after arrival , rats were maintained on a constant 12-h light/12-h dark cycle with free access to water and ad libitum fed with a standard chow diet and allowed to acclimate for at least a week . all procedures were approved by the institutional animal care and use committee ( iacuc ) of the university of pittsburgh , and were in accordance with the national research council 's guide for the care and use of laboratory animals . chronic indwelling catheters were implanted in the left common carotid artery and the right jugular vein ( 18 ) . three dietary groups were established 1 ) standard chow 2 ) high fat ( hf ; td 96001 , 45% of calories from fat ; harlan teklad , madison wi ) , and 3 ) high sucrose ( hs ; 68% of calories from sucrose and 11% of calories from fat ; research diet ) . each of the three dietary groups was further divided into two subgroups ; one subgroup was injected via the carotid artery with gadolinium chloride ( gdcl3 ; sigma , st . gdcl3 is a selective toxicant for kcs and is commonly used to deplete the liver of these cells ( 1921 ) . in the current study , depletion of kcs was evaluated by quantification of cd68 ( a macrophage marker)-positive cells by immunohistochemistry ( 19,20 ) and measurements of cd68 and f4/80 mrna levels using quantitative ( q)rt - pcr ( supplemental table 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0016/dc1 ) . one week prior to the beginning of diet exposures , all animals received two injections of a sterile filtered gdcl3 ( 10 mg / kg ) or saline solution into the carotid artery cannula ( at days 7 and 4 ) . four further injections were administered during dietary exposures ( at days 0 , + 3 , + 7 , and + 10 ) . at day + 14 ( i.e. , after 2 weeks of dietary exposure ) , animals were fasted overnight . subsequently , animals were killed and tissues ( blood , liver , adipose tissue ) were isolated or animals underwent a hyperinsulinemic - euglycemic clamp ( see below ) and were then killed and tissues isolated . for all experiments , caloric intake , weight gain during diet exposures , and the method for simultaneous isolation of hepatocytes and kcs from the same rat was adapted from smedsrd et al . briefly , livers were perfused through the portal vein in situ with an oxygenenated modified ( no ca , and kh4po4 was substituted with nah2po4 ) krebs - henseleit buffer plus 2.5 mmol / l egta at 37c at a rate of 18 ml / min for 10 min ( 24 ) . subsequently , 100 ml of krebs - henseleit buffer in the presence of ca ( 2.5 mmol / l ) and containing 0.04% collagenase ( type iv ; sigma ) was recirculated through the liver for 10 min . the resulting cell suspension was filtered through a sterile 150-mesh nylon screen and centrifuged three times at 50 g at 4c for 2 min , and the cell pellet containing hepatocytes was further purified in a 30% percoll / dulbecco 's modified eagle 's medium ( dmem ) solution ( ge healthcare bioscience ab , uppsala , sweden ) by centrifugation at 50 g for 10 min . the hepatocytes were plated at a density of 1.5  10 cells / well in collagen - coated ( sigma ) 6-well plates with complete dmem containing 10% fbs , 25 mmol / l glucose , 10 mmol / l hepes , 250 units / ml penicillin , and 250 g / ml streptomycin and incubated for 2 h at 37c in 5% co2 . nonattached cells were removed by washing with pbs after 1 h. for isolation of kcs , the combined supernatant from the non - percoll centrifugations above was centrifuged at 50 g for 2 min , and the resulting supernatant ( enriched in nonparenchymal cells ) was centrifuged at 350 g for 5 min . the cell pellet containing nonparenchymal cells was resuspended in 4 ml complete dmem medium and mixed with 6 ml 30% ( wt / vol ) histodenz ( sigma ) in pbs . the resulting suspension was layered under 10 ml dmem containing 10% fbs and centrifuged at 1300 g for 20 min at 22c with the brake off . subsequently , the cell pellet was suspended in dmem at a density of 1.0  10 cells per milliliter . approximately 1.0  10 cells were then plated on transwell inserts ( 0.4-m pore size membrane ; corning , lowell , ma ) in 2 ml to allow attachment of kcs . after 1 h , the medium containing non - kcs was replaced with fresh medium . cell viability as assessed by trypan blue exclusion was greater than 95% for kcs and greater than 90% for hepatocytes . 4 ) , kc attachment was assessed by cd68 immunofluorescence ( abd serotec , killington , oxford , u.k . ) . hepatocytes were incubated in the absence or presence of kcs , and/or lipopolysaccharide ( lps ; 500 ng / ml ) , and/or 0.4 mmol / l [ h]-palmitate ( 5 ci / ml ) for the measurement of fatty acid oxidation and esterification and triglyceride accumulation . all fatty acid incubations were for 24 h , except for the measurement of fatty acid esterification where labeled palmitate was present for only 2 h after the 24-h coculture period . at the end of incubations , media were collected and cells were washed two times in an excess volume of ice - cold pbs , briefly treated with 12.5% trypsin - edta , and collected by scraping into 2 ml of pbs , centrifugation , and retention of the pellet for analysis of lipids and proteins . for all conditions , triplicate ( 3-well ) measurements were made in each experiment . for neutralizing antibody experiments , anti - tnf , anti ifn- , at 5 g / ml . all antibodies were from r&d systems ( minneapolis , mn ) . for analysis of insulin signaling pathways , cells were serum deprived for 2 h after 24-h coculture , and were subsequently stimulated with 100 nm insulin for 10 min . after 2 washes with pbs containing 2 mmol / l protease inhibitors and phosphatase inhibitors , cells were scraped into phosphatidylinositol 3 kinase ( pi 3-kinase ) assay buffer or cell lysis buffer for subsequent analysis . for the assessment of basal hepatic glucose output ( hgo ) , a 10-ci [ 3-h]-glucose bolus was administered intravenously followed by a 120-min infusion at a rate of 0.1 ci / min . arterial blood samples were taken at 60 , 90 , and 120 min for the determination of plasma glucose  specific activity . to assess insulin suppression of hgo , a