BACKGROUND
the arcuate nucleus  of the hypothalamus is a key integrative center for peripheral and central signals controlling appetite and metabolism  <cit> .
the arc is ideally situated to sense peripherally circulating humoral factors as it is located in a site in the central nervous system with an impaired blood-brain barrier, and arc neurons express high levels of cns receptors for circulating factors including leptin and insulin  <cit>  the best described anabolic neurons within the arcuate nucleus use neuropeptide y and agouti-related peptide  as neurotransmitters.
both npy and agrp stimulate food intake.
a parallel and opposing pathway is comprised of neurons expressing proopiomelanocortin  and cocaine- and amphetamine-regulated transcript .
both pomc and cart inhibit food intake.
efferent projections from npy/agrp and pomc/cart neurons to secondary energy homeostatic neurons in intra- and extra-hypothalamic sites are key features of energy homeostatic circuitry.
asb- <dig> belongs to the ankyrin repeat and suppressor of cytokine signaling  box containing protein family that has  <dig> members .
all asb proteins have two functional domains, a c-terminal socs box region of approximately  <dig> amino acids and an up stream ankyrin repeat region  <cit> .
the ankyrin repeat domain directs specific protein-protein interactions while the socs box region serves as an adapter to mediate the degradation of proteins targeted by the ankyrin repeat region  <cit> .
the  <dig> asb proteins vary in the numbers of ankyrin repeats, and contain other novel regions, corresponding to differing target proteins to which they bind.
asb- <dig> has nine ankyrin repeats n-terminal to its socs box  <cit> .
microarray analysis of arc rnas from fed and fasted rats revealed that asb- <dig> was down-regulated by fasting.
quantitative pcr showed that asb- <dig> mrna was also down-regulated in the arc of the genetically obese zucker rat, a model of long-term energy imbalance.
expression of asb- <dig> mrna was restricted to neuroanatomical areas in the hypothalamus and amygdala associated with energy homeostasis.
notably, asb- <dig> mrna was expressed in the two types of arc neurons critical to feeding behavior, pomc and npy neurons  <cit> .
however, the target of asb- <dig> in these neurons remains unknown.
insulin receptor substrate  proteins represent a family of adapter proteins that play a central role in signal transduction by insulin, igf-i, and a growing number of cytokines including leptin  <cit> .
to date, six irs proteins have been identified   <cit> .
although the different irs proteins vary in size, they share a number of structural and functional characteristics.
the nh <dig> -terminal of each protein consists of a homologous pleckstrin homology  domain and a phosphotyrosine binding region .
the ph and ptb domains mediate specific interactions with the insulin, igf-i and cytokine receptors.
the cooh-terminal portion of each protein contains a number of tyrosine phosphorylation sites  <cit> .
tyrosine phosphorylation sites in the c-terminal portion of irs are phosphorylated upon the activation of receptors by hormones or cytokines.
tyr-phosphorylated irs proteins serve as signaling scaffolds that propagate hormone action through binding of src homology  <dig>  domain-containing proteins, such as the p <dig> regulatory subunit of pi3k  <cit> .
the different members of irs proteins have different tissue distributions.
irs <dig> mrna is found to be highly expressed in the hypothalamus and amygdala of the brain  <cit> .
however, the neuronal types that express irs <dig> remain to be identified.
in the present study we first examined whether pomc and npy neurons express irs <dig> mrna.
we then examined the colocalization and interaction of asb- <dig> with irs <dig> in neurons and the involvement of asb- <dig> in the modulation of insulin signaling.
RESULTS
colocalization of asb- <dig> with irs4
using in situ hybridization irs <dig> mrna was found to be expressed in the arcuate nucleus, ventromedial nucleus , dorsomedial nucleus, lateral hypothalamus and amygdala, which is consistent with that reported by numan et al  <cit> .
except for the vmh, which exhibits no asb- <dig> expression, the expression pattern of asb- <dig> was consistent with that of irs <dig> .
dark field photomicrograph showed that in the arcuate nucleus the expression pattern of asb- <dig> was similar to that of irs <dig> .
interaction of asb- <dig> with irs4
colocalization of asb- <dig> with irs <dig> in the hypothalamus implies that these molecules may have functional interactions.
in order to examine this possibility we first performed co-immunoprecipitation  experiments using hek <dig> cells.
