neuropeptide y ( npy ) is one of the most common peptides in the brain and an abundant neurotransmitter in the peripheral sympathetic nervous system ( sns ) .
it plays a well - established role in the hypothalamic control of body energy balance by promoting feeding and lipid storage in white adipose tissue ( wat ) .
however , in pair - fed rats , central npy administration still leads to increased fat accumulation , which suggests that npy has an important role in promoting adiposity independent of food intake .
npy outside the hypothalamus in the regulation of energy homeostasis has not been widely studied , although npy and its receptors are located in key peripheral tissues , such as wat , liver , and pancreas .
npy inhibits lipolysis in adipocytes via y1 receptors , and could modulate adipose tissue expansion by regulating angiogenesis .
npy also inhibits insulin release via pancreatic y1 receptors . on the other hand , npy in the brainstem
could modulate sympathetic tone , which is known to have multiple effects on energy homeostasis .
to address the role of npy colocalized with noradrenaline in sns and brain noradrenergic neurons , we created a transgenic mouse model ( oe - npy mouse ) , where npy is overexpressed under the dopamine - betahydroxylase ( dbh ) promoter .
this resulted in moderately increased levels of npy protein ( 1.31.8-fold ) peripherally in adrenal glands and centrally in noradrenergic neurons of the brainstem , but not in the hypothalamus .
the oe - npy mice displayed increased adiposity and liver triglyceride accumulation without changes in body weight , food consumption , or physical activity on a normal chow diet .
the increased adiposity was observed in both genders at all ages studied , but the major metabolic complications such as impaired glucose tolerance , hyperinsulinemia , and hepatosteatosis were only present in male oe - npy mice .
furthermore , the oe - npy mice showed elevated sympathetic tone and increased responses to stress , and increased susceptibility to arterial thickening after vascular injury .
others have shown that stress - induced activation of the npy system combined with a high - calorie diet results in augmented obesity .
the primary finding of kuo et al . was increased fat mass without change in food intake or body weight after two weeks of daily stress and high fat diet
. prolonged stress and diet led to liver steatosis , impaired glucose tolerance and obesity , which were attenuated by local y2r antagonist administration and fat - targeted y2-gene knockdown procedure .
these data combined show that extrahypothalamic npy regulates adiposity independent of food intake and that npy together with stress are potent factors in several pathways leading to a complex disorder called the metabolic syndrome . in humans ,
association studies of a polymorphism in the npy gene have linked npy to metabolic disturbances .
the npy1228 t > c polymorphism ( rs16139 ) , which causes an amino acid substitution of leucine 7 to proline 7 ( l7p ) in the signal peptide   is functional , enhancing the secretion of npy [ 10 , 11 ] .
it is associated with an earlier onset of type 2 diabetes in carriers of l7p , as well as with other traits of the metabolic syndrome   especially in obese subjects .
recently , it was reported that there is a gender difference in the npy - mediated effects of the proline 7 allele .
the associations of the l7p seem to be more pronounced in men than women , which is in agreement with our findings in oe - npy mice indicating that males develop more severe metabolic changes .
human obesity and metabolic syndrome are often due to lifestyle changes including increased sedentary work and decreased physical activity , and increased accessibility and affordability of dense , high - calorie foods .
an increase in dietary fat and sucrose content has been shown to produce hyperglycaemia and obesity in various strains of mice and rats . in the present study
, we exposed male and female oe - npy and wildtype ( wt ) control mice to an energy - dense western - type diet for seven weeks , and compared the genotype and sex differences in the impacts of the diet on the development of obesity , insulin resistance , and impaired glucose tolerance . based on the findings in these mice ,
the possible reasons , that is , food intake and physical activity , were studied further in the oe - npy female mice .
eight - week - old male and female oe - npy mice heterozygous for the npy transgene   and their wt littermates on a c57bl/6n genetic background were used .
animal care was in accordance with the guidelines of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ( council of europe no .