constant rate insulin ( humulin ; eli lilly ) infusion ( 4 mu  kg  min ) and a variable rate glucose infusion commenced at + 120 min , and the [ 3-h]-glucose infusion continued at a rate of 0.18 ci / min for another 90 min . blood glucose concentration was monitored at 10-min intervals to determine the exogenous glucose infusion rate necessary to maintain euglycemia . blood samples taken during the final 30 min of the clamp were used to assess plasma radioactivity for the determination of hgo and the glucose disposal rate ( gdr ) ( 25,26 ) . for immunohistochemistry , tissues ( liver and epididymal fat ) cd68-positive cells were detected using -cd68 ( abd serotec ) and were quantified on 89 fields of 40 sections using an olympus ( new york , ny ) light microscope . for immunofluorescence , cd68 and tnf were detected in liver sections after antigen retrieval by heating sections in 1.6 mmol / l edta in a high - pressure cooker for 7 min . subsequently , sections were immunostained with -cd68 ( 1:200 ) and -tnf ( 1:100 ; mbl , woburn , ma ) followed by incubation with alexa 488conjugated or cyanin 3conjugated secondary antibodies , respectively . images were visualized using an olympus provis fluorescence microscope and modified with magnafire software ( olympus , melville , ny ) . triglycerides , diacylglycerol ( dag ) , ceramide , fatty acid oxidation , and fatty acid esterification were determined as described previously ( 27,28 ) . cholesterol was assessed using the infinity cholesterol kit ( thermo , rockford , il ) . insulin receptor substrate 1 ( irs-1)associated pi 3-kinase activity , and irs-1 , insulin receptor  ( ir ) , acetyl - coa carboxylase ( acc ) , amp - activated protein kinase ( ampk ) , and akt phosphorylation were determined as previously described ( 24,29 ) . tnf ( by elisa ; r&d systems ) , lps ( limulus amebocyte lysate assay ; lonza bioscience , walkersville , md ) , insulin ( by elisa ; alpco , salem , nh ) , and free fatty acids ( roche , indianapolis , in ) were measured using commercial kits according to the protocols provided by the manufacturer . gene expression was measured using quantitative real - time pcr ( supplemental table 1 ) as previously described ( 30 ) . statistical significance was determined by t test and , where appropriate , one - way anova ( bonferroni post hoc test ) was performed using the systat statistical program ( evanston , il ) . after arrival , rats were maintained on a constant 12-h light/12-h dark cycle with free access to water and ad libitum fed with a standard chow diet and allowed to acclimate for at least a week . all procedures were approved by the institutional animal care and use committee ( iacuc ) of the university of pittsburgh , and were in accordance with the national research council 's guide for the care and use of laboratory animals . chronic indwelling catheters were implanted in the left common carotid artery and the right jugular vein ( 18 ) . three dietary groups were established 1 ) standard chow 2 ) high fat ( hf ; td 96001 , 45% of calories from fat ; harlan teklad , madison wi ) , and 3 ) high sucrose ( hs ; 68% of calories from sucrose and 11% of calories from fat ; research diet ) . each of the three dietary groups was further divided into two subgroups ; one subgroup was injected via the carotid artery with gadolinium chloride ( gdcl3 ; sigma , st . gdcl3 is a selective toxicant for kcs and is commonly used to deplete the liver of these cells ( 1921 ) . in the current study , depletion of kcs was evaluated by quantification of cd68 ( a macrophage marker)-positive cells by immunohistochemistry ( 19,20 ) and measurements of cd68 and f4/80 mrna levels using quantitative ( q)rt - pcr ( supplemental table 1 , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0016/dc1 ) . one week prior to the beginning of diet exposures , all animals received two injections of a sterile filtered gdcl3 ( 10 mg / kg ) or saline solution into the carotid artery cannula ( at days 7 and 4 ) . four further injections were administered during dietary exposures ( at days 0 , + 3 , + 7 , and + 10 ) . at day + 14 ( i.e. , after 2 weeks of dietary exposure ) , animals were fasted overnight . subsequently , animals were killed and tissues ( blood , liver , adipose tissue ) were isolated or animals underwent a hyperinsulinemic - euglycemic clamp ( see below ) and were then killed and tissues isolated . for all experiments , caloric intake , weight gain during diet exposures , and the method for simultaneous isolation of hepatocytes and kcs from the same rat was adapted from smedsrd et al . , livers were perfused through the portal vein in situ with an oxygenenated modified ( no ca , and kh4po4 was substituted with nah2po4 ) krebs - henseleit buffer plus 2.5 mmol / l egta at 37c at a rate of 18 ml / min for 10 min ( 24 ) . subsequently , 100 ml of krebs - henseleit buffer in the presence of ca ( 2.5 mmol / l ) and containing 0.04% collagenase ( type iv ; sigma ) was recirculated through the liver for 10 min . the resulting cell suspension was filtered through a sterile 150-mesh nylon screen and centrifuged three times at 50 g at 4c for 2 min , and the cell pellet containing hepatocytes was further purified in a 30% percoll / dulbecco 's modified eagle 's medium ( dmem ) solution ( ge healthcare bioscience ab , uppsala , sweden ) by centrifugation at 50 g for 10 min . the hepatocytes were plated at a density of 1.5  10 cells / well in collagen - coated ( sigma ) 6-well plates with complete dmem containing 10% fbs , 25 mmol / l glucose , 10 mmol / l hepes , 250 units / ml penicillin , and 250 g / ml streptomycin and incubated for 2 h at 37c in 5% co2 . nonattached cells were removed by washing with pbs after 1 h. for isolation of kcs , the combined supernatant from the non - percoll centrifugations above was centrifuged at 50 g for 2 min , and the resulting supernatant ( enriched in nonparenchymal cells ) was centrifuged at 350 g for 5 min . the cell pellet containing nonparenchymal cells was resuspended in 4 ml complete dmem medium and mixed with 6 ml 30% ( wt / vol ) histodenz ( sigma ) in pbs . the resulting suspension was layered under 10 ml dmem containing 10% fbs and centrifuged at 1300 g for 20 min at 22c with the brake off . subsequently , the cell pellet was suspended in dmem at a density of 1.0  10 cells per milliliter . approximately 1.0  10 cells were then plated on transwell inserts ( 0.4-m pore size membrane ; corning , lowell , ma ) in 2 ml to allow attachment of kcs . after 1 h , the medium containing non - kcs was replaced with fresh medium . cell viability as assessed by trypan blue exclusion was greater than 95% for kcs and greater than 90% for hepatocytes . in preliminary experiments ( supplemental fig . 