myc-tagged full length mouse asb- <dig>  and flag-tagged full length mouse irs <dig>  were co-expressed in hek <dig> cells and co-immunoprecipitated using either anti-flag or anti-myc antibody conjugated to agarose.
as shown in figure 3a, myc-asb- <dig> co-precipitated with flag-irs <dig> when flag-irs <dig> was precipitated with anti-flag antibody, and myc-asb- <dig> was detected with anti-myc antibody by western blotting; similarly flag-irs <dig> co-precipitated with myc-asb- <dig> when myc-asb- <dig> was precipitated with anti-myc antibody, and flag-irs <dig> was detected with anti-flag antibody .
normal igg was used as a negative control.
these results demonstrate that asb- <dig> interacts with irs <dig> in hek <dig> cells.
it has been reported that hek <dig> cells have endogenous irs <dig> expression  <cit> .
we further examined the interaction between asb- <dig> and irs <dig> by co-immunoprecipitating endogenous irs <dig> with transfected myc-asb- <dig> in these cells.
when myc-asb- <dig> was precipitated with myc antibody, irs <dig> could be detected with anti irs <dig> antibody .
these results showed that myc-asb- <dig> interacted with endogenous irs <dig> in hek cells.
finally we performed co-immunoprecipitation with rat hypothalamic extracts to confirm the interaction in vivo since the transfected experiments have limitations.
as shown in figure 3d, when irs <dig> was precipitated with anti irs <dig> antibody, asb- <dig> could be detected using anti asb- <dig> antibody, indicating that asb- <dig> interacts with irs <dig> in the rat hypothalamus.
effect of asb- <dig> on the levels, ubiquitination of irs <dig> and insulin signaling
we next examined the effect of asb- <dig> expression on irs <dig> levels.
when myc-asb- <dig> and flag-irs <dig> were co-expressed in hek <dig> cells, asb- <dig> expression reduced irs <dig> protein levels dose-dependently.
deletion of the socs box region  abolished the ability of asb- <dig> to affect irs <dig> levels .
when asb- <dig> was over expressed in these cells it also decreased endogenous irs <dig> levels dose-dependently .
the general model of action of asb proteins involves ubiquitination of proteins targeted by the ankyrin repeat region of the molecule.
we next examined the effects of asb- <dig> on ubiquitination of irs <dig>  as shown in figure 4e, using chinese hamster overy  cells, asb- <dig> increased the ubiquitination of irs <dig>  deletion of the socs box region abolished this effect.
since irs <dig> has been reported to be involved in the insulin signaling pathway  <cit> , and our data showed that asb- <dig> decreased irs <dig> level, we next examined the effect of asb- <dig> on down-stream signaling of isr <dig>  in hek <dig> cells expression of asb- <dig> decreased both basal and insulin-stimulated phosphorylation of akt at thr <dig> .
discussion
the results of the current study demonstrate that insulin receptor substrate  <dig> co-localizes with asb- <dig> within areas of the hypothalamus and amygdala that regulate caloric intake and energy expenditure.
asb- <dig> regulates irs <dig> levels by directing its degradation via ubiquitination and decreasing the down-stream signal of irs <dig>  five separate observations support this conclusion: 1) asb- <dig> and irs <dig> are both expressed in the arcuate nucleus, the dorsomedial nucleus of the hypothalamus and the amygdala; 2) asb- <dig> colocalizes with irs <dig>  and both molecules are expressed in pomc and npy neurons of the hypothalamus; 3) the molecules co-precipitate in vitro and in vivo; 4) increased expression of full-length asb- <dig> causes decreased levels of irs4; 5) expression of full-length asb- <dig> increases ubiquitination of irs <dig> and decreased phospho-akt at thr <dig>
although irs <dig> is thought to have a role in signal transduction similar to other member of the irs family, the tissue distribution of irs <dig> is substantially different than that of other irs molecules.
the restricted expression of irs <dig> mrna in the hypothalamus of the brain implies that irs <dig> might be involved in the regulation of energy homeostasis.
the current study further demonstrated that irs <dig> is expressed in both npy and pomc neurons of the arcuate nucleus.
these two types of neurons regulate food intake and metabolism when activated by insulin and leptin  <cit> .
the expression of irs <dig> in npy and pomc neurons implies that irs <dig> may function to mediate the action of insulin and leptin in these neuronal types.
the co-localization of asb- <dig> and irs <dig> provides an opportunity for these molecules to interact and modulate the signal transduction of insulin and/or leptin.
