document: alan aderem ( institute for systems biology , seattle , washington ) , which were originally generated by dr . 
   shizuo akira ( research institute for microbial diseases , osaka university , osaka , japan ) . 
   lcn2 mice were generated by gene targeting in embryonic stem ( es ) cells from mouse strain 129 , and targeted es cells were injected into c57bl/6 blastocysts as described previously ( 12 ) . 
   lcn2-null mice were backcrossed onto the b6 background for 10 generations before mice were used for the experiments . 
   heterozygous mating scheme was used to generate wild - type and lcn2 mice for the experiments . 
   aminals were housed in a specific pathegon - free facility at the university of minnesota . 
   animal handling followed the national institutes of health guidelines , and experimental procedures were approved by the university of minnesota animal care and use committee . 
   age - matched male wild - type and lcn2 mice were allocated into groups ( 34 mice / cage ) and fed an hfd ( fat calories : 60% ) obtained from bio - serv ( f3282 ; new brunswick , nj ) or a regular chow diet ( rcd ) , with free access to water for all studies . in the adaptive thermogenesis study , 
   age - matched wild - type and lcn2 mice on an rcd were exposed to 4c , with free acess to diet and water . 
   rectal temperature of the mice was measured at the indicated time points using a microtherma thermometer with rectal probe for mice ( braintree scientific , braintree , ma ) . during the experiemental period of hfd feeding , 
   glucose and insulin tolerance tests were conducted by the intraperitoneal injection of glucose and insulin . 
   mice were fasted overnight ( 12 h ) for the glucose tolerance test ( gtt ) and for 6 h for insulin tolerance tests ( itts ) . 
   gtts and itts were conducted by intraperitoneal injection of glucose ( 1 mg / g body wt ) or insulin ( 0.75 units / kg body wt ) with blood collection via the tail vein at 0 , 15 , 30 , 60 , 90 , and 120 min . 
   briefly , frozen liver tissue ( 100 mg ) was homogenized in 1 ml water . 
   an aliquot of the organic phase was collected and dried with nitrogen and then dissolved in isopropanol alcohol containing 1% triton . 
   preparation of isolated adipose cells from wild - type and lcn2 mice was performed as described previously ( 14,15 ) . 
   after mincing , epididymal fat pads were digested with collagenase ( 2 mg / ml solution ) in digestion vials containing krebs - ringer bicarbonate hepes buffer , ph 7.4 ; 200 nmol / l adenosine ; and 3.5% bsa . after a 2-h digestion , adipose cells were separated by centrifugation at 1,200 rpm for 10 min and washed twice with krebs - ringer bicarbonate hepes buffer . 
   adipose tissue was obtained from the epididymal and inguinal fat pad of wild - type and lcn2 mice fed an rcd . 
   tissue samples ( 2030 mg ) were immediately fixed in osmium tetroxide and incubated in a water bath at 37c for 48 h , as described previously ( 16 ) , and then adipose cell size was determined by a beckman coulter multisizer iii with a 400-m aperture . 
   the range of cell sizes that can effectively be measured using this aperture is 20240 m . 
   after collection of pulse sizes , the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the x - axis . 
   the figures were generated using graphpad prism 5.01 for windows ( graphpad software , la jolla , ca ) . 
   differences in parameters between wild - type and lcn2 mice were evaluated using a two - group t test with a 0.05 two - sided significance level . 
   during the experiemental period of hfd feeding , glucose and insulin tolerance tests were conducted by the intraperitoneal injection of glucose and insulin . 
   mice were fasted overnight ( 12 h ) for the glucose tolerance test ( gtt ) and for 6 h for insulin tolerance tests ( itts ) . 
   gtts and itts were conducted by intraperitoneal injection of glucose ( 1 mg / g body wt ) or insulin ( 0.75 units / kg body wt ) with blood collection via the tail vein at 0 , 15 , 30 , 60 , 90 , and 120 min . 
   briefly , frozen liver tissue ( 100 mg ) was homogenized in 1 ml water . 
   an aliquot of the organic phase was collected and dried with nitrogen and then dissolved in isopropanol alcohol containing 1% triton . 
   preparation of isolated adipose cells from wild - type and lcn2 mice was performed as described previously ( 14,15 ) . after mincing , 
   epididymal fat pads were digested with collagenase ( 2 mg / ml solution ) in digestion vials containing krebs - ringer bicarbonate hepes buffer , ph 7.4 ; 200 nmol / l adenosine ; and 3.5% bsa . after a 2-h digestion , adipose cells were separated by centrifugation at 1,200 rpm for 10 min and washed twice with krebs - ringer bicarbonate hepes buffer . after the final wash , adipose cells were collected for rna extraction . 
   adipose tissue was obtained from the epididymal and inguinal fat pad of wild - type and lcn2 mice fed an rcd . 
   tissue samples ( 2030 mg ) were immediately fixed in osmium tetroxide and incubated in a water bath at 37c for 48 h , as described previously ( 16 ) , and then adipose cell size was determined by a beckman coulter multisizer iii with a 400-m aperture . 
   the range of cell sizes that can effectively be measured using this aperture is 20240 m . 
   after collection of pulse sizes , the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the x - axis . 
   the figures were generated using graphpad prism 5.01 for windows ( graphpad software , la jolla , ca ) . 
   differences in parameters between wild - type and lcn2 mice were evaluated using a two - group t test with a 0.05 two - sided significance level . 
   at 3 weeks of age , male wild - type and lcn2 mice were fed either an hfd for 12 weeks or rcd . throughout the experimental period , 
   body weight was significantly different between wild - type and lcn2 mice fed an hfd ( fig . 
   lcn2 mice were significantly heavier than wild - type mice on either an hfd or rcd . 
   the weight of white and brown fat pads was significantly higher in hfd - fed ( fig . 