4 ) , kc attachment was assessed by cd68 immunofluorescence ( abd serotec , killington , oxford , u.k . ) . hepatocytes were incubated in the absence or presence of kcs , and/or lipopolysaccharide ( lps ; 500 ng / ml ) , and/or 0.4 mmol / l [ h]-palmitate ( 5 ci / ml ) for the measurement of fatty acid oxidation and esterification and triglyceride accumulation . all fatty acid incubations were for 24 h , except for the measurement of fatty acid esterification where labeled palmitate was present for only 2 h after the 24-h coculture period . at the end of incubations , media were collected and cells were washed two times in an excess volume of ice - cold pbs , briefly treated with 12.5% trypsin - edta , and collected by scraping into 2 ml of pbs , centrifugation , and retention of the pellet for analysis of lipids and proteins . for all conditions , triplicate ( 3-well ) measurements were made in each experiment . for neutralizing antibody experiments , anti - tnf , anti ifn- , at 5 g / ml . all antibodies were from r&d systems ( minneapolis , mn ) . for analysis of insulin signaling pathways , cells were serum deprived for 2 h after 24-h coculture , and were subsequently stimulated with 100 nm insulin for 10 min . after 2 washes with pbs containing 2 mmol / l protease inhibitors and phosphatase inhibitors , cells were scraped into phosphatidylinositol 3 kinase ( pi 3-kinase ) assay buffer or cell lysis buffer for subsequent analysis . hyperinsulinemic - euglycemic clamps were performed as previously described ( 18 ) . for the assessment of basal hepatic glucose output ( hgo ) , a 10-ci [ 3-h]-glucose bolus was administered intravenously followed by a 120-min infusion at a rate of 0.1 ci / min . arterial blood samples were taken at 60 , 90 , and 120 min for the determination of plasma glucose  specific activity . to assess insulin suppression of hgo , a constant rate insulin ( humulin ; eli lilly ) infusion ( 4 mu  kg  min ) and a variable rate glucose infusion commenced at + 120 min , and the [ 3-h]-glucose infusion continued at a rate of 0.18 ci / min for another 90 min . blood glucose concentration was monitored at 10-min intervals to determine the exogenous glucose infusion rate necessary to maintain euglycemia . blood samples taken during the final 30 min of the clamp were used to assess plasma radioactivity for the determination of hgo and the glucose disposal rate ( gdr ) ( 25,26 ) . for immunohistochemistry , tissues ( liver and epididymal fat ) were fixed overnight in a 4% paraformaldehyde solution , and subsequently embedded in paraffin . cd68-positive cells were detected using -cd68 ( abd serotec ) and were quantified on 89 fields of 40 sections using an olympus ( new york , ny ) light microscope . for immunofluorescence , cd68 and tnf were detected in liver sections after antigen retrieval by heating sections in 1.6 mmol / l edta in a high - pressure cooker for 7 min . subsequently , sections were immunostained with -cd68 ( 1:200 ) and -tnf ( 1:100 ; mbl , woburn , ma ) followed by incubation with alexa 488conjugated or cyanin 3conjugated secondary antibodies , respectively . images were visualized using an olympus provis fluorescence microscope and modified with magnafire software ( olympus , melville , ny ) . triglycerides , diacylglycerol ( dag ) , ceramide , fatty acid oxidation , and fatty acid esterification were determined as described previously ( 27,28 ) . cholesterol was assessed using the infinity cholesterol kit ( thermo , rockford , il ) . insulin receptor substrate 1 ( irs-1)associated pi 3-kinase activity , and irs-1 , insulin receptor  ( ir ) , acetyl - coa carboxylase ( acc ) , amp - activated protein kinase ( ampk ) , and akt phosphorylation were determined as previously described ( 24,29 ) . tnf ( by elisa ; r&d systems ) , lps ( limulus amebocyte lysate assay ; lonza bioscience , walkersville , md ) , insulin ( by elisa ; alpco , salem , nh ) , and free fatty acids ( roche , indianapolis , in ) were measured using commercial kits according to the protocols provided by the manufacturer . gene expression was measured using quantitative real - time pcr ( supplemental table 1 ) as previously described ( 30 ) . statistical significance was determined by t test and , where appropriate , one - way anova ( bonferroni post hoc test ) was performed using the systat statistical program ( evanston , il ) . to begin to address the role of kcs in dysregulated hepatic metabolism , we first determined the metabolic effects of 2 weeks of hf or hs feeding in rats in the absence and presence of kcs . 1a ) , increased liver cholesterol and dag , but not ceramide levels ( fig . d ) , and induced systemic hypertriglyceridemia ( table 1 ) without increasing adiposity or body weight gain ( table 1 ) , compared with standard chow ( sc)fed controls . there was a trend toward an increase in systemic tnf levels on the hf diet , but this increase did not attain statistical significance ( table 1 ) . finally , although fasting plasma insulin and glucose concentrations were similar in all diet groups , hf and hs induced hepatic insulin resistance as measured by the glucose clamp ( fig . thus , hgo during the glucose clamp was elevated 40% in hs- and hf - fed animals compared with controls ( fig . this corresponded to a decrease in the capacity of insulin to suppress hgo of 29 in hf- and 28% in hs - fed animals compared with 58% in controls ( fig . basal hgo and clamp gdr were similar in sc- , hf- , and hs - fed animals ( supplemental fig . depletion of kcs by gdcl3 decreased liver mrna levels of the kc - specific markers cd68/ed1 and f4/80 by 50 and 80% , respectively ( fig . histochemical analysis of liver confirmed an 70% decrease in cd68/ed1-positive cells ( fig . 