the co-expression of asb- <dig> and irs <dig> in amygdala is worth noting.
the amygdala is primarily involved in emotions such as fear, anger and pleasure, memory etc  <cit> .
the amygdala may also be involved in the role of motivation and reward in food intake  <cit> .
the amygdala sends projections to the lateral hypothalamic area  <cit> , which forms part of the feeding circuit and has been linked to initiation of feeding  <cit> .
the neuronal type that express asb- <dig> and irs <dig>  and the roles these two proteins play in amygdala remain unknown.
ankyrin repeat and socs box-containing protein- <dig>  was originally identified by a database search using a socs box consensus sequence  <cit> .
there are five major protein families that have socs boxes.
they differ in the type of domain that is upstream of the socs box.
while several asb proteins have been identified within the setting of a physiological process, no common functional theme is obvious.
in the context of metabolic regulation, socs- <dig> down-regulates leptin receptor and insulin receptor signaling  <cit> .
brain-specific socs- <dig> knockout mice showed elevated leptin sensitivity, were resistant to high fat diet induced obesity and had attenuated insulin resistance  <cit> .
socs- <dig> belongs to the sh <dig> domain protein family.
asb- <dig> is an adipocyte-specific protein that modulates aps interaction with the insulin receptor  <cit> .
utilizing a transgenic approach, we have recently reported that over-expression of asb- <dig> in hypothalamic pomc neurons of mice produced a lean phenotype by increasing locomotive activity and metabolic rate in spite of the fact that the transgenic animals consumed significantly more food than wildtype littermates  <cit> .
pomc-asb- <dig> mice had higher rates of oxygen consumption and output of carbon dioxide than wildtype littermates, indicating that over expression of asb- <dig> in pomc neurons increased energy expenditure.
consistent with the lean phenotype, blood leptin levels were lower in transgenic mice.
the cellular basis for these observations is currently unknown and prompted a search for potential protein binding partners for asb- <dig>  in the current study, insulin receptor substrate- <dig> was observed to co-precipitate with asb- <dig>  asb- <dig> increased degradation of irs <dig> via ubiquitination in cell lines.
like all irs family members, irs <dig> acts as an adaptor and is involved in the signal transduction of insulin, leptin and other cytokines and growth factors.
in hek cells and neuronal n <dig> cells, exposure to insulin or leptin has been reported to increase tyrosine phosphorylation of irs <dig>  <cit> .
irs <dig> interacts with the regulatory p <dig> subunit of phosphatidylinositol 3-kinase which then activates its downstream signal, akt/pkb  <cit> .
the inhibition of insulin signaling by asb- <dig> via interacting with irs <dig> may partially explain the hyperphagia in the pomc-asb- <dig> transgenic mice since insulin can inhibit food intake in the brain.
all members of asb protein family have two basic domains: the ankyrin repeat and the socs box.
the ankyrin repeat interacts specifically with targeted proteins.
the socs box region binds elongin bc in a protein complex  <cit> .
this protein complex has e <dig> ubiquitin ligase activity that facilitates polyubiquitination and proteasomal degradation of proteins bound by the upstream regions of the socs proteins  <cit> .
asb- <dig> is expected to follow this mechanism of action to increase ubiquitination of its target.
the current study showed that asb- <dig> interacts with irs <dig>  increases irs <dig> ubiquitination and decreases irs <dig> protein levels, probably by increasing its degradation.
deletion of the socs box of asb- <dig> abolishes both the decrement in irs <dig> levels and ubiquitination of irs <dig>  indicating that the socs box plays a key role in leading irs <dig> to the degradation pathway.
these observations are consistent with the general model of action of asb proteins.
the action of asb- <dig> to direct degradation of irs <dig> may be a mechanism by which npy and pomc neurons modulate the sensitivity to circulating levels of insulin.
CONCLUSIONS
this study demonstrated that asb- <dig> co-localizes and interacts with irs <dig> in hypothalamic neurons.
the interaction of asb- <dig> with irs <dig> in cell lines mediates the degradation of irs <dig> and decreases insulin signaling.

