were obtained and the plasma glucose concentration was measured as described above . the constant rate for plasma glucose disappearance ( kitt ) was calculated using the formula 0.693(t1/2 ) . the plasma glucose half - life ( t1/2 ) was calculated from the slope of the least squares analysis of the plasma glucose concentrations during the linear phase of decline after insulin injection in fed mice , as previously reported . pancreatic islets were isolated using a collagenase technique as described previously , where three milliliters of hanks buffered saline solution was injected into the mouse 's common bile duct . eight mice from at least 4 different litters were used for each experimental procedure per group ( n = 4 ) . to adapt the isolated islets to a baseline glucose concentration ( 5.6  mmol  l ) , the islets ( four islets per well ) were preincubated for 60  min with 1  ml of normal krebs - ringer solution [ concentration in mmol  l : nacl , 115 ; nahco3 , 24 ; kcl , 1.6 ; mgcl6h2o , 1 ; cacl22h2o , 1 ; and bovine serum albumin , bsa , 15 ] at ph 7.4 containing 5.6  mmol  l glucose . this solution was gassed with 95% o2 mixed with 5% co2 to maintain a ph of 7.4 . after the preincubation step , the islets were incubated with 2 different glucose concentrations , 5.6 and 16.7  mmol  l , for an additional 60  min . the supernatants from these incubations were collected and stored for further insulin measurements by the radioimmunoassay method . the limit of detection for insulin levels was 1.033  pmol  l , as previously described . to evaluate the obesity parameters , at 90 days of age , after each one of the experimental procedures 18 mice from 5 different litter for all groups were used ( n = 5 ) . the body length and weight were used to calculate the rodent body mass index , or lee index . all data were subjected to a d'agostino - pearson normality test to assess the gaussian distribution . afterward , all data were subjected to regular one - way anova with a bonferroni multiple comparisons posttest using graphpad prism software version 6.01 for windows ( graphpad software , inc . , san diego , ca , usa ) . neither food intake nor body weight gain throughout period of life was affected by neonatal treatment with msg and/or the maternal flax - diet , as shown in figures 1(a ) and 1(b ) , respectively . while treatment with the flax - diet did not influence the lee index in mice from the control - flax group , it was reduced by 8% in msg - flax mice , when compared to the msg - com group ( p < 0.05 ; table 2 ) . fat accretion in the retroperitoneal tissue was 3-fold higher in the msg - com group compared to the control - com group , p < 0.001 , whereas it was decreased in both groups treated perinatally with the flax - diet ; fat accretion was reduced by 39% in the control - flax group and by 25% in the msg - flax group ( p < 0.01 ; table 2 ) . as expected , the msg - com treated mice exhibited hyperglycemia throughout the ipgtt , with an observed increase of 33% in the auc compared to control - com mice ( p < 0.001 ; figure 2(a ) ) . the maternal flax - diet treatment , however , caused a 16% reduction in glucose intolerance compared to mice whose mothers did not receive n-6/n-3 fatty acids , allowing the msg - flax mice to normalize their glucose concentrations . as showed in figure 2(b ) , msg - com mice exhibited a low capacity for glucose uptake after insulin injection . the decrease in kitt was 53% compared to control - com mice , p < 0.001 . by contrast , the msg - flax treated mice displayed an increased glucose disappearance rate of 155% , p < 0.001 . however , glucose uptake in the control - flax treated mice was not affected compared to the control - com treated mice . isolated pancreatic islets from the msg - com mice oversecreted insulin when stimulated with 5.6  mmol  l glucose ; a 5-fold increase was observed compared to islets from control - com animals ( p < 0.05 ; figure 3(a ) ) . perinatal treatment with the flax - diet increased glucose - induced insulin secretion ( giis ) up to 189% in the islets isolated from the control - flax mice group , while it was slightly increased by 18% in islets isolated from the msg - flax mice ( p = 0.336 ; figure 3(a ) ) . glucose concentrations of 16.7  mmol  l also induced insulin oversecretion in islets isolated from the msg - com mice , a 2-fold increase in the giis compared to the control - com mice ( p < 0.001 ) . < 0.01 , increase in giis that was stimulated by a high glucose concentration in the islets isolated from the control - flax group compared to the control - com group . by contrast , no change was observed in the msg - flax group ( p > 0.999 ; figure 3(b ) ) . while the global brain incorporation of essential n-3 and n-6 pufas in the weaned msg - treated mice was not impaired , it was increased by the maternal flax - diet treatment in both control and msg - treated mice ( p < 0.01 ; table s2 , supplementary material ) . interestingly , the pancreas of weaned msg - com mice displayed less prominent incorporation of essential n-3 pufas associated with high incorporation of n-6 pufas in relation to control - com ones ( p < 0.01 ; table s3 , supplementary material ) . maternal flax - diet treatment was able to induce an essential n-6 pufas reduction of 31% in eicosadienoic acid ( eda ) and a reduction of 9% in arachidonic acid ( aa ) in control - flax , while it was reduced in 67% and 71% , respectively , in the msg - flax mice ( p < 0.01 ; table s3 , supplementary material ) . on the other hand , the pancreatic n-3 pufas incorporation was increased by 320% to alpha - linolenic acid ( lna ) and 229% to