123 , strasbourg 1985 ) , and all experimental procedures were approved by the institutional animal care and use committee .
oe - npy mice are stress - sensitive , and therefore the mice in this study were housed with their same - sex siblings instead of single cages and maintained on a 12-h light / dark cycle ( lights on at 6 am ) with free access to food and water .
the number of animals in the male groups was 9 - 10 , and in the female groups 15 - 16 .
the mice were placed on a western - type diet ( 42% kcal fat from milk fat , 43% carbohydrates from sucrose and corn starch , 15% protein , and 0.15% supplementary cholesterol , code 829100 , special diets services , essex , uk ) and fed ad libitum for seven weeks .
after five weeks on the diet , the mice were fasted from 6:00  h to 10:00  h , and administered intraperitoneally ( ip ) with glucose ( 10% w / v , 1  g  kg body weight ) for glucose tolerance test ( gtt ) .
tail vein blood glucose was measured immediately before the injection and at 20 , 40 , 60 , and 90  min with a glucose analyzer ( precision xtra , abbott diabetes care , abbott park , il , usa ) .
areas under the resultant curves ( auc ) were calculated with the trapezoidal method in graphpad prism 5.01 software .
mmol  l ( 250  mg  dl ) in mice following a fast started on the morning of the experiment as guided by mouse metabolic phenotyping centers ( http://www.mmpc.org/ ) established by the national institutions of health ( nih ) and by the animal models of diabetic complications consortium ( amdcc , http://www.amdcc.org/ ) .
the same level of fasted blood glucose in determination of hyperglycaemia in mice has been used by others as well [ 16 , 17 ] .
after six weeks on the diet , the mice were fasted for 1  h. recombinant human insulin ( protaphane , novo nordisk ) at a dose of 0.5 or 1.0  iu  kg was injected ip for insulin tolerance test ( itt ) .
blood glucose was measured immediately before the injection and at 20 , 40 , and 60  min after the injection .   at sacrifice ,
the mice were fasted from 6:00  h to 10:00  h , the rectal temperature was measured ( ellab , roedovre , denmark ) , and the animals were anesthetized with ketamine ( ketalar 75  mg  kg ) and medetomidine ( domitor 1  mg  kg ) .
terminal blood samples were obtained from inferior vena cava with 1  ml syringe and 23  g needle into heparinized tubes .
plasma was centrifuged ( 4000  rpm , 10  min ) and stored at 70c until analyzed .
wat weight was determined by collecting the subcutaneous , epididymal / gonadal , and retroperitoneal fat pads .
in addition , brown fat and liver weights were determined , and the tissues were snap frozen in liquid nitrogen for further analyses .
brown fat samples were cryo - sectioned on microscopic slides and stained with hematoxylin and eosin ( h&e ) for standard morphology .
plasma concentrations of insulin ( mercodia ultrasensitive mouse insulin elisa , mercodia ab , uppsala , sweden ) and total cholesterol ( biovision cholesterol quantitation kit , biovision inc .
, mountain view , ca , usa ) were determined according to the manufacturers ' instructions . total rna in bat
was extracted with trizol reagent ( invitrogen , carlsbad , ca , usa ) combined with dnase treatment ( turbo dna - free kit , ambion inc . ,
rna was converted to cdna with high capacity rna - to - cdna kit ( applied biosystems ) according to the manufacturer 's instructions .
predesigned taqman gene expression assay ( applied biosystems , assay i d mm01244861_m1 ) for uncoupling protein-1 ( ucp-1 ) was used to analyze the mrna levels in bat .
ucp-1 levels were quantitated relatively to the housekeeping gene -actin , ( mouse actb , vic / mgb probe , primer limited with 7300 real - time pcr system applied biosystems ) .
relative ct method and the formula 2 were used as the quantitation method . in order to study the genotype differences in the female gender in more detail , another set of female mice age - matched to experiment 1 were studied .
this time we used oe - npy mice homozygous for the transgene and c57bl/6n wt female mice .
heterozygous oe - npy mice were bred together in order to produce wt , heterozygous and homozygous pups , which were genotyped by qpcr .
genomic dna was isolated from tail biopsies with a commercial kit ( puregene dna purification kit , gentra , minneapolis , mn , usa ) . forward
( 5-tggctggagtgcgatcttc-3 ) and reverse ( 5-gagtttgaccgtctacgtgc-3 ) primers , and the taqman - mgb probe ( 6fam - ccgatactgtcgtcgtc - mgb ) were designed for the lacz reporter gene in the transgene construct with the primer express 3.0 software ( applied biosystems ) .
lacz levels were quantitated relatively to the housekeeping gene -actin , ( mouse actb , vic / mgb probe , primer limited ) .
homozygous oe - npy and littermate wt mice were selected for breeding ( oe - npy  oe - npy and wt  wt ) , thus creating purely transgenic or wt lines housed separately .
the first litters from the homozygous and wt mouse lines were genotyped as described above to verify all pups had the same genotype for the transgene .  
the female mice ( n = 712 ) were placed on the western - type diet for five weeks and their weight gain and food consumption were measured on a weekly basis .