   1d ) mice than that in wild - type mice . both hfd - fed and aged lcn2 mice showed a significantly higher weight of liver , heart , kidney , and spleen ( fig . 
   similar metabolic phenotypes were also observed in female lcn2 mice ( data not shown ) . 
   however , the overall growth did not appear to be significantly affected , as the body length and bone length ( trabecular ) were not significantly different between the genotypes ( data not shown ) . 
   figure 1 g and h demonstrated the fat - cell size distributions of wild - type and lcn2 mice on an rcd at 15 weeks of age , as measured by multisizer analysis using cells isolated from osmium - fixed epididymal and inguinal adipose tissue . 
   the average peak diameter of the large - cell population was significantly larger for lcn2 mice compared with wild - type mice , indicating that lcn2 adipose tissue contains larger adipose cells . 
   growth curve , weight of white and brown fat pads and organs in lcn2 mice . 
   growth curve of lcn2 mice on an hfd ( n = 1014 ) ( a ) and rcd ( n = 13 ) ( b ) . 
   fat pad weight of lcn2 mice on an hfd at 16 weeks of age ( c ) and on rcd at 30 weeks of age ( d ) . 
   organ weight of lcn2 mice on hfd at 16 weeks of age ( e ) and on rcd at 30 weeks of age ( f ) . 
   * p < 0.05 ; * * p < 0.01 . fat - cell size distribution of mice on rcd diet at 15 weeks of age ( g and h ) . 
   epi : epididymal ; ing : inguinal . because lcn2-deficient mice gained more body weight and increased adiposity , particularly when mice were fed an hfd or aged , we examined food intake and energy expenditure in lcn2 mice . 
   an analysis of indirect calorimetry measurements showed that food intake , ambulatory activity , vo2 , and rq were not significantly different between rcd - fed wild - type and lcn2 mice at 18 weeks of age ( fig . 
   we then assessed thermogenic activity of brown adipose tissue ( bat ) under thermoneutral and thermal stress conditions . 
   body temperature was measured in an ambient temperature of 28c and 22c as well as during acute exposure to 4c . 
   wild - type and lcn2 mice at 12 weeks of age exhibited a similar body temperature in an ambient temperature of 28c ( wild type , 38.34  0.34 ; lcn2 , 38.2  0.30 ) . 
   however , female lcn2 mice had a significantly lower rectal temperature than wild - type mice ( fig . 
   2a ) , while male lcn2 mice had a trend toward decrease in rectal temperature when kept in an ambient temperature of 22c . 
   more strikingly , lcn2 mice displayed cold sensitivity and could not survive when exposed to 4c for > 10 h. lcn2 mice ( seven of seven ) died after being exposed to 4c for 12 h , while all of the wild - type mice ( n = 7 ) in the experiment survived . 
   when acutely exposed to 4c , rectal temperature of lcn2 mice dropped significantly within 3 h compared with wild - type mice ( fig . 
   . five - hour cold exposure caused a 10-fold increase in lcn2 gene expression in adipose tissue in wild - type mice ( fig . 
   a : basal rectal body temperature of 8-week - old male and female wild - type and lcn2 mice on an rcd ( n = 710 ) measured during the daytime in an ambient temperature of 22c . 
   b : body temperature curve of lcn2 mice ( n = 8) and wild - type mice ( n = 7 ) exposed to 4c . 
   c : gene expression of lcn2 in wat of wild - type mice exposed to 4c for 5 h. d : gene expression of ucp-1 and pgc-1 in bat and ppar , cpt-1 , tfam , and nrf1 in skeletal muscle of hfd - fed mice ( n = 6 ) . 
   g : gene expression of hsl in bat and wat of mice exposed to 4c for 5 h. h : gene expression of mitochondrial genes in bat of lcn2 mice under the hfd condition . 