 the arcuate nucleus of the hypothalamus is a key integrative center for peripheral and central signals controlling appetite and metabolism. the arc is ideally situated to sense peripherally circulating humoral factors as it is located in a site in the central nervous system with an impaired blood-brain barrier, and arc neurons express high levels of cns receptors for circulating factors including leptin and insulin the best described anabolic neurons within the arcuate nucleus use neuropeptide y and agouti-related peptide as neurotransmitters. both npy and agrp stimulate food intake. a parallel and opposing pathway is comprised of neurons expressing proopiomelanocortin and cocaine- and amphetamine-regulated transcript. both pomc and cart inhibit food intake. efferent projections from npy/agrp and pomc/cart neurons to secondary energy homeostatic neurons in intra- and extra-hypothalamic sites are key features of energy homeostatic circuitry. asb- belongs to the ankyrin repeat and suppressor of cytokine signaling box containing protein family that has members. all asb proteins have two functional domains, a c-terminal socs box region of approximately amino acids and an up stream ankyrin repeat region. the ankyrin repeat domain directs specific protein-protein interactions while the socs box region serves as an adapter to mediate the degradation of proteins targeted by the ankyrin repeat region. the asb proteins vary in the numbers of ankyrin repeats, and contain other novel regions, corresponding to differing target proteins to which they bind. asb- has nine ankyrin repeats n-terminal to its socs box. microarray analysis of arc rnas from fed and fasted rats revealed that asb- was down-regulated by fasting. quantitative pcr showed that asb- mrna was also down-regulated in the arc of the genetically obese zucker rat, a model of long-term energy imbalance. expression of asb- mrna was restricted to neuroanatomical areas in the hypothalamus and amygdala associated with energy homeostasis. notably, asb- mrna was expressed in the two types of arc neurons critical to feeding behavior, pomc and npy neurons. however, the target of asb- in these neurons remains unknown. insulin receptor substrate proteins represent a family of adapter proteins that play a central role in signal transduction by insulin, igf-i, and a growing number of cytokines including leptin. to date, six irs proteins have been identified. although the different irs proteins vary in size, they share a number of structural and functional characteristics. the nh -terminal of each protein consists of a homologous pleckstrin homology domain and a phosphotyrosine binding region. the ph and ptb domains mediate specific interactions with the insulin, igf-i and cytokine receptors. the cooh-terminal portion of each protein contains a number of tyrosine phosphorylation sites. tyrosine phosphorylation sites in the c-terminal portion of irs are phosphorylated upon the activation of receptors by hormones or cytokines. tyr-phosphorylated irs proteins serve as signaling scaffolds that propagate hormone action through binding of src homology domain-containing proteins, such as the p regulatory subunit of pi3k. the different members of irs proteins have different tissue distributions. irs mrna is found to be highly expressed in the hypothalamus and amygdala of the brain. however, the neuronal types that express irs remain to be identified. in the present study we first examined whether pomc and npy neurons express irs mrna. we then examined the colocalization and interaction of asb- with irs in neurons and the involvement of asb- in the modulation of insulin signaling.  colocalization of asb- with irs4 using in situ hybridization irs mrna was found to be expressed in the arcuate nucleus, ventromedial nucleus, dorsomedial nucleus, lateral hypothalamus and amygdala, which is consistent with that reported by numan et al. except for the vmh, which exhibits no asb- expression, the expression pattern of asb- was consistent with that of irs. dark field photomicrograph showed that in the arcuate nucleus the expression pattern of asb- was similar to that of irs. interaction of asb- with irs4 colocalization of asb- with irs in the hypothalamus implies that these molecules may have functional interactions. in order to examine this possibility we first performed co-immunoprecipitation experiments using hek cells. myc-tagged full length mouse asb- and flag-tagged full length mouse irs were co-expressed in hek cells and co-immunoprecipitated using either anti-flag or anti-myc antibody conjugated to agarose. as shown in figure 3a, myc-asb- co-precipitated with flag-irs when flag-irs was precipitated with anti-flag antibody, and myc-asb- was detected with anti-myc antibody by western blotting; similarly flag-irs co-precipitated with myc-asb- when myc-asb- was precipitated with anti-myc antibody, and flag-irs was detected with anti-flag antibody. normal igg was used as a negative control. these results demonstrate that asb- interacts with irs in hek cells. it has been reported that hek cells have endogenous irs expression. we further examined the interaction between asb- and irs by co-immunoprecipitating endogenous irs with transfected myc-asb- in