nucleobindin precursor protein , which was identified for the first time in the human 
 and mouse cell lines , is known to have two isotypes , nucleobindin 1 ( nucb1 ) and 
 nucleobindin 2 ( nucb2 ) ( miura et al . , 1992 ; 
 kanai & tanuma , 1992 ; barnikol - watanabe et al . , 1994 ) . however , nucb2 
 only functions physiologically in humans and rodents ( miura et al . , 1992 ) . nucb2 produces nesfatin-1 ( residues 1 - 82 ) , 
 nesfatin-2 ( residues 85 - 163 ) , and nesfatin-3 ( residues 166 - 396 ) by the enzyme 
 pro - hormone convertase ( pc)-1/3 after putative post - translational processing . until 
 now , a physiological activity has only been demonstrated for nesfatin-1 ( oh - i et al . , 2006 ; stengel et al . , 2012 ) . nesfatin-1 is initially known to be expressed in the hypothalamic nuclei such as 
 arcuate nucleus , paraventricular nucleus , supraoptic nucleus , lateral hypothalamic 
 area and zona incerta in rats ( brailoiu et al . , 
 2007 ; chen et al . , 2012 ; foo et al . , 2008 ; fort et al . , 2008 ; goebel et al . , 
 2009 ; goebel et al . , 2011 ; recent studies showed that a large amount of the nesfatin-1 
 expression was detected in the digestive system as well as in the brain . it has also 
 been reported that the nesfatin-1 generated in the gastric organs , by acting in the 
 brain , reduces food intake and suppresses the motilities of the stomach - duodenum 
 ( prinz et al . , 2016 ; jiang et al . , 2016 ) . nesfatin-1 immunoreactive 
 cells are also co - localized with insulin in pancreatic beta - cells of mice and rats , 
 suggesting a potential role for nesfatin-1 in pancreatic islet and glucose 
 homeostasis ( shimizu & osaki , 2014 ; gonzalez et al . , 2009 ) . furthermore , nesfatin-1 
 was detected in the adipose tissues of humans and rodents , showing that it was 
 expressed more in the subcutaneous than in the visceral fat ( shimizu et al . recent studies have demonstrated that nesfatin-1/ nucb2 are expressed in the 
 reproductive system ( garcia - galiano et al . , 
 2010 ) . we have also been shown that nesfatin-1/ nucb2 mrna and protein 
 are expressed in the mouse hypothalamus and pituitary and its expression levels were 
 regulated by the gonadotropin ( kim et al . , 
 2014 ; chung et al . , 2015 ) . moreover , we showed that the expression of nesfatin-1/nucb2 in the pituitary and 
 hypothalamus is regulated by estradiol and progesterone secreted by the ovary ( chung et al . , 2015 ) . therefore , we investigated 
 whether the expression of nesfatin-1/nucb2 in the pituitary and hypothalamus is 
 affected by the testosterone produced by the testis in the male mouse . eight - week - old male icr mice were purchased from koatech ( south korea ) and housed 
 in groups of five per cage under controlled illumination ( 12:12 h light / dark 
 cycle , lights on / off : 6 h/18 h ) and temperature ( 222 ) . animal care and 
 experimental procedures were approved by the institutional animal care and the 
 use committee at the seoul women s university in accordance with guidelines 
 established by the korea food and drug administration . mice were castrated and rested for 48 h followed by intraperitoneal injection 
 ( 100 l / mouse ) subcutaneously with sesame oil alone and with testosterone ( 300 
 ng/100 l ; sigma , st . mice were euthanized 
 by co2 anesthesia followed by cervical dislocation , and then the 
 cerebrum , hypothalamus , pituitary gland , and stomach were collected 24 h after 
 treatment . we obtained the fundus part of the stomach and the front part of 
 cerebrum . for dissection of the hypothalamus and pituitary gland , we removed 
 brain from skull and placed ventral side up . using curved forceps , we took out 
 the pituitary gland from hypophyseal fossa , and then pushed the curved part of 
 forceps down around the hypothalamus and pinched it out . the hypothalamus and pituitary glands were gently dissected away from the other 
 tissues , put on a nylon mesh sheet in a 24-well culture plate ( becton dickinson 
 labware , franklin lakes , nj ) , and cultured in 200 l / well serum - free dmem / f12 
 medium ( invitrogen , groningen , the netherlands ) containing antibiotics 
 ( invitrogen ) . the hypothalamus and pituitary glands were treated with 
 10 , 10 , and 10 m testosterone 
 ( sigma , st . louis , mo ) for 24 h.   the cerebrum , hypothalamus , pituitary gland , and stomach were homogenized with 
 rna isoplus ( takara bio , shiga , japan ) . after chloroform extraction and 
 isopropyl alcohol precipitation , rna was dissolved in rnase - free depc ( takara 
 bio , shiga , japan ) solution . the rna concentrations were measured with the 
 nano - drop ( thermo fisher scientific inc . , first strand cdna 