   * p < 0.05 ; * * p < 0.01 . to explore the possible mechanisms for cold intolerance in lcn2 mice , uncoupling protein ( ucp)-1mediated thermogenic activity and capacity of bat 
   we observed that upon hfd feeding , lcn2 mice expressed significantly lower levels of ucp-1 and peroxisome proliferator  activated receptor ( ppar )  coactivator ( pgc-1) genes in bat than wild - type mice mice ( fig . 
   however , ucp-1 and pgc-1 expression in bat were markedly stimulated at an even higher level in lcn2 mice , as wild - type mice after exposure to 4c for 5 h ( fig . 
   figure 2f showed that the protein expression levels of adipose triglyceride lipase ( atgl ) in white adipose tissue ( wat ) were slightly reduced in lcn2 mice under the hfd and cold condition compared with wild - type mice . however , cold - induced hsl gene expression in bat and wat was completely diminished in lcn2 mice ( fig . 
   lastly , we investigated the mitochondrial oxidative capacity of bat , as this function of bat is critical for providing the energy for thermogenesis . 
   interestingly , we observed that there was a significant reduction in the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function , such as nuclear respiratory factor ( nrf)1 , transcription factor a , mitochondrial ( tfam ) , atp synthase 5 ( atp5 ) , cytochrome c oxidase ( cox ) 4 , and carnitine palmitoyltransferase ( cpt)-1 in bat of lcn2 mice fed an hfd ( fig . 
   in addition , the expression of transcription factors ppar , nrf1 and tfam , and cpt-1 gene in skeletal muscle was also significantly downregulated in lcn2 mice fed an hfd ( fig . 
   2d ) . to assess the effects of lcn2 deficiency on systemic insulin sensitivity , gtts and itts were performed . 
   glucose clearance curves in response to glucose administration were significantly increased in hfd - fed ( fig . 
   3b ) , indicating that lcn2 mice are more glucose intolerant upon hfd feeding . in the itt , 
   increased insulin - stimulated glucose disposal curve was observed in lcn2 mice , indicating that lcn2 mice are more insulin resistant than wild - type controls when fed an hfd ( fig . 
   3d ) . analysis of serum metabolic parameters , as shown in table 1 , demonstrated that levels of fasting serum glucose and insulin were significantly elevated , while adiponectin levels were reduced in hfd - fed lcn2 mice when compared with wild - type controls . 
   serum levels of leptin and plasminogen activator inhibitor-1 were not significantly changed in lcn2 mice on an rcd , although serum leptin levels had a trend toward a decrease in hfd - fed lcn2 mice . 
   after adjustment to body weight , the serum levels of glucose , insulin , and adiponectin were consistently different at the statistically significant level , indicating that the changes in glucose tolerance and insulin sensitivity after hfd in lcn2 mice is not simply due to body weight effects . to further confirm that lcn2 mice have decreased insulin sensitivity , in vivo insulin - stimulated akt phosphorylation was evaluated . 
   wild - type and lcn2 mice on rcd were injected intraperitoneally with insulin , killed after 10 min , and tissues were collected for evaluation of akt phosohorylation . 
   consistently , insulin - stimulated akt phosphorylation in liver , muscle , and adipose cells was significantly reduced in lcn2 mice as compared with wild - type mice ( fig . 
   the examination of lcn2 effect on upstream signaling components of insulin signaling pathway demonstrated that the expression levels of insulin receptor substrate-1 protein were markedly reduced in wat of lcn2 mice on an hfd compared with wild - type mice ( fig . 
   this data further support that lcn2 mice developed more insulin resistance and lcn2 regulates insulin signaling activity likely at the upstream level . 
   gtts ( a ) and itts ( c ) conducted in lcn2 mice on an hfd ( n = 1012 , age 1415 weeks ) . 
   gtts ( b ) and itts ( d ) conducted in lcn2 mice on an rcd ( n = 1012 , age 2829 weeks ) . 
   serum metabolic parameters in wild - type and lcn2 mice data are means  se . 
   measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . 
   * p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . in vivo insulin - stimulated akt phosphorylation in lcn2 mice . 
   representative western blot analysis of serine - phosphorylated akt in muscle , liver , and isolated primary adipose cells from lcn2 ( n = 4 per group ) and wild - type mice ( n = 4 per group ) under basal and in vivo insulin - stimulated conditions . to test the role of lcn2 in inflammatory response , expression of lcn2 and inflammatory molecules was examined in adipose tissue and liver of hfd - induced wild - type and lcn2 mice . 
   the gene expression of proinflammatory cytokine tnf and monocyte chemoattractant protein ( mcp)-1 were upregulated , while expression of arginase 1 , an anti - inflammatory marker , was downregulated in adipose tissue of hfd - fed lcn2 mice compared with wild - type control mice ( fig . 
   downregulation of arginase 1 and interleukin ( il)-10 was also observed in the liver of hfd - fed lcn2 mice ( fig . 
   a : protein expression levels of lcn2 in adipose tissues of wild - type mice fed an rcd and hfd . 
   b : protein expression levels of lcn2 in bone marrow macrophages ( bm - mac ) isolated from wild - type mice fed an rcd and hfd . 
   c : gene expression of inflammatory molecules in adipose tissue and liver of wild - type and lcn2 mice fed on hfd . 
   * p < 0.05 . to investigate whether increased liver weight in lcn2 mice is associated with the development of fatty liver and dyslipidemia , liver triglyceride content and blood lipid profiles were measured . in comparison with wild - type controls , hfd - fed and 
   aged lcn2 mice demonstrated a significant increase in lipid accumulation as detected by oil - red o staining of the liver section ( fig . 
   the development of more severe fatty liver disease , together with elevated fasting blood glucose levels , suggests hepatic insulin resistance in lcn2 mice . 
   to elucidate hepatic glucose production in wild - type and lcn2-deficient mice fed an hfd , mice were fasted for 18 h and gene expression of two key gluconeogenic enzymes pepck and glucose-6-phosphatase ( g6pase ) were detected by quantitative pcr . 