these cells. when myc-asb- was precipitated with myc antibody, irs could be detected with anti irs antibody. these results showed that myc-asb- interacted with endogenous irs in hek cells. finally we performed co-immunoprecipitation with rat hypothalamic extracts to confirm the interaction in vivo since the transfected experiments have limitations. as shown in figure 3d, when irs was precipitated with anti irs antibody, asb- could be detected using anti asb- antibody, indicating that asb- interacts with irs in the rat hypothalamus. effect of asb- on the levels, ubiquitination of irs and insulin signaling we next examined the effect of asb- expression on irs levels. when myc-asb- and flag-irs were co-expressed in hek cells, asb- expression reduced irs protein levels dose-dependently. deletion of the socs box region abolished the ability of asb- to affect irs levels. when asb- was over expressed in these cells it also decreased endogenous irs levels dose-dependently. the general model of action of asb proteins involves ubiquitination of proteins targeted by the ankyrin repeat region of the molecule. we next examined the effects of asb- on ubiquitination of irs as shown in figure 4e, using chinese hamster overy cells, asb- increased the ubiquitination of irs deletion of the socs box region abolished this effect. since irs has been reported to be involved in the insulin signaling pathway, and our data showed that asb- decreased irs level, we next examined the effect of asb- on down-stream signaling of isr in hek cells expression of asb- decreased both basal and insulin-stimulated phosphorylation of akt at thr. discussion the results of the current study demonstrate that insulin receptor substrate co-localizes with asb- within areas of the hypothalamus and amygdala that regulate caloric intake and energy expenditure. asb- regulates irs levels by directing its degradation via ubiquitination and decreasing the down-stream signal of irs five separate observations support this conclusion: 1) asb- and irs are both expressed in the arcuate nucleus, the dorsomedial nucleus of the hypothalamus and the amygdala; 2) asb- colocalizes with irs and both molecules are expressed in pomc and npy neurons of the hypothalamus; 3) the molecules co-precipitate in vitro and in vivo; 4) increased expression of full-length asb- causes decreased levels of irs4; 5) expression of full-length asb- increases ubiquitination of irs and decreased phospho-akt at thr although irs is thought to have a role in signal transduction similar to other member of the irs family, the tissue distribution of irs is substantially different than that of other irs molecules. the restricted expression of irs mrna in the hypothalamus of the brain implies that irs might be involved in the regulation of energy homeostasis. the current study further demonstrated that irs is expressed in both npy and pomc neurons of the arcuate nucleus. these two types of neurons regulate food intake and metabolism when activated by insulin and leptin. the expression of irs in npy and pomc neurons implies that irs may function to mediate the action of insulin and leptin in these neuronal types. the co-localization of asb- and irs provides an opportunity for these molecules to interact and modulate the signal transduction of insulin and/or leptin. the co-expression of asb- and irs in amygdala is worth noting. the amygdala is primarily involved in emotions such as fear, anger and pleasure, memory etc. the amygdala may also be involved in the role of motivation and reward in food intake. the amygdala sends projections to the lateral hypothalamic area, which forms part of the feeding circuit and has been linked to initiation of feeding. the neuronal type that express asb- and irs and the roles these two proteins play in amygdala remain unknown. ankyrin repeat and socs box-containing protein- was originally identified by a database search using a socs box consensus sequence. there are five major protein families that have socs boxes. they differ in the type of domain that is upstream of the socs box. while several asb proteins have been identified within the setting of a physiological process, no common functional theme is obvious. in the context of metabolic regulation, socs- down-regulates leptin receptor and insulin receptor signaling. brain-specific socs- knockout mice showed elevated leptin sensitivity, were resistant to high fat diet induced obesity and had attenuated insulin resistance. socs- belongs to the sh domain protein family. asb- is an adipocyte-specific protein that modulates aps interaction with the insulin receptor. utilizing a transgenic approach, we have recently reported that over-expression of asb- in hypothalamic pomc neurons of mice produced a lean phenotype by increasing locomotive activity and metabolic rate in spite of the fact that the transgenic animals consumed significantly more food than wildtype littermates. pomc-asb- mice had higher rates of oxygen consumption and output of carbon dioxide than wildtype littermates, indicating that over expression of asb- in pomc neurons increased energy expenditure. consistent with the lean phenotype, blood leptin levels were lower in transgenic mice. the cellular basis for these