3c and e ) . interestingly , adipose tissues levels of the same markers were unaltered by gdcl3 ( fig . furthermore , the effects of gdcl3 were independent of diet , whereas a hf or hs diet had no effects on liver kcs or adipose tissue macrophage ( atm ) numbers in control ( saline - treated ) animals ( supplemental fig . 2 ) . although the depletion of kcs had no effects on liver triglyceride levels in sc - fed animals , the development of hepatic steatosis was prevented in hf- and hs - fed animals ( fig . however , plasma hypertriglyceridemia was not resolved by kc depletion ( table 1 ) , and indeed , there was a trend toward increased triglycerides ( tgs ) in kc - depleted hs- and hf - fed , but not sc - fed , animals . liver peroxisome proliferator  activated receptor- ( ppar ) and acc1 gene expression were increased in kc - depleted hf and hs , and cpt1 expression trended upward , with no changes in mttp , lcad , or dgat2 expression ( fig . hepatic insulin resistance induced by hf and hs diets was prevented in kc - depleted animals ( fig . 2 ) , and there were no effects on fasting glucose or insulin concentrations ( table 1 ) , or basal hgo and clamp gdr ( supplemental fig . 1 ) . the effects of kc depletion on liver triglycerides , dag , ceramide , and cholesterol in response to a high - fat or high - sucrose diet . male wistar rats depleted of kcs or control rats were exposed to a high - fat or high - sucrose diet for 2 weeks as described in research design and methods . subsequently , animals were killed after an overnight fast , livers were isolated , and triglyceride ( a ) , dag ( b ) , cholesterol ( c ) , and ceramide ( d ) content of the liver was determined . body weight , epididymal fat , and plasma measurements in the saline- or gdcl3-treated rats exposed to nc , hf , or hs diets for 2 weeks data are means  se ; n = minimum 5/group except post - clamp sc insulin where n = 3 . values for tgs , ffas , tnf , and lps are from overnight - fasted , preclamp blood samples . * significantly different from nc saline . the effects of kc depletion on the development of liver insulin resistance on a high - fat or high - sucrose diet . male wistar rats depleted of kcs or control rats were exposed to a high - fat ( a and b ) or high - sucrose ( c and d ) diet for 2 weeks . subsequently , animals were fasted overnight and then underwent 4 mu  kg  min euglycemic - hyperinsulinemic clamp in the presence of [ 3-h]-glucose for the measurement of hepatic insulin sensitivity as described in research design and methods . hepatic glucose output ( a and c ) and insulin suppression of hepatic glucose output ( b and d ) were assessed . male wistar rats were depleted of kcs by administration of gdcl3 as described in research design and methods . subsequently , liver and adipose tissue were isolated and the presence of macrophages was determined by qrt - pcr assessment of cd68/ed1 and f4/80 mrna levels ( a and b ) and counting of cd68/ed1-positive cells after immunohistochemical staining of liver and adipose tissue sections ( c  f ) as described in research design and methods . n = a minimum of six in each group for qrt - pcr and n = 3 for immunohistochemical analysis of cd68/ed1-positive cells . ( a high - quality digital representation of this figure is available in the online issue . ) expression of lipid metabolism genes in the liver of sc- , hf- , and hs - fed rats with or without gdcl3 administration . male wistar rats were depleted of kcs by administration of gdcl3 as described in research design and methods . subsequently , liver was isolated , rna was extracted , and the expression of a number of lipid metabolism genes was determined by qrt - pcr . the demonstration that a liver depleted of kcs is protected against diet - induced steatosis and insulin resistance implicates a mechanistic role for these cells in the determination of hepatic insulin sensitivity and the regulation of lipid metabolism . however , this in vivo study does not directly assess the effects of kcs on hepatocyte metabolism and insulin action . studies provide supportive evidence in this regard , demonstrating that lps injection rapidly induces steatosis ( 31 ) , whereas mice with macrophages lacking the ability to m2 polarize have decreased hepatocyte oxidative metabolism and steatosis ( 15,16 ) . again , however , these data implicate , rather then demonstrate , a role for kcs in hepatocyte metabolic abnormalities . to more directly address this issue , a coculture system composed of kcs and hepatocytes isolated from the same rat liver was used . 4 ) were incubated in the presence of lps and free fatty acids to induce m1 polarization and , subsequently , readouts of lipid metabolism and insulin action in hepatocytes were assessed . importantly , lps alone had no effect on hepatocyte triglyceride , fatty acid esterification , or fatty acid oxidation ( fig . c ) . however , when kcs were present , hepatocyte triglycerides and fatty acid esterification were increased , and fatty acid oxidation was decreased . furthermore , phospho - acc was decreased , with a trend toward decreased phospho - ampk ( fig . f ) . lipogenesis ( as measured by c - acetate incorporation into total lipids ) , was not increased by kcs ( supplemental fig . thus , m1 activation of kcs resulted in a marked decrease in insulin 's capacity to stimulate phosphorylation of the insulin receptor and irs-1 , pi 3-kinase activity , and akt phosphorylation ( fig . notably , saturated fatty acids alone in the presence of kcs induced decreases in hepatocyte fatty acid oxidation , and increases in fatty acid esterification , but did not alter triglyceride levels ( supplemental fig . the effects of m1-polarized kcs on hepatocyte fatty acid oxidation and esterification and