   6c , expression levels of pepck1 , pepck2 , and g6pase genes were significantly higher in the liver of lcn2 mice than that in wild - type controls , suggesting that increased hepatic glucose production is attributed to hyperglycemia in lcn2 mice . 
   we next examined the hepatic capabilities for fatty acid synthesis and oxidation to explore the possible mechanism for the development of fatty liver in lcn2 mice . 
   6d , the lipogenic genes sterol regulatory element - binding protein-1c ( srebp-1c ) , acetyl - coa carboxylase 1 ( acc1 ) , spot 14 ( s14 ) , and scd-1 were expressed at a significantly higher level in hfd - fed lcn2 mice compared with wild - type controls , while genes involved in fatty acid oxidation such as ppar , cpt-1 , and acetyl - coa carboxylase 2 were similarly expressed between genotypes ( data not shown ) . therefore , the development of fatty liver results primarily from the increased capability for fatty acid synthesis in the liver in lcn2 mice . 
   b : liver triacyglyceride levels in lcn2 mice on an rcd ( n = 68 , age 30 weeks ) and an hfd ( n = 11 , age 1516 weeks ) . 
   c : the mrna expression of gluocneogenic genes in liver ( n = 6 ) . 
   d : the mrna expression of lipogenic genes in liver ( n = 6 ) . 
   the data are represented as means  se . * p < 0.05 ; * * p < 0.01 . 
   ( a high - quality digital color representation of this figure is available in the online issue . ) 
   compared with wild - type mice , lcn2 mice on an rcd had significantly higher serum levels of triglycerides , cholesterol , and free fatty acids . 
   the hfd feeding did not induce a further increase in serum levels of triglyceride and cholesterol but significantly augmented the levels of ldl and -hydroxybutyrate in lcn2 mice compared with wild - type controls . 
   the above data have suggested that lcn2-deficient mice became more insulin resistant upon hfd feeding and age . 
   since adipose tissue is a key site for insulin - regulated glucose / lipid metabolism and glucose disposal , we examined the expression of genes involved in lipid metabolism in primary adipose cells isolated from lcn2 mice . 
   compared with wild - type adipose cells , expression levels of genes involved lipid metabolism and adipokines , including ppar , lipoprotein lipase ( lpl ) , glut4 , adiponectin , and leptin were markedly reduced in adipose tissue of hfd - fed lcn2 mice ( online appendix fig . 
   plasma lipid profile in wild - type and lcn2-null mice data are means  se . 
   measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . * 
   p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . 
   at 3 weeks of age , male wild - type and lcn2 mice were fed either an hfd for 12 weeks or rcd . throughout the experimental period , 
   body weight was significantly different between wild - type and lcn2 mice fed an hfd ( fig . 
   lcn2 mice were significantly heavier than wild - type mice on either an hfd or rcd . 
   the weight of white and brown fat pads was significantly higher in hfd - fed ( fig . 
   1d ) mice than that in wild - type mice . both hfd - fed and aged lcn2 mice showed a significantly higher weight of liver , heart , kidney , and spleen ( fig . 
   similar metabolic phenotypes were also observed in female lcn2 mice ( data not shown ) . 
   however , the overall growth did not appear to be significantly affected , as the body length and bone length ( trabecular ) were not significantly different between the genotypes ( data not shown ) . 
   figure 1 g and h demonstrated the fat - cell size distributions of wild - type and lcn2 mice on an rcd at 15 weeks of age , as measured by multisizer analysis using cells isolated from osmium - fixed epididymal and inguinal adipose tissue . 
   the average peak diameter of the large - cell population was significantly larger for lcn2 mice compared with wild - type mice , indicating that lcn2 adipose tissue contains larger adipose cells . 
   growth curve , weight of white and brown fat pads and organs in lcn2 mice . 
   growth curve of lcn2 mice on an hfd ( n = 1014 ) ( a ) and rcd ( n = 13 ) ( b ) . 
   fat pad weight of lcn2 mice on an hfd at 16 weeks of age ( c ) and on rcd at 30 weeks of age ( d ) . 
   organ weight of lcn2 mice on hfd at 16 weeks of age ( e ) and on rcd at 30 weeks of age ( f ) . 
   * p < 0.05 ; * * p < 0.01 . fat - cell size distribution of mice on rcd diet at 15 weeks of age ( g and h ) . 
   because lcn2-deficient mice gained more body weight and increased adiposity , particularly when mice were fed an hfd or aged , we examined food intake and energy expenditure in lcn2 mice . 
   an analysis of indirect calorimetry measurements showed that food intake , ambulatory activity , vo2 , and rq were not significantly different between rcd - fed wild - type and lcn2 mice at 18 weeks of age ( fig . 
   we then assessed thermogenic activity of brown adipose tissue ( bat ) under thermoneutral and thermal stress conditions . 
   body temperature was measured in an ambient temperature of 28c and 22c as well as during acute exposure to 4c . 
   wild - type and lcn2 mice at 12 weeks of age exhibited a similar body temperature in an ambient temperature of 28c ( wild type , 38.34  0.34 ; lcn2 , 38.2  0.30 ) . 
   however , female lcn2 mice had a significantly lower rectal temperature than wild - type mice ( fig . 