 synthesis was performed using the extracted rna and oligo dt , followed by 
 double - strand synthesis in rt buffer ( invitrogen , carlsbad , ca ) with dntp ( bio 
 basic inc . , ontario , canada ) and rtase ( invitrogen , carlsbad , ca ) .   conventional pcr was performed in buffer solution containing template cdna , taq 
 polymerase ( bionics , korea ) , dntps ( bio basic inc . primers were designed for nucb2 and -actin on the basis of the mouse 
 cdna sequences . the following primer pairs were used : nucb2 forward 
 5-tttgaa - cacctgaaccacca-3 ; reverse 5-tggtcttcgtgcttcctctt-3 and -actin forward 
 5-ctctttgatgtcacgcacgatttc-3 ; reverse 5-atcgtgggccgctctaggcacc-3 primers 
 ( bionics , korea ) . the optimum temperature cycling protocol was used as 95 for 
 15 s , 60 for 30 s and 72 for 30 min , using the gene pro thermal cycler ( bioer , 
 china ) . the reaction products were run on a 2% agarose gel and visualized with 
 ethidium bromide to check the length of the amplified cdna . qrt - pcr was performed in buffer solution containing template cdna , sybr green 
 ( roche , manheim , germany ) , and each primer . primer pairs were as follows : nucb2 
 forward 5-aaaaccttggcctgtctgaa-3 ; reverse 5-catcgataggaacagcttcca-3 and gapdh 
 forward 5-ttgatggcaacaatctccac-3 ; reverse 5-cgtcccgtgacaaaa - tggt-3 ( bionics , 
 korea ) . the optimum temperature cycling protocol was determined to be 95 for 10 
 s , 60 for 10 s and 72 for 10 s using the light cycler 480 real - time pcr system 
 ( roche , manheim , germany ) . hypothalamus , pituitary , and stomach , and fat were quickly removed and extracted 
 the protein with edta homogenization buffer , the samples were sds - page and 
 transferred to the pvdf membrane . the membrane was treated in a blocking 
 solution and incubated with rabbit anti - rat nesfatin-1 antibody ( h-003 - 22 , 
 phoenix pharmaceuticals ) / anti - mouse -actin antibody ( sc-47778 , santa cruz 
 biotechnology ) followed by incubation with donkey anti - rabbit igg - hrp ( sc-2313 , 
 santa cruz biotechnology ) / donkey anti - mouse igg - hrp ( sc-2096 , santa cruz 
 biotechnology ) , respectively . by using the ecl plus western blotting detection 
 reagents ( amersham ; ge healthcare ) , the membrane was detected to investigate the 
 expression level of nesfatin-1 protein . the tissues of hypothalamus and pituitary gland were fixed in 4% paraformaldehyde 
 buffer saline for 2 h. the tissues were rinsed in ethanol series to remove 
 fixative residues , embedded in paraffin block . the tissues blocks were cut 10 m 
 sections using a microtome , deparaffinized , and rehydrated with graded 
 xylene - alcohol series , and then washed with pbs before immunostaining . the 
 sections were incubated with rabbit anti - rat nesfatin-1 polyclonal antibody 
 ( phoenix pharmaceuticals , inc . , burlingame , ca ) , followed by incubation with 
 alexa fluor 488 conjugated goat anti - rabbit igg ( jackson immunoresearch 
 laboratory , west grove , pa ) . the sections were counter stained with dapi 
 ( 4',6-diamidino-2-phenylindole ; sigma , st . louis , mo ) for 10 min and mounted on 
 the slides with mounting medium ( vector laboratories , inc . , burlingame , ca ) , and 
 then observed under fluorescence microscopy ( axioskop2 , carl zeiss , 
 germany ) . the results were presented as the mean and the standard error of the mean(sem ) . nucb2 mrna expression in the cerebrum , hypothalamus , pituitary , and stomach was 
 detected by conventional pcr and qrt - pcr . we found that the nucb2 gene was 
 expressed in all tissues and its mrna expression was significantly higher in the 
 pituitary gland than in the cerebrum , hypothalamus , and stomach ( fig . western blots confirmed that , 
 similar to nucb2 mrna expression , nesfatin-1/nucb2 protein was highly expressed 
 in the pituitary gland ( fig . immunohistochemical staining showed that nesfatin-1/nucb2 protein was localized 
 in many cells within the anterior pituitary gland ( fig . ( a ) nucb2 mrna expression was confirmed in the hypothalamus and pituitary 
 gland by conventional pcr . ( b ) western blots show that nesfatin-1 
 protein is highly expressed in the pituitary gland . ( c ) the levels of 
 nucb2 mrna in cerebrum , hypothalamus , pituitary gland and stomach were 
 analyzed by qrt - pcr . the nucb2 mrna was significantly higher in the 
 pituitary than in the other tissues . ( a ) hypothalamus section was stained with nesfatin-1 antibody to localize 
 nesfatin-1 protein . ( b ) pituitary gland section was stained with 
 nesfatin-1 antibody to observe the nesfatin-1 protein expression . nesfatin-1 protein was localized in many cells within the anterior 
 pituitary gland a ; 600 , b ; 1,800. to assess whether nucb2 mrna expression in the hypothalamus and pituitary gland 