eicosapentaenoic acid ( epa ) in control - flax mice , while in the msg - flax mice the pancreatic incorporation of the lna and epa was increased by 433% and 577% , respectively ( p < 0.001 ; table s3 , supplementary material ) . tissue incorporation of essential n-6/n-3 pufas ratio was examined in both brain and pancreas from weaned mice ( figure 4 ) . while the neonatal treatment with msg did not impair the n-6/n-3 essential pufas ratio in brain from weaned mice , when compared with control ones , perinatal treatment with flax - diet induced a reduction in this ratio in the brain tissue from both control ( 18% ) and msg - treated mice ( 14% , p < 0.001 ; figure 4(a ) ) . the n-6/n-3 essential pufas ratio incorporated in the pancreatic tissue from msg - com was 36% higher when compared with control - com group ( p < 0.001 ; figure 4(b ) ) . however , the magnitude of improvement in the n-6/n-3 essential pufas ratio was strongly observed in the msg - flax mice ( 79% ) , when compared with the control - flax mice ( 50% , p < 0.001 ; figure 4(b ) ) . this study demonstrates that metabolic dysfunction is attenuated , or even blocked , by n-6/n-3 essential fatty acid ( at a ratio of 1.2  :  1.0 ) supplementation in the maternal diet . to our knowledge , this is the first study showing that metabolic alterations , reflected by the lee index , pancreatic islets dysfunction , and glucose intolerance , are restored in msg - obese mice whose mothers were exposed to fatty acid supplementation during pregnancy and lactation . a well documented experimental model of cardiometabolic syndrome , the msg - obesity model is characterized by hallmarks such as high fasting insulinemia , dyslipidemia , glucose intolerance , obesity , hypertension , and insulin resistance [ 2528 ] . once the regulation of growth hormone release in msg - obese rodents is deficient due to a relative loss of growth hormone - releasing factor , the body weight in this model 's rodents is lower than in their counterparts , even though the fat stores as well as lee index are tightly huge compared to control ones , which is an unusual characteristic due to the lack of growth hormone in this experimental model . the growth hormone weakness is due to hypothalamic neuronal connection failure [ 30 , 31 ] . considering the early use of msg injection in high concentrations , it acts as a powerful excitatory neurotransmitter by destroying the hypothalamic neuronal circuitry involved in the energy balance ; it is the major mechanism behind the induction of obesity in this model . once in early age the blood - brain barrier is not yet completed , the obesity - induction is promoted in neonatal rodents by partial destruction of the hypothalamus arcuate nucleus that contains neuronal circuitry controlling body weight and energy metabolism , as classically and long reported [ 13 , 14 , 32 ] . indeed , beside the classical use of msg as an obesity and metabolic syndrome model inductor in early life , it is important to keep in mind that there is a lot of misunderstanding about the msg function on metabolism , especially regarding the dietary ingestion by adult human being ( as recently revised ) . in fact , glutamate food ingestion does not influence negatively the energy and/or metabolism homeostasis [ 33 , 34 ] . beyond umami - savory taste , through the basic taste function , the msg from diet does not disturb brain or metabolic function , once it does not go to blood flow nor cross the blood - brain - barrier , thus not inducing obesity by this way . here we show a significant interaction pointing to amelioration of the metabolic dysfunction in msg - obesity model associated with maternal diet supplementation with a mixture of n-6/n-3 fatty acids in a ratio of 1.2  :  1.0 . according to our data , the flax - diet treatment increased the insulin secretion in the islets isolated from control mice , while no change was observed in islets isolated from msg - flax mice . interestingly , this finding may be associated with a mechanism of long - term protection against exhaustion by insulin oversecretion in the pancreatic islets of msg - obese mice . in accordance with metabolic improvement , as observed in the present study , the incorporation of essential pufas , especially an increase in n-3 , and a reduction in the n-6 essential pufas were higher in pancreatic tissue from weaned msg - flax mice that can reflect an important antioxidant role influencing the improvement in pancreatic function . indeed , the beneficial effect of n-3 pufas in the cell membrane have been shown by displaying anti - inflammatory and antioxidant action as well as by protecting cells ' metabolic function against the oxidative stress [ 36 , 37 ] . herein , we speculate that the high incorporation of n-3 and low n-6 essential fatty acids into pancreatic tissue from weaned mice , induced by flax - diet , may be implicated in programming amelioration of pancreatic function in msg - treated mice . even if we did not have studied the beta - cells nor isolated pancreatic islets , our data can suggest lower ratio of n-6/n-3 essential pufas can exert some antioxidant action protecting the pancreatic islets metabolism and function against the oxidative stress . on this line , it is important to remark that pancreatic islet cells are important target of proinflammatory cytokines in obesity that induce beta - cell dysfunction . dietary supplementation with sunflower oil and golden flaxseed flour as sources of n-6/n-3 fatty acids in a 1.2  :  1.0 ratio in the maternal diet during pregnancy and lactation improved the metabolism of msg - obese mice adult offspring , including