   2a ) , while male lcn2 mice had a trend toward decrease in rectal temperature when kept in an ambient temperature of 22c . 
   more strikingly , lcn2 mice displayed cold sensitivity and could not survive when exposed to 4c for > 10 h. lcn2 mice ( seven of seven ) died after being exposed to 4c for 12 h , while all of the wild - type mice ( n = 7 ) in the experiment survived . 
   when acutely exposed to 4c , rectal temperature of lcn2 mice dropped significantly within 3 h compared with wild - type mice ( fig . 
   five - hour cold exposure caused a 10-fold increase in lcn2 gene expression in adipose tissue in wild - type mice ( fig . 
   a : basal rectal body temperature of 8-week - old male and female wild - type and lcn2 mice on an rcd ( n = 710 ) measured during the daytime in an ambient temperature of 22c . 
   b : body temperature curve of lcn2 mice ( n = 8) and wild - type mice ( n = 7 ) exposed to 4c . 
   c : gene expression of lcn2 in wat of wild - type mice exposed to 4c for 5 h. d : gene expression of ucp-1 and pgc-1 in bat and ppar , cpt-1 , tfam , and nrf1 in skeletal muscle of hfd - fed mice ( n = 6 ) . 
   g : gene expression of hsl in bat and wat of mice exposed to 4c for 5 h. h : gene expression of mitochondrial genes in bat of lcn2 mice under the hfd condition . 
   * p < 0.05 ; * * p < 0.01 . to explore the possible mechanisms for cold intolerance in lcn2 mice , uncoupling protein ( ucp)-1mediated thermogenic activity and capacity of bat 
   we observed that upon hfd feeding , lcn2 mice expressed significantly lower levels of ucp-1 and peroxisome proliferator  activated receptor ( ppar )  coactivator ( pgc-1) genes in bat than wild - type mice mice ( fig . 
   however , ucp-1 and pgc-1 expression in bat were markedly stimulated at an even higher level in lcn2 mice , as wild - type mice after exposure to 4c for 5 h ( fig . 
   figure 2f showed that the protein expression levels of adipose triglyceride lipase ( atgl ) in white adipose tissue ( wat ) were slightly reduced in lcn2 mice under the hfd and cold condition compared with wild - type mice . 
   however , cold - induced hsl gene expression in bat and wat was completely diminished in lcn2 mice ( fig . 
   lastly , we investigated the mitochondrial oxidative capacity of bat , as this function of bat is critical for providing the energy for thermogenesis . interestingly 
   , we observed that there was a significant reduction in the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function , such as nuclear respiratory factor ( nrf)1 , transcription factor a , mitochondrial ( tfam ) , atp synthase 5 ( atp5 ) , cytochrome c oxidase ( cox ) 4 , and carnitine palmitoyltransferase ( cpt)-1 in bat of lcn2 mice fed an hfd ( fig . 
   in addition , the expression of transcription factors ppar , nrf1 and tfam , and cpt-1 gene in skeletal muscle was also significantly downregulated in lcn2 mice fed an hfd ( fig . 
   to assess the effects of lcn2 deficiency on systemic insulin sensitivity , gtts and itts were performed . 
   glucose clearance curves in response to glucose administration were significantly increased in hfd - fed ( fig . 
   3b ) , indicating that lcn2 mice are more glucose intolerant upon hfd feeding . in the itt , 
   increased insulin - stimulated glucose disposal curve was observed in lcn2 mice , indicating that lcn2 mice are more insulin resistant than wild - type controls when fed an hfd ( fig . 
   3d ) . analysis of serum metabolic parameters , as shown in table 1 , demonstrated that levels of fasting serum glucose and insulin were significantly elevated , while adiponectin levels were reduced in hfd - fed lcn2 mice when compared with wild - type controls . 
   serum levels of leptin and plasminogen activator inhibitor-1 were not significantly changed in lcn2 mice on an rcd , although serum leptin levels had a trend toward a decrease in hfd - fed lcn2 mice . 
   after adjustment to body weight , the serum levels of glucose , insulin , and adiponectin were consistently different at the statistically significant level , indicating that the changes in glucose tolerance and insulin sensitivity after hfd in lcn2 mice is not simply due to body weight effects . to further confirm that lcn2 mice have decreased insulin sensitivity , in vivo insulin - stimulated akt phosphorylation was evaluated . 
   wild - type and lcn2 mice on rcd were injected intraperitoneally with insulin , killed after 10 min , and tissues were collected for evaluation of akt phosohorylation . 
   consistently , insulin - stimulated akt phosphorylation in liver , muscle , and adipose cells was significantly reduced in lcn2 mice as compared with wild - type mice ( fig . 