 was regulated by testosterone secreted by the testis , mice were castrated and 
 injected with testosterone . the expression of nucb2 mrna in the hypothalamus was 
 not changed after castration , but its expression was significantly decreased 
 after injection with testosterone ( fig . the expression of nucb2 mrna was 
 significantly decreased after castration and increased after injection with 
 testosterone ( fig . the expression of nucb2 
 mrna in the hypothalamus was not changed after castration , but decreased 
 after injection with testosterone . by contrast , the expression of nucb2 
 mrna in the pituitary gland was decreased after castration and raised 
 after injection with testosterone . * indicates p<0.05 vs. non - treated 
 control . to investigate whether testosterone directly regulated expression of nucb2 mrna , 
 we treated testosterone to mouse hypothalamus and pituitary gland tissues 
 cultured in vitro . nucb2 mrna expression in the hypothalamus 
 was significantly decreased with 10 and 10 m 
 testosterone ( fig . by contrast , nucb2 
 mrna expression in the pituitary gland was increased in dose - dependent manner , 
 showing significant increase in 10 m testosterone treatment ( fig . ( a ) in the cultured 
 hypothalamus , nucb2mrna expression was significantly decreased in 
 10and 10 m testosterone treatment . ( b ) by 
 contrast , nucb2 mrna expression was significantly increased in 106 
 m testosterone treatment . nesfatin-1 protein produced by neurons in the hypothalamus is known to be secreted 
 into cerebrospinal fluid , and be controlling of appetite and energy metabolism 
 ( oh - i et al . , 2006 ) . recently , it has 
 been reported that nesfatin-1/nucb2 mrna is expressed in gastric organs such as 
 stomach and pancreas ( stengel et al . , 2009 ) , 
 in adipose tissues of humans and rodents ( garcia - galiano et al . , 2010 ) and in the male and female mouse 
 reproductive organs ( kim et al . , 2010 ; kim et al . , 2011 ) our previous study has 
 demonstrated that nesfatin-1/nucb2 expression level is higher in the pituitary gland 
 compared to other organs and its expression is regulated by 17-estradiol and 
 progesterone secreted from the ovary ( chung et al . , 
 2015 ) . however , currently no data exist on the expression of 
 nesfatin-1/nucb2 and its regulation mechanism in the pituitary of male mouse . 
 therefore , in the present study we examined whether nesfatin-1/nucb2 is present in 
 the male mouse pituitary and if its expression is regulated by testosterone .   to examine the relative expression of nesfatin-1/nucb2 mrna and protein in the 
 pituitary , we performed conventional pcr using total rna extracted from the 
 cerebrum , hypothalamus , pituitary , and stomach . the conventional pcr analysis showed 
 that nucb2 mrna expression is detected in all tissues . the qrt - pcr and western 
 blotting data showed that the expression of nesfatin-1 protein and nucb2 mrna were 
 higher in the pituitary gland than in other tissues . it is well known that 
 nesfatin-1/ nucb2 mrna is abundantly expressed in the hypothalamus ( oh - i et al . , 2006 ) . furthermore , recent reports 
 have shown that nesfatin-1/nucb2 is also expressed in the pituitary ( foo et al . , 2008 ) . these results raise the 
 possibility that nesfatin-1 produced in the brain may control the metabolic 
 homeostasis not only through the hypothalamus , but also through the pituitary . in 
 other words , nesfatin-1 protein produced by the pituitary may move to target cells 
 in the various organs through the bloodstream , similar to other hormones produced by 
 the pituitary gland . we also investigated whether testosterone regulates nesfatin-1/nucb2 expression in 
 hypothalamus and pituitary gland . in our in vivo experiment , nucb2 mrna expression 
 was dramatically decreased in the pituitary gland after castration and significantly 
 increased with testosterone , suggesting testosterone regulate nucb2 mrna expression 
 in the pituitary gland . in the hypothalamus gland , nucb2 mrna expression was not 
 changed after castration , but significantly decreased with testosterone , suggesting 
 testosterone also regulate nucb2 mrna expression in the hypothalamus . many cells in 
 the hypothalamus and pituitary gland contain testosterone receptors ( jin and yang , 2014 ) , indicating that injected 
 testosterone may affect the activity of both the hypothalamus and pituitary gland by 
 binding to the receptor . next , we conducted an in vitro experiment to examine the direct 
 effects of testosterone on nucb2 mrna expression in the cultured hypothalamus and 
 pituitary gland . the expression of nucb2 mrna in the cultured hypothalamus tissues 
 was significantly decreased with testosterone in dose - dependent manner . these 