observations is currently unknown and prompted a search for potential protein binding partners for asb- in the current study, insulin receptor substrate- was observed to co-precipitate with asb- asb- increased degradation of irs via ubiquitination in cell lines. like all irs family members, irs acts as an adaptor and is involved in the signal transduction of insulin, leptin and other cytokines and growth factors. in hek cells and neuronal n cells, exposure to insulin or leptin has been reported to increase tyrosine phosphorylation of irs. irs interacts with the regulatory p subunit of phosphatidylinositol 3-kinase which then activates its downstream signal, akt/pkb. the inhibition of insulin signaling by asb- via interacting with irs may partially explain the hyperphagia in the pomc-asb- transgenic mice since insulin can inhibit food intake in the brain. all members of asb protein family have two basic domains: the ankyrin repeat and the socs box. the ankyrin repeat interacts specifically with targeted proteins. the socs box region binds elongin bc in a protein complex. this protein complex has e ubiquitin ligase activity that facilitates polyubiquitination and proteasomal degradation of proteins bound by the upstream regions of the socs proteins. asb- is expected to follow this mechanism of action to increase ubiquitination of its target. the current study showed that asb- interacts with irs increases irs ubiquitination and decreases irs protein levels, probably by increasing its degradation. deletion of the socs box of asb- abolishes both the decrement in irs levels and ubiquitination of irs indicating that the socs box plays a key role in leading irs to the degradation pathway. these observations are consistent with the general model of action of asb proteins. the action of asb- to direct degradation of irs may be a mechanism by which npy and pomc neurons modulate the sensitivity to circulating levels of insulin.  this study demonstrated that asb- co-localizes and interacts with irs in hypothalamic neurons. the interaction of asb- with irs in cell lines mediates the degradation of irs and decreases insulin signaling. 
the arcuate nucleus of the hypothalamus regulates food intake. ankyrin repeat and socs box containing protein is expressed in neuropeptide y and proopiomelanocortin neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. in hek cells co-transfected with myc-tagged asb- and flag-tagged irs asb- co-immunoprecipitated with irs4; in these cells endogenous irs also co-immunoprecipitated with transfected myc-asb-4; furthermore, asb- co-immunoprecipitated with irs in rat hypothalamic extracts. these data demonstrated that asb- co-localizes and interacts with irs in hypothalamic neurons. insulin receptor substrate is an adaptor molecule involved in the signal transduction by both insulin and leptin. the interaction of asb- with irs in cell lines mediates the degradation of irs and decreases insulin signaling. double in situ hybridization showed that irs colocalized with asb- and both asb- and irs mrna were expressed in proopiomelanocortin and neuropeptide y neurons within the arcuate nucleus of the hypothalamus. in the present study we examined the colocalization and interaction of asb- with irs and the involvement of asb- in insulin signaling. in hek cells over expression of asb- decreased irs protein levels and deletion of the socs box abolished this effect. asb- increased the ubiquitination of irs4; deletion of socs box abolished this effect. in situ hybridization showed that the expression pattern of asb- was consistent with that of irs in the rat brain.
 the arcuate nucleus of the hypothalamus regulates food intake. ankyrin repeat and socs box containing protein is expressed in neuropeptide y and proopiomelanocortin neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. however, the target protein of asb- in these neurons remains unknown. insulin receptor substrate is an adaptor molecule involved in the signal transduction by both insulin and leptin. in the present study we examined the colocalization and interaction of asb- with irs and the involvement of asb- in insulin signaling.  in situ hybridization showed that the expression pattern of asb- was consistent with that of irs in the rat brain. double in situ hybridization showed that irs colocalized with asb- and both asb- and irs mrna were expressed in proopiomelanocortin and neuropeptide y neurons within the arcuate nucleus of the hypothalamus. in hek cells co-transfected with myc-tagged asb- and flag-tagged irs asb- co-immunoprecipitated with irs4; in these cells endogenous irs also co-immunoprecipitated with transfected myc-asb-4; furthermore, asb- co-immunoprecipitated with irs in rat hypothalamic extracts. in hek cells over expression of asb- decreased irs protein levels and deletion of the socs box abolished this effect. asb- increased the ubiquitination of irs4; deletion of socs box abolished this effect. expression of asb- decreased both basal and insulin-stimulated phosphorylation of akt at thr  these data demonstrated that asb- co-localizes and interacts with irs in hypothalamic neurons. the interaction of asb- with irs in cell lines mediates the degradation of irs and decreases insulin signaling. 