   the examination of lcn2 effect on upstream signaling components of insulin signaling pathway demonstrated that the expression levels of insulin receptor substrate-1 protein were markedly reduced in wat of lcn2 mice on an hfd compared with wild - type mice ( fig . 
   this data further support that lcn2 mice developed more insulin resistance and lcn2 regulates insulin signaling activity likely at the upstream level . 
   gtts ( a ) and itts ( c ) conducted in lcn2 mice on an hfd ( n = 1012 , age 1415 weeks ) . 
   gtts ( b ) and itts ( d ) conducted in lcn2 mice on an rcd ( n = 1012 , age 2829 weeks ) . 
   serum metabolic parameters in wild - type and lcn2 mice data are means  se . 
   measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . * 
   p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . in vivo insulin - stimulated akt phosphorylation in lcn2 mice . 
   representative western blot analysis of serine - phosphorylated akt in muscle , liver , and isolated primary adipose cells from lcn2 ( n = 4 per group ) and wild - type mice ( n = 4 per group ) under basal and in vivo insulin - stimulated conditions . to test the role of lcn2 in inflammatory response , expression of lcn2 and inflammatory molecules 
   was examined in adipose tissue and liver of hfd - induced wild - type and lcn2 mice . 
   5b ) . furthermore , the gene expression of proinflammatory cytokine tnf and monocyte chemoattractant protein ( mcp)-1 were upregulated , while expression of arginase 1 , an anti - inflammatory marker , was downregulated in adipose tissue of hfd - fed lcn2 mice compared with wild - type control mice ( fig . 
   downregulation of arginase 1 and interleukin ( il)-10 was also observed in the liver of hfd - fed lcn2 mice ( fig . 
   a : protein expression levels of lcn2 in adipose tissues of wild - type mice fed an rcd and hfd . 
   b : protein expression levels of lcn2 in bone marrow macrophages ( bm - mac ) isolated from wild - type mice fed an rcd and hfd . 
   c : gene expression of inflammatory molecules in adipose tissue and liver of wild - type and lcn2 mice fed on hfd . 
   to investigate whether increased liver weight in lcn2 mice is associated with the development of fatty liver and dyslipidemia , liver triglyceride content and blood lipid profiles were measured . in comparison with wild - type controls , hfd - fed and 
   aged lcn2 mice demonstrated a significant increase in lipid accumulation as detected by oil - red o staining of the liver section ( fig . 
   the development of more severe fatty liver disease , together with elevated fasting blood glucose levels , suggests hepatic insulin resistance in lcn2 mice . to elucidate hepatic glucose production in wild - type and 
   lcn2-deficient mice fed an hfd , mice were fasted for 18 h and gene expression of two key gluconeogenic enzymes pepck and glucose-6-phosphatase ( g6pase ) were detected by quantitative pcr . as illustrated in fig . 
   6c , expression levels of pepck1 , pepck2 , and g6pase genes were significantly higher in the liver of lcn2 mice than that in wild - type controls , suggesting that increased hepatic glucose production is attributed to hyperglycemia in lcn2 mice . 
   we next examined the hepatic capabilities for fatty acid synthesis and oxidation to explore the possible mechanism for the development of fatty liver in lcn2 mice . 
   6d , the lipogenic genes sterol regulatory element - binding protein-1c ( srebp-1c ) , acetyl - coa carboxylase 1 ( acc1 ) , spot 14 ( s14 ) , and scd-1 were expressed at a significantly higher level in hfd - fed lcn2 mice compared with wild - type controls , while genes involved in fatty acid oxidation such as ppar , cpt-1 , and acetyl - coa carboxylase 2 were similarly expressed between genotypes ( data not shown ) . 
   therefore , the development of fatty liver results primarily from the increased capability for fatty acid synthesis in the liver in lcn2 mice . 
   b : liver triacyglyceride levels in lcn2 mice on an rcd ( n = 68 , age 30 weeks ) and an hfd ( n = 11 , age 1516 weeks ) . 
   c : the mrna expression of gluocneogenic genes in liver ( n = 6 ) . 
   d : the mrna expression of lipogenic genes in liver ( n = 6 ) . 
   the data are represented as means  se . * p < 0.05 ; * * p < 0.01 . 
   ( a high - quality digital color representation of this figure is available in the online issue . ) 
   compared with wild - type mice , lcn2 mice on an rcd had significantly higher serum levels of triglycerides , cholesterol , and free fatty acids . 
   the hfd feeding did not induce a further increase in serum levels of triglyceride and cholesterol but significantly augmented the levels of ldl and -hydroxybutyrate in lcn2 mice compared with wild - type controls . 
   the above data have suggested that lcn2-deficient mice became more insulin resistant upon hfd feeding and age . since adipose tissue is a key site for insulin - regulated glucose / lipid metabolism and glucose disposal , we examined the expression of genes involved in lipid metabolism in primary adipose cells isolated from lcn2 mice . compared with wild - type adipose cells , expression levels of genes involved lipid metabolism and adipokines , including ppar , lipoprotein lipase ( lpl ) , glut4 , adiponectin , and leptin were markedly reduced in adipose tissue of hfd - fed lcn2 mice ( online appendix fig 
   plasma lipid profile in wild - type and lcn2-null mice data are means  se . 