triglyceride accumulation . hepatocytes and kcs were isolated from rat livers , plated , and cultured as described in research design and methods . subsequently , the effects of the indicated exposures on fatty acid oxidation , fatty acid esterification , and triglyceride levels ( a  c ) and acc and ampk phosphorylation ( d  f ) were assessed as described in research design and methods . were isolated from rat livers , plated , and cultured as described in research design and methods . / l insulin for 10 min , and protein extracts were prepared and insulin receptor ( ir ) phosphorylation , irs-1 phosphorylation , irs-1associated pi 3-kinase activity , and akt phosphorylation were assessed as described in research design and methods . the data presented above demonstrate metabolic cross talk between kcs and hepatocytes , which results in altered lipid metabolism and insulin sensitivity , but do not address the biochemical mechanisms of these effects . an obvious candidate mechanism is tnf , because it has a well - described role in the pathogenesis of insulin resistance , has been implicated in the development of dyslipidemia and steatosis , and is a prototypical m1 cytokine produced by macrophages when they are activated ( 3239 ) . to address this possibility , we first evaluated the effects of overnutrition on tnf production by kcs in our model systems . importantly , in both hs- and hf - fed animals kcs had increased tnf , and this increase was not present in kcs of sc - fed animals as assessed by immunofluorescence ( fig . we next directly evaluated the potential role of this cytokine in the adverse effects of kcs on hepatocyte lipid metabolism and insulin responsiveness , using fatty acid oxidation , triglyceride accumulation , and pi 3-kinase activity as readouts , respectively . in response to lps exposure , kcs increased production of tnf , an effect that was substantially greater than that observed when hepatocytes were exposed to lps alone ( fig . 7b ) . importantly , blocking of tnf activity using a tnf-neutralizing antibody resulted in an improvement of the adverse effects of kcs on hepatocyte fatty acid oxidation and accumulation of tgs ( fig . notably , neutralizing antibodies against a panel of other m1-associated cytokines ( il-1 , il-1 , il-6 , ifn- , ifn- , il-12 , il-18 ) were unable to prevent the effects of m1-polarized kcs on hepatocyte lipid metabolism ( supplemental fig . 7 ) . tnf expression in kcs in liver in response to standard chow , high - fat , and high - sucrose diets and tnf secretion from isolated m1-polarized kcs . male wistar rats were exposed to a high - fat or high - sucrose diet for 2 weeks . subsequently , the livers were isolated , sections were prepared , and immunofluorescent staining for kcs and tnf was performed ( a ) , as described in research design and methods . the left panel shows cd68/ed1-positive cells ( green ) , the center panels show tnf-positive cells ( red ) , and the right panels show the merged images . in all panels , n = a minimum of three . for in vitro experiments ( b ) , hepatocytes and kcs were isolated from rat livers , plated , and cultured as shown . after the indicated exposures , media were taken and tnf was quantified by enzyme - linked immunosorbent assay ( elisa ) . ( a high - quality digital representation of this figure is available in the online issue . ) the effects of tnf depletion on hepatocyte lipid metabolism and insulin - stimulated pi 3-kinase activity . hepatocytes and kcs were isolated from rat livers , plated , and cultured as described in research design and methods . subsequently , the effects of the indicated exposures on fatty acid oxidation and triglycerides in the absence or presence of a tnf-neutralizing antibody were assessed as described in research design and methods ( a ) . in separate experiments , hepatocytes were incubated with 100 nmol / l insulin for 10 min after similar exposures to those in ( a ) , proteins extracts were then prepared , and irs-1associated pi 3-kinase activity was assessed as described in research design and methods ( b ) . n = a minimum of six for each experimental condition , except for triglycerides ( n = 3 ) . to begin to address the role of kcs in dysregulated hepatic metabolism , we first determined the metabolic effects of 2 weeks of hf or hs feeding in rats in the absence and presence of kcs . 1a ) , increased liver cholesterol and dag , but not ceramide levels ( fig . d ) , and induced systemic hypertriglyceridemia ( table 1 ) without increasing adiposity or body weight gain ( table 1 ) , compared with standard chow ( sc)fed controls . there was a trend toward an increase in systemic tnf levels on the hf diet , but this increase did not attain statistical significance ( table 1 ) . finally , although fasting plasma insulin and glucose concentrations were similar in all diet groups , hf and hs induced hepatic insulin resistance as measured by the glucose clamp ( fig . thus , hgo during the glucose clamp was elevated 40% in hs- and hf - fed animals compared with controls ( fig . this corresponded to a decrease in the capacity of insulin to suppress hgo of 29 in hf- and 28% in hs - fed animals compared with 58% in controls ( fig . basal hgo and clamp gdr were similar in sc- , hf- , and hs - fed animals ( supplemental fig . depletion of kcs by gdcl3 decreased liver mrna levels of the kc - specific markers cd68/ed1 and f4/80 by 50 and 80% , respectively ( fig . histochemical analysis of liver confirmed an 70% decrease in cd68/ed1-positive cells ( fig . 3c and e ) . interestingly , adipose tissues levels of the same markers were unaltered by gdcl3 ( fig . furthermore , the effects of gdcl3 were independent of diet , whereas a hf or hs diet had no effects on liver kcs or adipose tissue macrophage ( atm ) numbers in control ( saline - treated ) animals ( supplemental fig . 