 results suggest that nucb2 mrna expression in the hypothalamus is directly 
 down - regulated by testosterone . by contract , in the cultured pituitary gland 
 tissues , the present results show the possibility that the reduced nesfatin-1/nucb2 
 expression in the hypothalamus in response to testosterone , which is produced and 
 secreted by the leydig cells in testis , may contribute to feeding behavior in male 
 and even in female mice . testosterone has been known to influence appetite control 
 and feeding behavior ( iwasa et al . , 2016 ; 
 bautista et al . , 2012 ) . hypothalamic mrna 
 levels of er , which plays pivotal roles in regulation of body weight and 
 metabolism , were decreased by chronic testosterone administration in rats ( iwasa et al . , 2016 ) . in female rats following 
 ovariectomy , a stabilization of food intake was not observed , but energy intake was 
 increased . by contrast , castration in males induced loss of body weight , whereas 
 castrated males given testosterone showed complete restoration of eating activity 
 ( ferreira et al . , we also have 
 previously reported that nucb2 expression in the pituitary gland of female mice was 
 dramatically decreased after ovariectomy and increased with injection of 
 progesterone and estradiol , respectively . the in vitro 
 experiment to elucidate the direct effect of progesterone and estradiol on nucb2 
 mrna expression showed nucb2 mrna expression was significantly increased with 
 estradiol and decreased with progesterone alone and progesterone and estradiol in 
 cultured pituitary tissue ( chung et al . , 
 2015 ) . the present study demonstrated that nesfatin-1/nucb2 was highly 
 expressed in the mouse pituitary and was regulated by testosterone . recently , it is 
 reported that nesfatin-1 production is greater in the reproductive organs , such as 
 the seminal gland , testis , uterus , and ovary in rats with metabolic syndrome ( kim et al . these results suggest that metabolic syndrome could also influence 
 the nesfatin-1/nucb2 expression in the pituitary gland regulating appetite and 
 energy homeostasis . estradiol has been known to inhibit feeding in rats with a 
 reduction of food intake around the time of ovulation , when estradiol presents its 
 highest levels . men with higher testosterone levels compared to women accumulate 
 more fat in the intraabdominal depot , suggesting that testosterone stimulates 
 feeding ( bautista et al . , 2012 ) . the present study demonstrated that nesfatin-1/nucb2 was expressed in the 
 hypothalamus and pituitary gland of male mouse and its mrna expression in the 
 hypothalamus and pituitary gland was regulated by testosterone . this data suggests 
 that reproductive - endocrine regulation through hypothalamus - pituitary - testis axis 
 may contribute to nesfatin-1/nucb2 expression in the hypothalamus and pituitary 
 gland .
abstractnesfatin-1/nucb2 is known to take part in the control of the appetite and energy 
 metabolism . recently 
 , many reports have shown nesfatin-1/nucb2 expression and 
 function in various organs . 
 we previously demonstrated that nesfatin-1/nucb2 
 expression level is higher in the pituitary gland compared to other organs and 
 its expression is regulated by 17-estradiol and progesterone secreted from the 
 ovary . 
 however , currently no data exist on the expression of nesfatin-1/nucb2 
 and its regulation mechanism in the pituitary of male mouse . therefore , we 
 examined whether nesfatin-1/nucb2 is expressed in the male mouse pituitary and 
 if its expression is regulated by testosterone . as a result of pcr and western 
 blotting 
 , we found that a large amount of nesfatin-1/nucb2 was expressed in the 
 pituitary and hypothalamus . 
 the nucb2 mrna expression level in the pituitary was 
 decreased after castration , but not in the hypothalamus . 
 in addition , its mrna 
 expression level in the pituitary was increased after testosterone treatment in 
 the castrated mice , whereas , the expression level in the hypothalamus was 
 significantly decreased after the treatment with testosterone . 
 the in 
 vitro experiment to elucidate the direct effect of testosterone on 
 nucb2 mrna expression showed that nucb2 mrna expression was significantly 
 decreased with testosterone in cultured hypothalamus tissue , but increased with 
 testosterone in cultured pituitary gland . 
 the present study demonstrated that 
 nesfatin-1/nucb2 was highly expressed in the male mouse pituitary and was 
 regulated by testosterone . 
 this data suggests that reproductive - endocrine 
 regulation through hypothalamus - pituitary - testis axis may contribute to nucb2 
 mrna expression in the mouse hypothalamus and pituitary gland .