in addition , after two weeks on the diet , a 24-h spontaneous physical activity along with food intake was monitored with a photo - beam recording system ( san diego instruments , san diego , ca , usa ) in 10 minute intervals .
mice were placed in single cages and had a 23-h adjustment period before monitoring the 24-h activity .
whole body fat mass was measured in vivo after the five - week period with an echomri-700 ( echo medical systems , houston , tx , usa ) .
the reproduction history of all three genotype lines was followed and the number of total pups as well as female versus male pups was analyzed .
the number of wt , heterozygous oe - npy , and homozygous oe - npy dames used in breeding was 45 , 26 , and 34 , respectively .  
the results were analyzed with a student 's parametric t - test or with a mann - whitney 's nonparametric u - test to compare the groups of oe - npy and wt mice .
weight gain , gtt and itt were analyzed with two - way anova for repeated measurements and bonferroni posthoc tests . 
  test was used to calculate the statistical difference in the number of male mice with hyperglycaemia as 0 = fasting glucose
< 13.8  mmol  l or 1 = fasting glucose > 13.8  mmol  l. statistical analyses were carried out using graphpad prism 5.01 ( graphpad software , san diego , ca , usa ) .
female oe - npy mice gained more weight compared with their wt littermates on the western diet ( figures 1(a ) and 1(c ) ) . in males ,
prominent weight gain was observed in both genotypes , but no difference in the weight gain pattern was observed between the oe - npy and wt mice ( figures 1(b ) and 1(d ) ) .
the female transgenic mice also had larger wat and bat mass as measured by individual fat pads or the sum of wat pads compared with wt female mice ( figures 1(e ) and 1(g ) ) . in males , a significant difference between the genotypes was only observed in the epididymal wat weight ( figures 1(f ) and 1(h ) ) .
glucose tolerance was significantly impaired in female oe - npy mice compared with wt mice , as shown by the much greater rise in blood glucose in oe - npy mice over the studied 90  min time period following administration of glucose ( figure 2(a ) ) .
in addition , the auc value in oe - npy mice was significantly higher than in wt controls ( table 1 ) .
in male mice , the glucose tolerance was similar between the genotypes and thus no statistical significance between the groups was reached ( figure 2(b ) ) .
however , four out of ten oe - npy male mice , but none of the nine wt controls , had a 4-h fasting glucose value over 13.8  mmol  l. thus , they were considered to show severe hyperglycaemia [ 16 , 17 ] .
this difference reached statistical difference of p < 0.05 with the    test .
there was no difference in gtt auc values between the genotypes in males ( table 1 ) .
insulin sensitivity was assessed by investigating the action of exogenous insulin on blood glucose levels .
blood glucose levels were higher in female oe - npy mice compared with their wt controls at all investigated time points after intraperitoneal injection of insulin ( figure 2(c ) ) .
insulin was first administered with 0.5  iu / kg , which was sufficient in females but had no effect on circulating glucose levels in males .
hence , the experiment was repeated with a higher dose of 1.0  iu  kg .
this dose decreased blood glucose on average by 35% in eight out of nine wt and seven out of ten oe - npy male mice ( figure 2(d ) ) , whereas the rest of the mice ( 1/9 of wt and 3/10 of oe - npy ) were resistant to the effect of insulin , that is , the blood glucose levels were the same or even higher than at baseline .
no significant difference between the genotypes in blood glucose values after insulin dosing in males was observed . circulating insulin and total cholesterol levels were determined after a 4-h fast .
no difference in either parameter was observed between the genotypes in males or females ( table 1 ) .
the female oe - npy mice had significantly heavier livers per se ( wt : 1.1  0.07 ; oe - npy : 1.3  0.06 grams ; p < 0.05 ) , but when the weights were corrected with body weights , the statistical difference was lost ( data not shown ) . in males ,
the actual ( wt : 2.5  0.21 ; oe - npy : 2.8  0.22 grams ; p = ns ) and normalized liver weights were identical between the genotypes .
gross visual examination of the livers suggested steatosis as observed by a pale abnormal color with naked eyes .
no difference in triglyceride levels between the genotypes in males or females was observed ( table 1 ) .
rectal body temperatures did not differ between the genotypes in males ( wt : 35.4  0.24 ; oe - npy : 35.7  0.29 degrees centigrade ) or females ( wt : 35.9  0.36 ; oe - npy : 35.7  0.37 degrees centigrade ) .