   measurements were performed on 6-h  fasted mice fed an rcd and hfd ( n = 911 ) . * 
   p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . 
   we show here that lcn2 plays a critical role in lipid metabolism , adaptive thermoregulation , and diet - induced obesity and insulin resistance in vivo . 
   lack of lcn2 in mice potentiates hfd - induced obesity , dyslipidemia , fatty liver , glucose intolerance , and insulin resistance . 
   we also discovered that lcn2 is a critical regulator of adaptive thermogenesis and mitochondrial oxidative function in bat . 
   lcn2 deficiency impairs adaptive thermogenesis and increases the susceptibility of lcn2 mice to hfd - induced obesity . 
   our data demonstrated that lcn2 mice gained more body weight when they were fed hfd or aged . increased body fat mass together with enlarged multiple organs 
   in particular , increased organ weights may largely account for increased body weight in lcn2 mice under the rcd condition . 
   in general , wild - type and lcn2 mice at a young age did not demonstrate significantly different metabolic phenotypes when they were fed rcd . however , lcn2 mice developed more severe systemic insulin resistance , hyperglycemia , hyperinsulinemia , and hypoadiponectinemia under the hfd condition 
   . one of the most profound phenotypic features of lcn2 mice is cold sensitivity and lower body temperature under thermal stress conditions . 
   it has been known for a long time that nonshivering thermogenesis is used to maintain constant body temperature and energy balance , controlling body weight , especially in small mammals ( 1719 ) . 
   both neural and hormonal signals , including norepinephrine , epinephrine , thyroid hormones , glucagon , insulin , glucocorticoid , and leptin , are known to regulate thermogenesis ( 17,20 ) through different thermogenic mechanisms involving stimulating atp utilization , increasing ucp-1mediated proton leak and heat production and affecting substrate availability and utilization for energy production . in rodents , 
   bat is the main tissue that is responsible for sympathetic nervous system  regulated nonshivering themogenesis with the involvement of norepinephrine and epinephrine ( 17,20,21 ) . ucp-1 and pgc-1 are the key genes that mediate sympathetic thermogenic responses in bat . 
   for example , ucp-1 gene expression in bat of ob / ob mice was reduced ( 23 ) . however 
   , bat ucp-1 expression could be normally stimulated in response to cold environment in young ob / ob mice , albeit ob / ob mice show decreased capability of utilizing fat and cold intolerance ( 23 ) . in this study 
   , we found that 5-h cold exposure leads to a significant increase in lcn2 expression in wat , which is in agreement with the result in a recent study ( 24 ) . in the absence of lcn2 , mice were cold intolerant and failed to maintain the body temperature when acutely exposed to cold temperature . interestingly , cold exposure could still significantly stimulate gene expression of ucp-1 and pgc-1 in bat in lcn2 mice , suggesting that cold - induced sympathetic thermogenesis remained intact in the absence of lcn2 . 
   the role of leptin in thermogenesis has been well documented , and its thermogenic effect is considered to be primarily central ( 25 ) . 
   for example , leptin administration has been reported to rescue cold - induced hypothermia in the absence of ucp-1 in leptin and ucp-1 double knockout mice ( 26,27 ) , while the opposite results were perceived in the other two independent studies ( 28,29 ) . in lcn2 mice on rcd , 
   serum leptin levels were not different from that in wild - type mice , albeit serum leptin levels had a trend toward decreased in lcn2 mice in response to hfd feeding . 
   additionally , wild - type and lcn2 mice on an rcd did not exhibit a difference in body temperature in thermoneutral conditions . 
   therefore , it seems rather unlikely that leptin could be the major contributor to cold intolerance in lcn2 mice ; leptin may be only partially involved in lcn2-regulated thermogenesis . 
   in addition to hormonal signals , defects in key metabolic regulators of substrate availability for energy production , atp synthesis and mitochondrial biogenesis and oxidative function could all possibly affect thermogenic function . 
   indeed , studies in mice with the genetic disruption of key metabolic regulators have proved this assumption ; mice without expressing atgl could similarly result in cold intolerance ( 30 ) . in the present study 
   , the disruption of lcn2 completely diminished cold - induced hsl gene expression in bat and wat , whereas atgl expression in wat was only slightly affected in lcn2 mice under both the hfd and cold conditions . 
   since hsl mice are not cold sensitive ( 31 ) , hsl is not an important regulator for adaptive thermogenesis . 
   therefore , the effect of changes in atgl levels on the supply of substrates for energy production for thermogenesis is minimal in lcn2 mice . on the other hand 
   , the production of the energy necessary for thermogenesis requires normal mitochondrial oxidative function , and mitochondrial dysfunction could have a significant impact on adaptive thermogenesis . 
   this hypothesis has been supported by a previous study in estrogen - related receptor  ( eer ) knockout mice . in eer-deficient mice , mitochondrial biogenesis and oxidative function in bat 
   was disturbed , and mice failed to maintain body temperature when exposed to cold ( 32 ) . to this end 
   interestingly , we found that the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function such as nrf1 , tfam , atp5b , cox 4 , and cpt-1 were significantly reduced in bat of lcn2 mice fed an hfd . in the skeletal muscle , the expression of -oxidation genes ppar and cpt-1 , as well as mitochondrial genes nrf1 and tfam , were also downregulated in hfd - fed lcn2 mice . 