a correction of glucose intolerance , low insulin sensitivity in peripheral tissues , high fat tissue accumulation , and insulin oversecretion . maternal treatment with essential fatty acids was also able to improve the giis in the control mice . these data indicate that pufas are able to induce programming in the offspring that provides a certain resistance against metabolic deterioration . a previous study using a similar ratio of n-6/n-3 fatty acids in pregnant rats has shown that the offspring exhibited an improvement in early brain development , and amelioration in metabolic impairment has been reported to be induced by pufas dietary consumption [ 39 , 40 ] . the data presented herein suggest that n-6/n-3 fatty acids in a ratio of 1.2  :  1.0 exert by somehow a sort of protection attenuating the deleterious central effects of msg treatment . unfortunately , we did not study the brain of offspring from mothers who were treated with the flax - diet to assess , for instance , the arcuate nucleus structure or the expression of proteins involved in energy and metabolic control , which could suggest potential mechanisms underlying the resistance against obesity onset induced by maternal fatty acids treatment . according to our results , high incorporation of n-3 taken together with low incorporation of n-6 essential pufas into brain tissue of weaned mice whose mothers were fed a flax - diet during pregnancy and lactation is suggestive that this diet supplementation can prevent impairment in the hypothalamic central areas controlling body composition and energy balance , probably due to an action on reducing neuronal damage induced by msg treatment early in life . similar effects of n-3 essential pufas uptake in neurons from weaned mice of mothers fed a diet supplemented with flaxseed have been observed . indeed , the n-3 pufas in human diet have improved clinical symptoms regarding neurological impairment , which have been associated with beneficial implication in protecting against corticosterone - induced neuronal stress . in this regard , synaptic plasticity , dopaminergic and serotonergic neurotransmission , and hypothalamic - pituitary - adrenal axis activity among many other neurological functions have been improved by n-3 pufas dietary supplementation [ 4244 ] . as is known , hypothalamic - pituitary - adrenal axis hyperactivity is an important hallmark in obesity , like that in msg - obesity model [ 27 , 45 ] , and in many other human 's metabolic disorders [ 4648 ] . in fact , as previously reported , the importance of n-3 pufas role against the inflammatory process in obesity and metabolic disturbances was showed in transgenic mice . high n-3/n-6 pufas ratio was strongly implicated in improving obesity - linked metabolic disturbances [ 39 , 49 ] . given that , it is remarkable that this amelioration has been associated with n-3 pufas action on preventing adipose inflammatory process , as well as on modulating changes in gut microbiota [ 39 , 51 ] , which is known to be strongly involved in obesity and metabolic syndrome development both in human and in animal model . in this respect , on the fight against public health burden due to human 's metabolic diseases around the world , . it might be helpful and protect against the onset of metabolic syndrome in human population [ 39 , 53 , 54 ] . in the present study , beyond different possible mechanisms , it might also be a suggestive mechanism behind the metabolic amelioration observed in the msg - obese mice . in summary , our data show that dietary supplementation with n-6 and n-3 pufas in a ratio of 1.2  :  1.0 offered to mothers during pregnancy and lactation attenuates the metabolism malfunctions in msg - obese mice adult offspring by improving pancreatic islets function , peripheral glucose tolerance , and insulin sensitivity as well reducing the fat mass stores . considering that perinatal life is a fragile target to environmental aggression , which can induce metabolic syndrome later in life , early dietary supplementation with n-6 and n-3 pufas in a ratio of 1.2  :  1.0 can be powerfully used for protection against metabolic dysfunctions .
essential polyunsaturated fatty acids ( pufas ) prevent cardiometabolic diseases . 
 we aimed to study whether a diet supplemented with a mixture of n-6/n-3 pufas , during perinatal life , attenuates outcomes of long - term metabolic dysfunction in prediabetic and obese mice . 
 seventy - day - old virgin female mice were mated . from the conception day 
 , dams were fed a diet supplemented with sunflower oil and flaxseed powder ( containing an n-6/n-3 pufas ratio of 1.2  :  1.0 ) throughout pregnancy and lactation , while control dams received a commercial diet . 
 newborn mice were treated with monosodium l - glutamate ( msg , 4  mg  g1 body weight per day ) for the first 5 days of age . 
 a batch of weaned pups was sacrificed to quantify the brain and pancreas total lipids ; another batch were fed a commercial diet until 90 days of age , where glucose homeostasis and glucose - induced insulin secretion ( giis ) as well as retroperitoneal fat and lee index were assessed . 
 msg - treated mice developed obesity , glucose intolerance , insulin resistance , pancreatic islet dysfunction , and higher fat stores . 
 maternal flaxseed diet - supplementation decreased n-6/n-3 pufas ratio in the brain and pancreas and blocked glucose intolerance , insulin resistance , giis impairment , and obesity development . 
 the n-6/n-3 essential pufas in a ratio of 1.2  :  1.0 supplemented in maternal diet during pregnancy and lactation prevent metabolic dysfunction in msg - obesity model .