2 ) . although the depletion of kcs had no effects on liver triglyceride levels in sc - fed animals , the development of hepatic steatosis was prevented in hf- and hs - fed animals ( fig . however , plasma hypertriglyceridemia was not resolved by kc depletion ( table 1 ) , and indeed , there was a trend toward increased triglycerides ( tgs ) in kc - depleted hs- and hf - fed , but not sc - fed , animals . liver peroxisome proliferator  activated receptor- ( ppar ) and acc1 gene expression were increased in kc - depleted hf and hs , and cpt1 expression trended upward , with no changes in mttp , lcad , or dgat2 expression ( fig . hepatic insulin resistance induced by hf and hs diets was prevented in kc - depleted animals ( fig . 2 ) , and there were no effects on fasting glucose or insulin concentrations ( table 1 ) , or basal hgo and clamp gdr ( supplemental fig . 1 ) . the effects of kc depletion on liver triglycerides , dag , ceramide , and cholesterol in response to a high - fat or high - sucrose diet . male wistar rats depleted of kcs or control rats were exposed to a high - fat or high - sucrose diet for 2 weeks as described in research design and methods . subsequently , animals were killed after an overnight fast , livers were isolated , and triglyceride ( a ) , dag ( b ) , cholesterol ( c ) , and ceramide ( d ) content of the liver was determined . body weight , epididymal fat , and plasma measurements in the saline- or gdcl3-treated rats exposed to nc , hf , or hs diets for 2 weeks data are means  se ; n = minimum 5/group except post - clamp sc insulin where n = 3 . values for tgs , ffas , tnf , and lps are from overnight - fasted , preclamp blood samples . * significantly different from nc saline . the effects of kc depletion on the development of liver insulin resistance on a high - fat or high - sucrose diet . male wistar rats depleted of kcs or control rats were exposed to a high - fat ( a and b ) or high - sucrose ( c and d ) diet for 2 weeks . subsequently , animals were fasted overnight and then underwent 4 mu  kg  min euglycemic - hyperinsulinemic clamp in the presence of [ 3-h]-glucose for the measurement of hepatic insulin sensitivity as described in research design and methods . hepatic glucose output ( a and c ) and insulin suppression of hepatic glucose output ( b and d ) were assessed . male wistar rats were depleted of kcs by administration of gdcl3 as described in research design and methods . subsequently , liver and adipose tissue were isolated and the presence of macrophages was determined by qrt - pcr assessment of cd68/ed1 and f4/80 mrna levels ( a and b ) and counting of cd68/ed1-positive cells after immunohistochemical staining of liver and adipose tissue sections ( c  f ) as described in research design and methods . n = a minimum of six in each group for qrt - pcr and n = 3 for immunohistochemical analysis of cd68/ed1-positive cells . ( a high - quality digital representation of this figure is available in the online issue . ) expression of lipid metabolism genes in the liver of sc- , hf- , and hs - fed rats with or without gdcl3 administration . male wistar rats were depleted of kcs by administration of gdcl3 as described in research design and methods . subsequently , liver was isolated , rna was extracted , and the expression of a number of lipid metabolism genes was determined by qrt - pcr . the demonstration that a liver depleted of kcs is protected against diet - induced steatosis and insulin resistance implicates a mechanistic role for these cells in the determination of hepatic insulin sensitivity and the regulation of lipid metabolism . however , this in vivo study does not directly assess the effects of kcs on hepatocyte metabolism and insulin action . studies provide supportive evidence in this regard , demonstrating that lps injection rapidly induces steatosis ( 31 ) , whereas mice with macrophages lacking the ability to m2 polarize have decreased hepatocyte oxidative metabolism and steatosis ( 15,16 ) . again , however , these data implicate , rather then demonstrate , a role for kcs in hepatocyte metabolic abnormalities . to more directly address this issue , a coculture system composed of kcs and hepatocytes isolated from the same rat liver was used . 4 ) were incubated in the presence of lps and free fatty acids to induce m1 polarization and , subsequently , readouts of lipid metabolism and insulin action in hepatocytes were assessed . importantly , lps alone had no effect on hepatocyte triglyceride , fatty acid esterification , or fatty acid oxidation ( fig . c ) . however , when kcs were present , hepatocyte triglycerides and fatty acid esterification were increased , and fatty acid oxidation was decreased . furthermore , phospho - acc was decreased , with a trend toward decreased phospho - ampk ( fig . lipogenesis ( as measured by c - acetate incorporation into total lipids ) , was not increased by kcs ( supplemental fig . thus , m1 activation of kcs resulted in a marked decrease in insulin 's capacity to stimulate phosphorylation of the insulin receptor and irs-1 , pi 3-kinase activity , and akt phosphorylation ( fig . notably , saturated fatty acids alone in the presence of kcs induced decreases in hepatocyte fatty acid oxidation , and increases in fatty acid esterification , but did not alter triglyceride levels ( supplemental fig . the effects of m1-polarized kcs on hepatocyte fatty acid oxidation and esterification and triglyceride accumulation . hepatocytes and kcs were isolated from rat livers , plated , and cultured as described in research design and methods . subsequently , the effects of the indicated exposures on fatty acid oxidation , fatty acid esterification , and triglyceride levels ( a  c ) and acc and ampk phosphorylation ( d were isolated from rat livers , plated , and cultured as described in research design and methods . after the indicated exposures , hepatocytes were incubated with 100 nmol / l insulin for 10 min , and protein extracts were prepared and insulin receptor ( ir ) phosphorylation , irs-1 phosphorylation , irs-1associated pi 3-kinase activity , and akt phosphorylation were assessed as described in research design and methods . the data presented above demonstrate metabolic cross talk between kcs and hepatocytes , which results in altered lipid metabolism