   all the above data together suggest that mitochondrial metabolism is dysfunctional in bat and muscle of lcn2 mice , which mainly contributes to decreased thermogenic capacity , decreased energy expenditure , and the high susceptibility to diet - induced obesity and insulin resistance during the thermal stress conditions ( mice have been living at 22c ) . in acute cold , 
   muscle - shivering thermogenesis is considered to be the primary mechanism for a body temperature defense ( 21,33,34 ) . 
   bat nonshivering thermogenesis participates in combating the cold temperature only when the extra capacity is already recruited for using during the acute cold exposure ( 35 ) . therefore , cold intolerance ( acute exposure to 4c for 5 h ) in lcn2 mice may largely result from decreased oxidative capacity in skeletal muscle and bat as well as decreased atgl expression in wat . 
   our results clearly demonstrated that insulin responsiveness in muscle , adipose tissue , and liver was reduced , indicating systemic insulin resistance in lcn2 mice . 
   furthermore , fasting for 18 h induces significantly higher levels of expression for pepck and g6pase in the liver of hfd - fed lcn2 mice , supporting that the increased hepatic glucose production is an important contributor to fasting hyperglycemia in lcn2 mice . 
   this phenomenon is likely due to the impaired regulation of lipid metabolism in adipose tissue and liver in lcn2 mice . 
   we showed that lcn2 mice on an rcd had higher serum levels of triglycerides but lower serum triglyceride levels when fed an hfd as compared with wild - type mice . 
   lower serum triglycerides in hfd - fed lcn2 mice could be due to the increased triglycerides synthesis and decreased vldl ( or triglyceride ) secretion into the circulation resulting from the development of fatty liver in lcn2 mice . 
   this explanation is strengthened by a previous study ( 36 ) in lirko ( insulin receptor knockout ) mice with hepatic insulin resistance and 50% decrease in serum triglyceride levels . as reported in our previous in vitro study ( 6 ) , lcn2 is capable of suppressing inflammatory response of macrophages to lps stimulation . 
   we speculated that lcn2 possesses anti - inflammatory role in metabolic regulation . in this in vivo study , we show that lcn2 expression is downregulated in adipose tissue and bone marrow macrophages ( bm - mac ) in hfd - induced obese mice . in the absence of lcn2 , 
   the expression of proinflammatory cytokines such as mcp-1 and tnf are upregulated in adipose tissue , while expression levels of anti - inflammatory markers arginase 1 and il-10 are decreased in adipose tissue and liver , suggesting an increased inflammatory state in lcn2 mice . 
   these results further suggest that lcn2 mice are more susceptible to hfd - induced proinflammatory response than wild - type mice . in summary , herein we discovered a novel role for lcn2 in energy metabolism , adaptive thermogenesis , and insulin resistance . 
   mice lacking lcn2 display impaired adaptive thermogenesis and deteriorated diet - induced obesity and insulin resistance . 
   decreased mitochondrial oxidative capacity in bat and skeletal muscle may be the primary mechanisms for the development of diet - induced insulin resistance and cold sensitive in lcn2 mice . 
   however , the mechanisms for how lcn2 regulates mitochondrial function and lipid metabolism remain to be further identified . 
   it is likely that the binding of lcn2 to small hydrophobic molecules such as retinoic acids , fatty acids , and other unknown ligands or to the cell surface receptor such as megalin or ldl - related protein 2 ( 37 ) could potentially involve or mediate lcn2 effects in metabolism . 
   our findings provide new insight into the role of lcn2 in lipid metabolism and metabolic homeostasis . in this manner 
   , lcn2 may be a potential therapeutic target for controlling obesity - associated type 2 diabetes and metabolic complications . 
   
summary: objectivelipocalin ( lcn ) 2 belongs to the lipocalin subfamily of low  molecular mass  secreted proteins that bind small hydrophobic molecules . 
   lcn2 has been recently characterized as an adipose - derived cytokine , and its expression is upregulated in adipose tissue in genetically obese rodents . 
   the objective of this study was to investigate the role of lcn2 in diet - induced insulin resistance and metabolic homeostasis in vivo.research design and methodssystemic insulin sensitivity , adaptive thermogenesis , and serum metabolic and lipid profile were assessed in lcn2-deficient mice fed a high - fat diet ( hfd ) or regular chow diet.resultsthe molecular disruption of lcn2 in mice resulted in significantly potentiated diet - induced obesity , dyslipidemia , fatty liver disease , and insulin resistance . 
   lcn2/ mice exhibit impaired adaptive thermogenesis and cold intolerance . 
   gene expression patterns in white and brown adipose tissue , liver , and muscle indicate that lcn2/ mice have increased hepatic gluconeogenesis , decreased mitochondrial oxidative capacity , impaired lipid metabolism , and increased inflammatory state under the hfd condition.conclusionslcn2 has a novel role in adaptive thermoregulation and diet - induced insulin resistance .