ucp-1 expression levels in bat were measured with qpcr by using the average mrna expression of the wt group as a calibrator .
no difference in ucp-1 levels in males ( wt : 1.13  0.18 ; oe - npy : 1.16  0.15 fold mrna expression ; p = ns ) or females ( wt : 1.09  0.13 ; oe - npy : 1.24  0.16 fold mrna expression ; p = ns ) were observed .
the tissue morphology showed atypical bat tissue with lipid deposition in both male groups and in the oe - npy female group whereas the wt females showed normal bat morphology ( figure 3 ) . in order to verify and to find the reason for the findings in females , the early steps of the weight gain and adiposity were further studied in a homozygous oe - npy line .
a similar weight gain pattern was observed as in the heterozygous mice in comparison with wt controls , that is , the weights start to differ after four weeks on the diet and are significantly higher at five weeks from the beginning of the diet ( figure 4(a ) ) .
food intake measured weekly in group - housed ( food consumed divided by the number of animals ) ( figure 4(b ) ) or in 47-h individually housed mice ( wt : 6.7  0.7 ; oe - npy : 6.3  1.2 grams ; p = ns ) was not different between the genotypes .
a whole body echomri analysis was performed at week five to measure the amount of fat in vivo .
the oe - npy females showed increased adiposity compared with wt mice ( figure 4(c ) ) , which explains the difference in body weights .
24-h locomotor activity of female wt and oe - npy mice measured in photo - beam cages after two weeks on the diet revealed no differences between the genotypes either in horizontal or vertical movements ( data not shown ) .
total activity per hour calculated as the sum of these two parameters is presented in figure 5 .
based on 82 litters from 45 wt dames , wt female mice produced on average 3.5 female ( total 284 ) and 3.6 male ( total 294 ) pups .
heterozygous oe - npy female mice ( 35 litters from 26 dames ) produced 4.0 female ( total 141 ) and 3.8 male ( total 134 ) pups and homozygous oe - npy mice ( 55 litters from 34 dames ) 3.3 female ( total 176 ) and 3.7 male ( total 199 ) pups .
in our previous work , we observed that both the male and female transgenic mice overexpressing npy in noradrenergic and adrenergic neurons showed increased adiposity without significant increase in body weight or food intake on a normal chow diet . in the current study , we hypothesized that npy overexpression would render the transgenic mice even more susceptible to obesity induced by a western - type diet . in line with the hypothesis ,
the female oe - npy mice gained significantly more weight compared to their wt littermate controls ( 40% versus 26% of initial weight ) and showed an increased adipose tissue mass .
in contrast , the male oe - npy mice gained weight similarly to their wt controls ( 55% of initial weight ) .
thus , npy overexpression overrode the resistance of the c57bl/6 female mice   to the diet - induced obesity , but susceptibility of the male c57bl/6 mice to the diet induced obesity   overrode the npy 's adiposity inducing effect
. the mechanisms of increased weight gain in the female oe - npy mice were studied in more detail in a separate group of mice .
this time the mice were homozygous for the transgene and thus housed with same - sex , same - genotype siblings , which made it possible to study the feeding behavior between the genotypes in unstressed conditions ( group housing ) .
the results showed that enhanced weight gain is not due to increased food consumption during the time preceding the difference in body weights .
rodents resistant to the diet - induced obesity increase bat thermogenesis via increased ucp-1 activity to avoid weight gain .
similar to the oe - npy and wt male mice , the female oe - npy mice showed atypical bat morphology with white fat - like appearance and large lipid vacuoles in bat .
this implied the onset of brown adipocyte degeneration often associated with obesity and impaired thermogenesis in mice [ 20 , 21 ] .
however , the bat thermogenic capacity , as measured by ucp-1 expression levels in bat , did not differ between the genotypes .
we show in this study that there are no differences in the number of litters or litter sizes between the oe - npy or wt mice .
thus , prenatal and suckling conditions in this regard are similar for the pups . therefore , different challenges in the developmental and prepubertal environment are an unlikely explanation for the weight gain in the diet - induced obesity in the oe - npy mice .
sexually dimorphic responses to npy - associated metabolic changes have been reported in npy y1 receptor knock - out mice , which develop late - onset obesity without hyperphagia that is more pronounced in female than male mice .
the difference was attributed to decreased skeletal muscle mitochondrial oxidative capacity in female y1 knock - out mice .
gonadal steroid hormones seem to play a role in the fat accumulation in the y1 knock - out mice as weight gain does not start until puberty [ 24 , 25 ] .