and insulin sensitivity , but do not address the biochemical mechanisms of these effects . an obvious candidate mechanism is tnf , because it has a well - described role in the pathogenesis of insulin resistance , has been implicated in the development of dyslipidemia and steatosis , and is a prototypical m1 cytokine produced by macrophages when they are activated ( 3239 ) . to address this possibility , we first evaluated the effects of overnutrition on tnf production by kcs in our model systems . importantly , in both hs- and hf - fed animals kcs had increased tnf , and this increase was not present in kcs of sc - fed animals as assessed by immunofluorescence ( fig . we next directly evaluated the potential role of this cytokine in the adverse effects of kcs on hepatocyte lipid metabolism and insulin responsiveness , using fatty acid oxidation , triglyceride accumulation , and pi 3-kinase activity as readouts , respectively . in response to lps exposure , kcs increased production of tnf , an effect that was substantially greater than that observed when hepatocytes were exposed to lps alone ( fig . 7b ) . importantly , blocking of tnf activity using a tnf-neutralizing antibody resulted in an improvement of the adverse effects of kcs on hepatocyte fatty acid oxidation and accumulation of tgs ( fig . notably , neutralizing antibodies against a panel of other m1-associated cytokines ( il-1 , il-1 , il-6 , ifn- , ifn- , il-12 , il-18 ) were unable to prevent the effects of m1-polarized kcs on hepatocyte lipid metabolism ( supplemental fig . 7 ) . tnf expression in kcs in liver in response to standard chow , high - fat , and high - sucrose diets and tnf secretion from isolated m1-polarized kcs . male wistar rats were exposed to a high - fat or high - sucrose diet for 2 weeks . subsequently , the livers were isolated , sections were prepared , and immunofluorescent staining for kcs and tnf was performed ( a ) , as described in research design and methods . the left panel shows cd68/ed1-positive cells ( green ) , the center panels show tnf-positive cells ( red ) , and the right panels show the merged images . in all panels , n = a minimum of three . for in vitro experiments ( b ) , hepatocytes and kcs were isolated from rat livers , plated , and cultured as shown . after the indicated exposures , media were taken and tnf was quantified by enzyme - linked immunosorbent assay ( elisa ) . ( a high - quality digital representation of this figure is available in the online issue . ) the effects of tnf depletion on hepatocyte lipid metabolism and insulin - stimulated pi 3-kinase activity . hepatocytes and kcs were isolated from rat livers , plated , and cultured as described in research design and methods . subsequently , the effects of the indicated exposures on fatty acid oxidation and triglycerides in the absence or presence of a tnf-neutralizing antibody were assessed as described in research design and methods ( a ) . in separate experiments , hepatocytes were incubated with 100 nmol / l insulin for 10 min after similar exposures to those in ( a ) , proteins extracts were then prepared , and irs-1associated pi 3-kinase activity was assessed as described in research design and methods ( b ) . n = a minimum of six for each experimental condition , except for triglycerides ( n = 3 ) . the purpose of the current study was to address the potential role of kcs in the pathogenesis of two of the liver metabolic abnormalities associated with states of overnutrition , specifically hepatic steatosis and insulin resistance . the data demonstrate that 1 ) kcs are m1 activated in states of overnutrition ; 2 ) depletion of kcs protects against the development of diet - induced steatosis , increased dag , and insulin resistance ; 3 ) m1 activation of kcs in vitro induces changes in hepatocyte lipid metabolic pathways and insulin sensitivity that are consistent with the effects of diet in vivo , and 4 ) tnf plays a role in mediating the detrimental effects of kcs on hepatocyte lipid metabolism and insulin responsiveness . the number and activity of adipose tissue macrophages are increased in obesity in mice and humans ( 811 ) . however , to date it has been unclear whether there is a similar effect of obesity on the activity and/or number of macrophages in other metabolic tissues of relevance to obesity and type 2 diabetes . in the case of liver , resident kcs comprise 5% of the total cell mass and their anatomical position in the sinusoidal spaces of the liver permits them to act as immune sentinels for the portal ( gut ) and systemic circulations . as such , they are most likely to see potential mediators of macrophage activation in obesity . furthermore , their proximity to parenchymal cells presents opportunities both to influence glucose and lipid metabolism and potentially to respond to parenchymal cell  derived signals . data presented in the current study clearly demonstrate activation of kcs ( as measured by increased tnf expression ) in response to overnutrition , whether a high - fat diet or a high - sucrose diet . one issue that arises from these observations is the nature of the dietary signal responsible for the activation of the kcs . based on known activators of the m1 response in macrophages and a number of other recent studies , an increase in lps derived from gut bacteria may mediate the effects of overnutrition ( 4046 ) . indeed , raising lps to levels found in obese mice induces hepatic steatosis and insulin resistance ( 40 ) , whereas the treatment of obese mice with antibiotics reduces lps levels and improves steatosis and insulin resistance ( 41 ) . furthermore , it is now well established that obesity alters the composition of the gut bacterial population ( 4346 ) , that there is increased permeability of the gut to lps in obesity ( 41 ) , and that populating the gut of a lean mouse with bacteria derived from an obese mouse results in increased body fat ( 45 ) . however , a note of caution should be introduced regarding this discussion . although increases in systemic lps have previously been reported in obese mice ( 40,41 ) , significant increases in plasma lps were not observed in the current study , although a distinct trend was clear in sucrose - fed animals ( table 1 ) . it is possible that factors such as the diet length / sampling time point ( after 2 weeks of diet ) , the sampling site ( systemic arterial ) , the nutritional status of the animals ( fasted ) , or diurnal alterations may have masked changes . however , an alternative explanation is that the activation of kcs is mediated by other signals . in this regard , specifically , saturated fatty acids activate toll - like receptor 4 ( tlr-4 ) , the lps receptor , in macrophages ( 47 ) , obese tlr-4null mice have improved insulin sensitivity and lipid profiles ( 48 ) , tlr-4null mice are protected against lipid - induced insulin resistance ( 30 ) , and diabetes - induced obesity is a state of dyslipidemia . 7 ) that saturated free fatty acids can recapitulate the effect of kc - mediated alterations in hepatocyte fatty acid oxidation and esterification , but not triglyceride accumulation . therefore , it is possible that a combination of bacterial - derived lps and elevated free fatty acids may together be important mechanisms of activation of kcs in states of overnutrition . the current study builds on recent reports that have indirectly implicated a role for kcs in altering hepatic metabolism ( 15,16 ) . thus , myeloid deletion of ppar , which plays an important role in mediating the alternative activation ( the th-2 or m2 anti - inflammatory response ) of macrophages , increases susceptibility to insulin resistance , liver steatosis , and reduced hepatocyte oxidative metabolism in obesity . an implied , although indirect , implication of these data is that conditions that favor a proinflammatory m1/th-1 polarization ( the  classic  activation ) of kcs , such as states of overnutrition , play a role in the development of liver metabolic abnormalities . thus , overnutrition results in increased tnf production by kcs , and the rapid development of steatosis and insulin resistance . when kcs are depleted , the effects of diet on liver steatosis and insulin resistance are prevented furthermore , the m1 polarization of kcs in vitro induces changes in hepatocyte lipid metabolic pathways ( fatty acid oxidation , esterification , and triglyceride accumulation ) that would be expected to promote the development of steatosis . taken together with the studies discussed above ( 15,16 ) and other studies that have demonstrated hepatic metabolic alterations in response to the altered myeloid activity ( 12,14 ) , these data suggest that cross talk between kcs and parenchymal cells plays an important role in the regulation of hepatic metabolism . the current study implies a role for tnf in the pathogenesis of hepatic insulin resistance and dysregulated lipid metabolism induced by overnutrition . the source of tnf is most likely the kcs because high - fat and high - sucrose diets did not increase systemic tnf levels but did increase kc tnf levels in vivo , lps profoundly increased tnf production from kcs in vitro but far less so in hepatocytes , and blocking of tnf activity attenuated the effects of kcs on hepatocyte fatty acid oxidation , tg accumulation , and insulin activation of pi 3-kinase . tnf is the prototypical m1 cytokine and is rapidly produced by macrophages in response to proinflammatory signals . importantly , in the context of the current study , numerous studies have implicated tnf in the pathogenesis of insulin resistance and dyslipidemia . thus , obese tnf-null and tnf receptor  null mice have improved insulin sensitivity compared with obese controls ( 37 ) , and the induction of insulin resistance by tnf is well established ( 35,36,39 ) . in the context of lipid metabolism , the inhibition of tnf activity with infliximab decreases liver steatosis and improves insulin signaling in liver of high - fat  fed rats ( 32 ) , tnf bone marrow introduction into tnf mice induces hepatic steatosis on a high - fat diet ( 33 ) , and tnf injection induces hepatic steatosis in mice ( 34 ) . finally , lps injections in mice produce hepatic steatosis within hours , possibly by decreasing fatty acid oxidation ( 31 ) , and it is possible that the in vivo effects of lps are mediated by kc - derived tnf , although the contribution of other factors can not , and should not , be ruled out . an implication of our work and that of others ( 15,16 ) is that interventions that decrease m1 activity of kcs in the liver may have beneficial metabolic effects on insulin action , dyslipidemia , and elevated hepatic glucose output in situations of overnutrition . however , the removal of kcs has substantial effects on the innate immune response in liver ( 49,50 ) . thus , a more likely approach may be to target the activity of the specific pathways that are activated in kcs in response to overnutrition , while maintaining the other vital immune functions of kcs . furthermore , the long - term deletion of kcs from liver may lead to  off - target  effects , which could present as effects on liver function , or effects on peripheral tissue metabolism . again , however , it should be emphasized that complete removal of kcs from liver is most likely not a viable therapeutic intervention for the treatment or prevention of the metabolic abnormalities of obesity / type 2 diabetes . in conclusion , we have demonstrated a critical role for kcs in the pathogenesis of liver steatosis and insulin resistance in states of overnutrition and identified tnf as one potential mediator of these effects . the most important implications of these observations , taken with other studies in this area , is that the activity of components of the immune system such as macrophages , and potentially other immune cells type , is altered by dietary composition and/or excess nutrients and , furthermore , that cross - talk between immune cells and cells / tissues that are intimately involved in the regulation of systemic metabolism , such as adipose tissue and liver , have profound effects on systemic metabolic homeostasis .