estrogens seem to protect female mice from obesity as evidenced for instance by weight gain after gonadectomy , and their effects are at least in part mediated by hypothalamic npy [ 26 , 27 ] .
our results may imply that the overexpression of npy in adrenergic and noradrenergic neurons overrides the protective effects of estrogens in female mice , suggesting that npy in the brainstem and in the sns may also play a role in mediating estrogen action on body composition .
along with the increased adiposity , the oe - npy female mice displayed impaired glucose tolerance and altered insulin sensitivity or counterregulatory effect of glucose 40  min after the ip administration of glucose .
it is generally acknowledged that female mice are less likely to develop disturbances in glucose metabolism , which may be due to the antiobesity effects of estrogens in females [ 29 , 30 ] .
previously , chow - fed female oe - npy mice were shown to possess normal glucose tolerance despite of increased adiposity and sensitivity to stress [ 6 , 7 ] , and impairment in glucose tolerance in the current study occurred with increasing levels of obesity .
thus , disturbances in glucose metabolism in the female oe - npy mice seem to be caused by increased adiposity rather than by direct effects of npy or sympathoadrenal system on glucose metabolism , which is supported by a positive correlation with the area under the curve value for gtt and body weight in both female genotypes ( wt , r = 0.54 , p < 0.05 ; oe - npy , r = 0.45 , p = 0.08 ) .
in contrast , the diet - induced hepatosteatosis that similarly affected the wt and oe - npy mice is not likely to explain the impaired glucose tolerance in the female oe - npy mice .
however , increased glycogenolysis in the liver in response to the insulin - induced decline may be responsible for the altered insulin sensitivity presented in figure 3(c ) , which remains to be studied further . although gtt showed very high levels of blood glucose with no genotype differences in the male mice , the oe - npy mice had a tendency towards reduced insulin - induced decline in glucose levels as evidenced by the more numerous individuals that did not respond to insulin in itt .
in addition , forty per cent of the oe - npy males but none of the wt littermates developed severe hyperglycaemia defined as a blood glucose level over 13.8  mmol  l following a 4-h fast .
interestingly , these tendencies occurred without major differences in wat depot weights or at the level of hepatosteatosis in the oe - npy male mice .
this suggests that increased npy may have a diabetogenic effect in the context of obesity and supports the association of the l7p polymorphism with an earlier onset of diabetes in the obese human population .
oe - npy female mice showed a more pronounced diet - induced obesity , glucose intolerant , and insulin resistant phenotype on a western - type energy - dense diet than their wt littermates that could not be explained by increased feeding , decreased activity , or impaired thermogenesis .
this suggests that increased npy release may predispose women and females in general , to a greater risk of weight gain under high caloric conditions .
in contrast , there was no difference between the male oe - npy and wt mice in the degree of obesity , although the oe - npy mice seemed to be more susceptible to developing diabetes while on the diet .
interestingly , we have shown that the oe - npy males fed with chow and the oe - npy females with a western diet display similar traits with the humans who carry the proline 7 allele in their npy sequence , which support the associations between the rs16139 polymorphism and high npy levels and various metabolic risks .
furthermore , these results strengthen the hypothesis that npy has an important role in promoting adiposity via extrahypothalamic pathways .
 neuropeptide y ( npy ) is a neurotransmitter associated with feeding and obesity . 
 we have constructed an npy transgenic mouse model ( oe - npydbh mouse ) , where targeted overexpression leads to increased levels of npy in noradrenergic and adrenergic neurons . 
 we previously showed that these mice become obese on a normal chow . 
 now we aimed to study the effect of a western - type diet in oe - npydbh and wildtype ( wt ) mice , and to compare the genotype differences in the development of obesity , insulin resistance , and diabetes . 
 weight gain , glucose , and insulin tolerance tests , fasted plasma insulin , and cholesterol levels were assayed . 
 we found that female oe - npydbh mice gained significantly more weight without hyperphagia or decreased activity , and showed larger white and brown fat depots with no difference in ucp-1 levels . 
 they also displayed impaired glucose tolerance and decreased insulin sensitivity . 
 oe - npydbh and wt males gained weight robustly , but no difference in the degree of adiposity was observed . however , 40% of oe - npydbh but none of the wt males developed hyperglycaemia while on the diet . 
 the present study shows that female oe - npydbh mice were not protected from the obesogenic effect of the diet suggesting that increased npy release may predispose females to a greater risk of weight gain under high caloric conditions . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions