the human liposarcoma cell line lisa-2 ( 21 ) was used as a cellular model of visceral human adipocytes and was maintained in dulbeccos modified eagles medium ( dmem ) supplemented with 10% fetal serum and antibiotics , at 37c and 5% co2 . to induce insulin resistance , human adipocytes were cultured for 21 days in serum - free dmem / f12 ( 1:1 ) as previously described ( 22 ) . peripheral blood mononuclear cells , were isolated from blood samples with lymphoprep ( 99% were lymphocytes and monocytes ) as previously described ( 23 ) . the studies involving samples from patients were carried out in accordance with the helsinki declaration , and the protocol was approved by the ethical committee of the university clinic of navarra ( supplementary information , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0771/dc1 ) . mouse c2c12 myocytes ( american type culture collection ) and immortalized murine white adipocytes were maintained in dmem supplemented with 10% fetal serum and antibiotics , at 37c and 5% co2 . cells were transiently transfected according to the lipofectamine protocol ( invitrogen , paisley , u.k . ) , with 4 g of pcdna3-grk2-neo , pcdna3-grk2-k220r - hygro , or ad - shgrk2-rnai , to generate cell lines with increased or decreased grk2 expression , respectively , or with empty vector ( pcdna3-hygro ) as a control . glucose tolerance test ( gtt ) and insulin tolerance test ( itt ) were performed as we previously described ( 24 ) . glucose concentration ( mg / dl ) was determined in tail blood samples using an automatic analyzer ( accucheck from roche ) . other measurements were performed from blood collected from the tail and centrifuged at 2,500 g for 15 min to obtain the serum . insulin and leptin concentrations were assayed using a sensitive elisa kit ( linco research , st . triglycerides released were assayed by an enzymatic method using free glycerol determination kit ( sigma - aldrich , st . cells were stimulated for 30 min with insulin , and glucose uptake was measured during the last 10 min of culture by incorporation of 2-deoxy - d[13h]-glucose as previously described ( 22,24 ) . individual values were expressed as pmol of glucose per 10 min per mg of protein , and results were expressed as percentage of stimulation over basal ( control = 100 ) . male mice ( 12- to 14-week - old ) were subjected to anesthesia ( intraperitoneal injection of 60 mg / kg ketamine per 5 mg / kg xylazine ) , and 25 mg epididymal adipose tissue was removed from each mouse . insulin ( 1 iu / kg body wt ) was then injected via the cava vein , and a similar amount of epididymal adipose tissue was removed , after 1 , 3 , and 7 min . epididymal adipose tissues were homogenized with a polytron homogenizer in lysis buffer and centrifuged at 100,000 g for 15 min . the supernatants were collected , assayed for protein concentration , and stored at 80c until used . other methods were performed as specified in the supplementary information found in an online appendix . the human liposarcoma cell line lisa-2 ( 21 ) was used as a cellular model of visceral human adipocytes and was maintained in dulbeccos modified eagles medium ( dmem ) supplemented with 10% fetal serum and antibiotics , at 37c and 5% co2 . to induce insulin resistance , human adipocytes were cultured for 21 days in serum - free dmem / f12 ( 1:1 ) as previously described ( 22 ) . peripheral blood mononuclear cells , were isolated from blood samples with lymphoprep ( 99% were lymphocytes and monocytes ) as previously described ( 23 ) . the studies involving samples from patients were carried out in accordance with the helsinki declaration , and the protocol was approved by the ethical committee of the university clinic of navarra ( supplementary information , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0771/dc1 ) . mouse c2c12 myocytes ( american type culture collection ) and immortalized murine white adipocytes were maintained in dmem supplemented with 10% fetal serum and antibiotics , at 37c and 5% co2 . cells were transiently transfected according to the lipofectamine protocol ( invitrogen , paisley , u.k . ) , with 4 g of pcdna3-grk2-neo , pcdna3-grk2-k220r - hygro , or ad - shgrk2-rnai , to generate cell lines with increased or decreased grk2 expression , respectively , or with empty vector ( pcdna3-hygro ) as a control . glucose tolerance test ( gtt ) and insulin tolerance test ( itt ) were performed as we previously described ( 24 ) . glucose concentration ( mg / dl ) was determined in tail blood samples using an automatic analyzer ( accucheck from roche ) . other measurements were performed from blood collected from the tail and centrifuged at 2,500 g for 15 min to obtain the serum . insulin and leptin concentrations were assayed using a sensitive elisa kit ( linco research , st . triglycerides released were assayed by an enzymatic method using free glycerol determination kit ( sigma - aldrich , st . cells were stimulated for 30 min with insulin , and glucose uptake was measured during the last 10 min of culture by incorporation of 2-deoxy - d[13h]-glucose as previously described ( 22,24 ) . individual values were expressed as pmol of glucose per 10 min per mg of protein , and results were expressed as percentage of stimulation over basal ( control = 100 ) . male mice ( 12- to 14-week - old ) were subjected to anesthesia ( intraperitoneal injection of 60 mg / kg ketamine per 5 mg / kg xylazine ) , and 25 mg epididymal adipose tissue was removed from each mouse . insulin ( 1 iu / kg body wt ) was then injected via the cava vein , and a similar amount of epididymal adipose tissue was removed , after 1 , 3 , and 7 min . epididymal adipose tissues were homogenized with a polytron homogenizer in lysis buffer and centrifuged at 100,000 g for 15 min . the supernatants were collected , assayed for protein concentration , and stored at 80c until used . other methods were performed as specified in the supplementary information found in an online appendix . to study the modulation of insulin signaling by grk2 in relevant insulin - targeted cell types , we used murine white adipocytic and myocytic cell lines expressing enhanced or decreased grk2 protein levels , or overexpressing a catalytically inactive mutant of grk2 ( fig . the increase in glucose uptake elicited by insulin in both cell types was blocked in the presence of enhanced grk2 levels , whereas grk2 knockdown led to a slightly higher response ( fig . after acute insulin challenge , irs1 tyrosine phosphorylation and akt activation were impaired upon grk2 or grk2-k220r overexpression , both in adipocytes and in myoblasts ( fig . basal irs1 protein levels were higher in cells with silenced grk2 and decreased in cells with elevated grk2 , suggesting a functional relationship between grk2 and irs1 ( fig . co - immunoprecipitation experiments in adipocytes showed that both endogenous grk2 and irs1 proteins form a complex under basal conditions that is completely disrupted after 10 min of insulin treatment ( fig . grk2 overexpression enhances the amount of irs1 associated to the kinase by 87% and prevents the complete disruption of the complex by insulin . in contrast , grk2 downregulation decreases its basal association with irs1 and facilitates a complete disruption of this complex by insulin . immortalized murine white adipocytes and c2c12 myocytes were transiently transfected with empty vector ( control , c ) , pcdna3-grk2-neo ( + ) , the catalytically inactive pcdna3-grk2k220r ( k ) , or shgrk2-rnai (  ) to upregulate or knock - down grk2 expression , respectively . a : lysates were analyzed by western blot with the corresponding antibodies against grk2 and -actin . for examples of full - length blots , see supplementary fig . b : adipocytes and myocytes were cultured for 24 h in serum - free and low - glucose medium and stimulated in the absence or presence of 10 nmol glucose uptake was measured during the last 10 min by incorporation of 2-deoxyglucose into the cells . results were expressed as percentage of stimulation over basal of control cells ( adipocytes , 5.3  0.3 pmol glucose / mg protein/10 min ; myocytes , 45.8  3.2 pmol glucose / mg protein/10 min ) and are means  sem of five independent experiments . c : adipocytic and ( d ) myocytic cell lines were serum - starved overnight and then incubated in the absence or presence of 10 nmol / l insulin ( ins ) for 10 min . lysates were analyzed by western blot with the corresponding antibodies against total and/or phosphorylated forms of irs1 ( tyr 608 ) , akt ( ser473 ) , erk1/2 ( thr202/tyr204 ) , grk2 , and -actin . representative immunoblots of 46 independent experiments and densitometric analysis are shown in ( a , c , and d ) . * p < 0.01 . for examples of full - length blots , see supplementary fig . data in a , c , and d were normalized with the indicated controls , and results are expressed as percent over basal ( control cells transfected with empty vector ) . immortalized murine white adipocytes were transiently transfected with empty vector ( control , c ) , pcdna3-grk2-neo ( + ) , or shgrk2-rnai (  ) to upregulate or knock - down grk2 expression , respectively . a : adipocytic cell lines were serum - starved overnight and then incubated in the absence or presence of 10 nmol / l insulin for 10 min . total protein from adipocytic cells ( 1 mg ) was immunoprecipitated with the anti - irs1 or anti - igg antibodies , and the resulting immune complexes were analyzed by western blot with the corresponding antibodies against grk2 and irs1 . b : samples of ewat were obtained at the indicated times from wt and grk2 adult male mice undergoing insulin ( ins ) infusion ( 1 iu / kg body wt ) as detailed in research design and methods . lysates were immunoprecipitated with an anti - irs1 antibody , and the resulting immune complexes were analyzed by western blot with the corresponding antibodies against grk2 and irs1 . c : the same lysates were analyzed by western blot with the corresponding antibodies against total and/or phosphorylated forms of irs1 ( tyr 608 ) , akt ( ser473 ) , and -actin . data were normalized with the indicated controls , and results obtained in nonstimulated control cells ( a ) or wt animals ( b , c ) were taken as 100% . * p < 0.01 . interestingly , a similar picture emerges when investigating such parameters in adipose tissue from wild - type ( wt ) or grk2 hemizygous mice , expressing some 50% of grk2 protein ( 25,26 ) . the amount of basal irs1/grk2 complexes also depends on grk2 levels in vivo and insulin infusion disrupts such complexes ( fig . also , a faster kinetics in insulin - mediated irs1 and akt phosphorylation ( fig . overall , our data indicate that grk2 levels regulate insulin signaling in skeletal muscle and adipose tissue both in cultured cells and in vivo , most probably by mechanisms independent of kinase activity and involving the formation of dynamic grk2/irs1 complexes . if grk2 is to play a role in the modulation of insulin sensitivity in vivo , the expression of this kinase should be altered in conditions characterized by insulin resistance . obesity - associated metabolic disorders such as type 2 diabetes and insulin resistance can be induced by tnf- infusion , aging , or hfd ( 1 ) . therefore , we compared grk2 expression in epididymal white adipose tissue ( ewat ) , liver , and skeletal muscle obtained from 3- and 9-month - old mice , as well as from mice fed on a hfd or treated with tnf- , using only male mice ( fig . grk2 expression levels were enhanced in adipose tissue ( from 1.3- to 1.9-fold ) and muscle ( 1.4- to 1.7-fold ) in every experimental condition studied , and changes in liver expression were observed upon hfd treatment . grk2 expression was also enhanced by > 2-fold in ewat tissue of the ob / ob model of obese mice ( fig . 3b ) and by 2.5-fold in human adipocytes forced to develop an insulin - resistant state upon a chronic challenge with insulin ( fig . because lymphocyte grk2 levels have been reported to mirror changes in the kinase expression in other organs under several physiopathological circumstances ( 27 ) , we analyzed the levels of grk2 in peripheral blood mononuclear cells obtained from patients with diverse degrees of insulin resistance ( supplementary table 2 , available in an online appendix ) . in these cells , grk2 levels were higher in insulin - resistant patients compared with control individuals ( fig . although preliminary , our results indicate that grk2 levels are altered in murine experimental models and in human samples under insulin - resistant conditions , suggesting that this kinase might mediate the ability to induce insulin resistance of different neurohumoral factors that are altered in these situations ( 28,29 ) . grk2 protein expression is enhanced under insulin - resistance conditions . a : ewat , liver , and muscle from 3- or 9-month - old male mice , fed on a hfd for 12 weeks or treated with tnf- ( 0.1 g / kg body wt ) for 48 h ; ( b ) ewat from wt and ob / ob mice ; ( c ) insulin - sensitive and insulin - resistant human adipocytes ; and ( d ) peripheral blood mononuclear cells ( pbmcs ) collected from blood samples of insulin - sensitive individuals and insulin - resistant patients ( supplementary table 2 ) were lysed and subjected to immunoblot analysis with antibodies against grk2 and -actin as a loading control . data in a , b , and c are means  sem of 4 independent experiments . data in a were normalized with the indicated control , and results are expressed as percent over basal ( 3 month , wt mice ) . results in d represent the means  sem of 10 control subjects and 25 metabolic syndrome patients . * p < 0.01 . to test the hypothesis that such moderate changes in grk2 expression levels may indeed alter insulin signaling in vivo , we studied whole - body insulin action and glucose homeostasis in age - matched wild - type and grk2 male mice . these haploinsufficient mice constitute a good model to recapitulate differences in grk2 expression in the same range to those observed in pathological conditions , as well as to evaluate the potential of therapeutic strategies aimed at decreasing grk2 functionality in vivo . whereas grk2 knock - out mice die in utero with a marked cardiac hypoplasia ( 30 ) and the cardiac phenotype of grk2 mice has been well characterized ( 31,32 ) , insulin signaling has not been investigated so far in these animals . because tnf- infusion induces insulin resistance in murine models in vivo ( 24,33 ) , we examined insulin sensitivity by gtts and itts in wild - type and grk2 3-month - old male mice after a 48-h treatment with tnf- ( fig . , tnf- produced a significant 29% increase in fasting blood glucose levels ( table 1 ) . moreover , the additional pronounced hyperglycemic effect of tnf- after glucose injection observed in wild - type mice was attenuated in grk2 mice ( fig . the improved glucose tolerance observed was not due to an increase in circulating insulin because grk2 mice had lower circulating insulin levels than wild - type mice in both the fasted state ( table 1 ) and during the gtt ( fig . these results suggest that the more efficient glucose clearance observed in grk2 mice is caused by enhanced peripheral insulin sensitivity . in line with this notion , the hypoglycemic effect of insulin injection was impaired upon tnf- treatment in wild - type animals but not in grk2 mice ( fig . wt ( open symbols , solid lines ) and grk2 ( filled symbols , dotted lines ) adult male mice were treated with tnf- ( 0.1 g / g body wt , circles ) or vehicle ( 100 l pbs plus 0.1% bsa , squares ) for 48 h. a : gtts were performed on animals that had been fasted for 24 h and were given an injection of glucose ( 2 g / kg body wt ) . c : itts were performed on fed male mice that had received insulin ( 0.8 iu / kg body wt ) . d : wt and grk2 male mice were treated or not with insulin ( 1 iu / kg body wt ) for 15 min , and ewat , liver , and muscle were removed . lysates were subjected to western blot with the indicated antibodies against total and phosphorylated akt ( ser473 ) , grk2 , and -actin . s2 and supplementary table 1 for detailed quantification of these data . for examples of full - length blots , metabolic parameters of wt and grk2 male mice data are means  sem of measurements obtained for 818 animals for each group . 3 months vs. 9 months , fed a hfd or treated with tnf- ; p < 0.05 . serum from wt and grk2 3- and 9-month - old male mice , fed on a hfd for 12 weeks , or adult male mice treated with tnf- as in fig . 4 was collected , and the indicated metabolic parameters were measured as detailed in research design and methods . the homeostasis model assessment ( homa ) index was calculated as the product of the fasting plasma insulin level ( mu / ml ) and the fasting plasma glucose level ( mmol / l ) , divided by 22.5 . wild - type mice treated with tnf- showed a complete impairment in insulin - induced akt phosphorylation in skeletal muscle , adipose tissue , and liver ( fig . s2 , available in an online appendix , and supplementary table 1 ) that was not detected in grk2 mice . interestingly , tnf- substantially increased grk2 protein content in ewat and muscle of wild - type mice ( by 82 and 42% , respectively ) ( fig . 4d , supplementary fig . s2 , and supplementary table 1 ) , whereas the amount of grk2 remained unaltered ( in liver and muscle ) or below basal wild - type levels ( adipose ) upon tnf- treatment in grk2 mice ( fig . altogether , these results suggest that grk2 is a negative modulator of insulin sensitivity and that lower grk2 levels protect against the systemic insulin resistance induced by tnf-. in the model of aging - associated insulin resistance , grk2 downregulation caused no differences in the gtts or itts of young ( 3-month - old ) male mice ( fig . c ) , whereas in adult 9-month - old grk2 male mice , glucose tolerance was improved compared with wild - type ( fig . a similar tendency had already been observed at 5 months of age in grk2 mice ( supplementary fig . aging also induced a 57% increase in fasting glucose concentration in wild - type mice , limited to 29% in grk2 mice ( table 1 ) . circulating levels of insulin were higher in 9-month - old wild - type than in grk2 mice in response to an identical glucose load ( fig . consistently , grk2 mice exhibited an enhanced decline in blood glucose levels upon administration of insulin ( fig . 5f ) and lower fasting serum insulin levels ( table 1 ) . altogether , these results suggest increased whole - body insulin sensitivity in grk2 mice , in agreement with the values obtained for the homeostasis model assessment index ( table 1 ) . a : gtts were performed on wt ( open circles , solid lines ) and grk2 ( filled circles , dotted lines ) 3-month - old male mice as in fig . c : itts were performed on wt ( open circles ) and grk2 ( filled circles ) 3-month - old male mice as in fig . d  f : gtts and itts were performed as in previous panels in wt ( open circles ) and grk2 ( filled circles ) 9-month - old male mice . g : lysates from wt and grk2 3- and 9-month - old male mice treated or not with insulin were processed as in fig . insulin - stimulated akt phosphorylation was markedly impaired with age in ewat , liver , and muscle in wild - type mice , while grk2 animals maintained a robust activation of this pathway ( fig . interestingly , in adult animals , grk2 protein expression was enhanced by 33 and 68% in ewat and muscle , respectively , compared with young mice ( figs . s2 , and supplementary table 1 ) , while grk2 levels in adult grk2 mice were below the expression detected at 3 months of age in wild - type animals in all tissues examined ( fig . overall , these data suggest there is a threshold above which the enhanced grk2 expression observed with age markedly affects insulin sensitivity in vivo . after a hfd regime for 12 weeks , the overall increase in body weight was 67% in wild - type and only 53% in grk2 mice ( fig . consistently , even when adipocyte diameter was increased in both groups , adiposity in grk2 hemizygous mice did not go beyond a certain threshold that was exceeded only in wild - type mice , with the size of hfd - fed grk2 epididymal adipocytes being 45% inferior to that of wild - type mice ( fig . wt ( open symbols , solid lines ) and grk2 ( filled symbols , dotted lines ) male mice were fed a cd or hfd for 12 weeks . a : body weight evolution and ( b ) percentage of change in body weight during the 12 weeks of treatment . d : paraffin - embedded sections of epididymal fat pads from wt and grk2 male mice fed on cd or hfd and stained with hematoxylin and eosin ( magnification 10 ; scale bar 50 m for all pictures ) . e : gtts ( circles , hfd ; squares , cd ) and ( f ) itts were performed as in fig . g : lysates from wt and grk2 male mice fed cd or hfd for 12 weeks treated or not with insulin were processed as in fig . ( a high - quality color representation of this figure is available in the online issue . ) gtt analysis indicated a clear deterioration in glucose metabolism in wild - type but not in hfd - fed grk2 mice . in the hfd - fed state 6e ) , because we detect no statistically significant changes in glucose area between chow diet ( cd)-fed and hfd - fed grk2 mice , which showed lower blood glucose levels ( table 1 ) and an enhanced reduction in blood glucose induced by insulin ( fig . hfd caused a marked increase in circulating levels of glucose , insulin , and triglycerides after 12 weeks of feeding ( table 1 ) in wild - type mice , whereas these changes were much more modest in grk2 animals . these parameters were not altered with cd in either type of animal ( data not shown ) . in summary , hfd promoted a marked insulin - resistance state in wild - type mice , in accordance with the observed increase in homeostasis model assessment values ( table 1 ) , whereas insulin sensitivity was preserved in grk2 mice . insulin - induced akt phosphorylation was dramatically decreased in adipose tissue , liver , and muscle of wild - type but not hfd - fed grk2 mice ( fig . s2 , and supplementary table 1 ) . on the other hand , in agreement with data in fig . 3a , grk2 protein levels were significantly increased in ewat ( 84% ) , liver ( 26% ) , and muscle ( 63% ) of wild - type but not grk2 mice fed on a hfd ( fig . these results indicate that , even in different cellular settings , whenever glucose intolerance develops , grk2 mice always maintain the levels of grk2 protein below those detected in wild - type and thus below a given threshold that helps to prevent the development of insulin insensitivity . c , a detailed weekly analysis of the weight of wild - type and grk2 animals from weaning to 15 months of age ( fig . 7a ) revealed less weight gain for both male and female grk2 mice ( fig . body weight differences were detected as early as at 5 months of age ( fig . 7a ) , and at 9 months of age , when some wild - type mice are visibly obese ( fig . 7d ) , the average body weight gain was 65 and 56% in male and female wild - type mice , respectively , but only 56 and 43% in grk2 mice ( fig . body fat distribution was studied by magnetic nuclear resonance imaging ( mnri ) ( fig . 7e ) , showing that 9-month - old grk2 mice had 33% less overall fat and smaller fat depots than age - matched wild - type littermates ( fig . epididymal fat mass was reduced by 30% in grk2 compared with wild - type mice at 9 months of age ( fig . 7 g ) , and grk2 mice exhibited reduced adipocytic size in epididymal ( threefold ) , retroperitoneal ( 4.6-fold ) , and inguinal ( threefold ) fat ( fig . interestingly , a decrease in circulating leptin levels was also detected in grk2 animals at 9 months of age ( table 1 ) . these results indicate that downregulation of grk2 is sufficient to induce a lean phenotype , which uncovers a previously unidentified role for grk2 in the regulation of adiposity . a : body weight evolution in male or female wt ( open circles , solid lines ) and grk2 ( filled circles , dotted lines ) mice between 2 and 15 months of age . c : daily food intake in 3- and 9-month - old male wt and grk2 mice . e : adiposity in 9-month - old male mice measured by mnri ; white represents areas with > 50% fat . results were expressed as proportion between fat volumes vs. total mouse volume ( fat vol / total vol ) . g : ewat weight in 3- and 9-month - old male wt and grk2 mice . h : paraffin - embedded sections of wat : retroperitoneal , epididymal , and inguinal fat pads from 9-month - old male wt and grk2 mice stained with hematoxylin and eosin . ( a high - quality color representation of this figure is available in the online issue . ) to study the modulation of insulin signaling by grk2 in relevant insulin - targeted cell types , we used murine white adipocytic and myocytic cell lines expressing enhanced or decreased grk2 protein levels , or overexpressing a catalytically inactive mutant of grk2 ( fig . the increase in glucose uptake elicited by insulin in both cell types was blocked in the presence of enhanced grk2 levels , whereas grk2 knockdown led to a slightly higher response ( fig . after acute insulin challenge , irs1 tyrosine phosphorylation and akt activation were impaired upon grk2 or grk2-k220r overexpression , both in adipocytes and in myoblasts ( fig . basal irs1 protein levels were higher in cells with silenced grk2 and decreased in cells with elevated grk2 , suggesting a functional relationship between grk2 and irs1 ( fig . co - immunoprecipitation experiments in adipocytes showed that both endogenous grk2 and irs1 proteins form a complex under basal conditions that is completely disrupted after 10 min of insulin treatment ( fig . grk2 overexpression enhances the amount of irs1 associated to the kinase by 87% and prevents the complete disruption of the complex by insulin . in contrast , grk2 downregulation decreases its basal association with irs1 and facilitates a complete disruption of this complex by insulin . immortalized murine white adipocytes and c2c12 myocytes were transiently transfected with empty vector ( control , c ) , pcdna3-grk2-neo ( + ) , the catalytically inactive pcdna3-grk2k220r ( k ) , or shgrk2-rnai (  ) to upregulate or knock - down grk2 expression , respectively . a : lysates were analyzed by western blot with the corresponding antibodies against grk2 and -actin . for examples of full - length blots , see supplementary fig . b : adipocytes and myocytes were cultured for 24 h in serum - free and low - glucose medium and stimulated in the absence or presence of 10 nmol glucose uptake was measured during the last 10 min by incorporation of 2-deoxyglucose into the cells . results were expressed as percentage of stimulation over basal of control cells ( adipocytes , 5.3  0.3 pmol glucose / mg protein/10 min ; myocytes , 45.8  3.2 pmol glucose / mg protein/10 min ) and are means  sem of five independent experiments . c : adipocytic and ( d ) myocytic cell lines were serum - starved overnight and then incubated in the absence or presence of 10 nmol / l insulin ( ins ) for 10 min . lysates were analyzed by western blot with the corresponding antibodies against total and/or phosphorylated forms of irs1 ( tyr 608 ) , akt ( ser473 ) , erk1/2 ( thr202/tyr204 ) , grk2 , and -actin . representative immunoblots of 46 independent experiments and densitometric analysis are shown in ( a , c , and d ) . * p < 0.01 . for examples of full - length blots , see supplementary fig . data in a , c , and d were normalized with the indicated controls , and results are expressed as percent over basal ( control cells transfected with empty vector ) . immortalized murine white adipocytes were transiently transfected with empty vector ( control , c ) , pcdna3-grk2-neo ( + ) , or shgrk2-rnai (  ) to upregulate or knock - down grk2 expression , respectively . a : adipocytic cell lines were serum - starved overnight and then incubated in the absence or presence of 10 nmol / l insulin for 10 min . total protein from adipocytic cells ( 1 mg ) was immunoprecipitated with the anti - irs1 or anti - igg antibodies , and the resulting immune complexes were analyzed by western blot with the corresponding antibodies against grk2 and irs1 . b : samples of ewat were obtained at the indicated times from wt and grk2 adult male mice undergoing insulin ( ins ) infusion ( 1 iu / kg body wt ) as detailed in research design and methods . lysates were immunoprecipitated with an anti - irs1 antibody , and the resulting immune complexes were analyzed by western blot with the corresponding antibodies against grk2 and irs1 . c : the same lysates were analyzed by western blot with the corresponding antibodies against total and/or phosphorylated forms of irs1 ( tyr 608 ) , akt ( ser473 ) , and -actin . data were normalized with the indicated controls , and results obtained in nonstimulated control cells ( a ) or wt animals ( b , c ) were taken as 100% . * p < 0.01 . interestingly , a similar picture emerges when investigating such parameters in adipose tissue from wild - type ( wt ) or grk2 hemizygous mice , expressing some 50% of grk2 protein ( 25,26 ) . the amount of basal irs1/grk2 complexes also depends on grk2 levels in vivo and insulin infusion disrupts such complexes ( fig . also , a faster kinetics in insulin - mediated irs1 and akt phosphorylation ( fig . overall , our data indicate that grk2 levels regulate insulin signaling in skeletal muscle and adipose tissue both in cultured cells and in vivo , most probably by mechanisms independent of kinase activity and involving the formation of dynamic grk2/irs1 complexes . if grk2 is to play a role in the modulation of insulin sensitivity in vivo , the expression of this kinase should be altered in conditions characterized by insulin resistance . obesity - associated metabolic disorders such as type 2 diabetes and insulin resistance can be induced by tnf- infusion , aging , or hfd ( 1 ) . therefore , we compared grk2 expression in epididymal white adipose tissue ( ewat ) , liver , and skeletal muscle obtained from 3- and 9-month - old mice , as well as from mice fed on a hfd or treated with tnf- , using only male mice ( fig . grk2 expression levels were enhanced in adipose tissue ( from 1.3- to 1.9-fold ) and muscle ( 1.4- to 1.7-fold ) in every experimental condition studied , and changes in liver expression were observed upon hfd treatment . grk2 expression was also enhanced by > 2-fold in ewat tissue of the ob / ob model of obese mice ( fig . 3b ) and by 2.5-fold in human adipocytes forced to develop an insulin - resistant state upon a chronic challenge with insulin ( fig . 3c ) . finally , because lymphocyte grk2 levels have been reported to mirror changes in the kinase expression in other organs under several physiopathological circumstances ( 27 ) , we analyzed the levels of grk2 in peripheral blood mononuclear cells obtained from patients with diverse degrees of insulin resistance ( supplementary table 2 , available in an online appendix ) . in these cells , grk2 levels were higher in insulin - resistant patients compared with control individuals ( fig . although preliminary , our results indicate that grk2 levels are altered in murine experimental models and in human samples under insulin - resistant conditions , suggesting that this kinase might mediate the ability to induce insulin resistance of different neurohumoral factors that are altered in these situations ( 28,29 ) . grk2 protein expression is enhanced under insulin - resistance conditions . a : ewat , liver , and muscle from 3- or 9-month - old male mice , fed on a hfd for 12 weeks or treated with tnf- ( 0.1 g / kg body wt ) for 48 h ; ( b ) ewat from wt and ob / ob mice ; ( c ) insulin - sensitive and insulin - resistant human adipocytes ; and ( d ) peripheral blood mononuclear cells ( pbmcs ) collected from blood samples of insulin - sensitive individuals and insulin - resistant patients ( supplementary table 2 ) were lysed and subjected to immunoblot analysis with antibodies against grk2 and -actin as a loading control . data in a , b , and c are means  sem of 4 independent experiments . data in a were normalized with the indicated control , and results are expressed as percent over basal ( 3 month , wt mice ) . results in d represent the means  sem of 10 control subjects and 25 metabolic syndrome patients . to test the hypothesis that such moderate changes in grk2 expression levels may indeed alter insulin signaling in vivo , we studied whole - body insulin action and glucose homeostasis in age - matched wild - type and grk2 male mice . these haploinsufficient mice constitute a good model to recapitulate differences in grk2 expression in the same range to those observed in pathological conditions , as well as to evaluate the potential of therapeutic strategies aimed at decreasing grk2 functionality in vivo . whereas grk2 knock - out mice die in utero with a marked cardiac hypoplasia ( 30 ) and the cardiac phenotype of grk2 mice has been well characterized ( 31,32 ) , insulin signaling has not been investigated so far in these animals . because tnf- infusion induces insulin resistance in murine models in vivo ( 24,33 ) , we examined insulin sensitivity by gtts and itts in wild - type and grk2 3-month - old male mice after a 48-h treatment with tnf- ( fig . , tnf- produced a significant 29% increase in fasting blood glucose levels ( table 1 ) . moreover , the additional pronounced hyperglycemic effect of tnf- after glucose injection observed in wild - type mice was attenuated in grk2 mice ( fig . the improved glucose tolerance observed was not due to an increase in circulating insulin because grk2 mice had lower circulating insulin levels than wild - type mice in both the fasted state ( table 1 ) and during the gtt ( fig . these results suggest that the more efficient glucose clearance observed in grk2 mice is caused by enhanced peripheral insulin sensitivity . in line with this notion , the hypoglycemic effect of insulin injection was impaired upon tnf- treatment in wild - type animals but not in grk2 mice ( fig . wt ( open symbols , solid lines ) and grk2 ( filled symbols , dotted lines ) adult male mice were treated with tnf- ( 0.1 g / g body wt , circles ) or vehicle ( 100 l pbs plus 0.1% bsa , squares ) for 48 h. a : gtts were performed on animals that had been fasted for 24 h and were given an injection of glucose ( 2 g / kg body wt ) . c : itts were performed on fed male mice that had received insulin ( 0.8 iu / kg body wt ) . glucose concentration was determined in blood samples obtained from the tail vein . d : wt and grk2 male mice were treated or not with insulin ( 1 iu / kg body wt ) for 15 min , and ewat , liver , and muscle were removed . lysates were subjected to western blot with the indicated antibodies against total and phosphorylated akt ( ser473 ) , grk2 , and -actin . s2 and supplementary table 1 for detailed quantification of these data . for examples of full - length blots , see supplementary fig . metabolic parameters of wt and grk2 male mice data are means  sem of measurements obtained for 818 animals for each group . 3 months vs. 9 months , fed a hfd or treated with tnf- ; p < 0.05 . serum from wt and grk2 3- and 9-month - old male mice , fed on a hfd for 12 weeks , or adult male mice treated with tnf- as in fig . 4 was collected , and the indicated metabolic parameters were measured as detailed in research design and methods . the homeostasis model assessment ( homa ) index was calculated as the product of the fasting plasma insulin level ( mu / ml ) and the fasting plasma glucose level ( mmol / l ) , divided by 22.5 . wild - type mice treated with tnf- showed a complete impairment in insulin - induced akt phosphorylation in skeletal muscle , adipose tissue , and liver ( fig . s2 , available in an online appendix , and supplementary table 1 ) that was not detected in grk2 mice . interestingly , tnf- substantially increased grk2 protein content in ewat and muscle of wild - type mice ( by 82 and 42% , respectively ) ( fig . 4d , supplementary fig . s2 , and supplementary table 1 ) , whereas the amount of grk2 remained unaltered ( in liver and muscle ) or below basal wild - type levels ( adipose ) upon tnf- treatment in grk2 mice ( fig . altogether , these results suggest that grk2 is a negative modulator of insulin sensitivity and that lower grk2 levels protect against the systemic insulin resistance induced by tnf-. in the model of aging - associated insulin resistance , grk2 downregulation caused no differences in the gtts or itts of young ( 3-month - old ) male mice ( fig . c ) , whereas in adult 9-month - old grk2 male mice , glucose tolerance was improved compared with wild - type ( fig . a similar tendency had already been observed at 5 months of age in grk2 mice ( supplementary fig . aging also induced a 57% increase in fasting glucose concentration in wild - type mice , limited to 29% in grk2 mice ( table 1 ) . circulating levels of insulin were higher in 9-month - old wild - type than in grk2 mice in response to an identical glucose load ( fig . consistently , grk2 mice exhibited an enhanced decline in blood glucose levels upon administration of insulin ( fig . 5f ) and lower fasting serum insulin levels ( table 1 ) . altogether , these results suggest increased whole - body insulin sensitivity in grk2 mice , in agreement with the values obtained for the homeostasis model assessment index ( table 1 ) . a : gtts were performed on wt ( open circles , solid lines ) and grk2 ( filled circles , dotted lines ) 3-month - old male mice as in fig . c : itts were performed on wt ( open circles ) and grk2 ( filled circles ) 3-month - old male mice as in fig . d  f : gtts and itts were performed as in previous panels in wt ( open circles ) and grk2 ( filled circles ) 9-month - old male mice . g : lysates from wt and grk2 3- and 9-month - old male mice treated or not with insulin were processed as in fig . insulin - stimulated akt phosphorylation was markedly impaired with age in ewat , liver , and muscle in wild - type mice , while grk2 animals maintained a robust activation of this pathway ( fig . interestingly , in adult animals , grk2 protein expression was enhanced by 33 and 68% in ewat and muscle , respectively , compared with young mice ( figs . s2 , and supplementary table 1 ) , while grk2 levels in adult grk2 mice were below the expression detected at 3 months of age in wild - type animals in all tissues examined ( fig . overall , these data suggest there is a threshold above which the enhanced grk2 expression observed with age markedly affects insulin sensitivity in vivo . after a hfd regime for 12 weeks , wild - type mice gained significantly more weight than grk2 mice ( fig . the overall increase in body weight was 67% in wild - type and only 53% in grk2 mice ( fig . consistently , even when adipocyte diameter was increased in both groups , adiposity in grk2 hemizygous mice did not go beyond a certain threshold that was exceeded only in wild - type mice , with the size of hfd - fed grk2 epididymal adipocytes being 45% inferior to that of wild - type mice ( fig . wt ( open symbols , solid lines ) and grk2 ( filled symbols , dotted lines ) male mice were fed a cd or hfd for 12 weeks . a : body weight evolution and ( b ) percentage of change in body weight during the 12 weeks of treatment . d : paraffin - embedded sections of epididymal fat pads from wt and grk2 male mice fed on cd or hfd and stained with hematoxylin and eosin ( magnification 10 ; scale bar 50 m for all pictures ) . e : gtts ( circles , hfd ; squares , cd ) and ( f ) itts were performed as in fig . g : lysates from wt and grk2 male mice fed cd or hfd for 12 weeks treated or not with insulin were processed as in fig . ( a high - quality color representation of this figure is available in the online issue . ) gtt analysis indicated a clear deterioration in glucose metabolism in wild - type but not in hfd - fed grk2 mice . in the hfd - fed state 6e ) , because we detect no statistically significant changes in glucose area between chow diet ( cd)-fed and hfd - fed grk2 mice , which showed lower blood glucose levels ( table 1 ) and an enhanced reduction in blood glucose induced by insulin ( fig . hfd caused a marked increase in circulating levels of glucose , insulin , and triglycerides after 12 weeks of feeding ( table 1 ) in wild - type mice , whereas these changes were much more modest in grk2 animals . these parameters were not altered with cd in either type of animal ( data not shown ) . in summary , hfd promoted a marked insulin - resistance state in wild - type mice , in accordance with the observed increase in homeostasis model assessment values ( table 1 ) , whereas insulin sensitivity was preserved in grk2 mice . insulin - induced akt phosphorylation was dramatically decreased in adipose tissue , liver , and muscle of wild - type but not hfd - fed grk2 mice ( fig . s2 , and supplementary table 1 ) . on the other hand , in agreement with data in fig . 3a , grk2 protein levels were significantly increased in ewat ( 84% ) , liver ( 26% ) , and muscle ( 63% ) of wild - type but not grk2 mice fed on a hfd ( fig . these results indicate that , even in different cellular settings , whenever glucose intolerance develops , grk2 mice always maintain the levels of grk2 protein below those detected in wild - type and thus below a given threshold that helps to prevent the development of insulin insensitivity . c , a detailed weekly analysis of the weight of wild - type and grk2 animals from weaning to 15 months of age ( fig . 7a ) revealed less weight gain for both male and female grk2 mice ( fig . body weight differences were detected as early as at 5 months of age ( fig . 7a ) , and at 9 months of age , when some wild - type mice are visibly obese ( fig . 7d ) , the average body weight gain was 65 and 56% in male and female wild - type mice , respectively , but only 56 and 43% in grk2 mice ( fig . body fat distribution was studied by magnetic nuclear resonance imaging ( mnri ) ( fig . 7e ) , showing that 9-month - old grk2 mice had 33% less overall fat and smaller fat depots than age - matched wild - type littermates ( fig . epididymal fat mass was reduced by 30% in grk2 compared with wild - type mice at 9 months of age ( fig . 7 g ) , and grk2 mice exhibited reduced adipocytic size in epididymal ( threefold ) , retroperitoneal ( 4.6-fold ) , and inguinal ( threefold ) fat ( fig . interestingly , a decrease in circulating leptin levels was also detected in grk2 animals at 9 months of age ( table 1 ) . these results indicate that downregulation of grk2 is sufficient to induce a lean phenotype , which uncovers a previously unidentified role for grk2 in the regulation of adiposity . a : body weight evolution in male or female wt ( open circles , solid lines ) and grk2 ( filled circles , dotted lines ) mice between 2 and 15 months of age . c : daily food intake in 3- and 9-month - old male wt and grk2 mice . e : adiposity in 9-month - old male mice measured by mnri ; white represents areas with > 50% fat . results were expressed as proportion between fat volumes vs. total mouse volume ( fat vol / total vol ) . g : ewat weight in 3- and 9-month - old male wt and grk2 mice . h : paraffin - embedded sections of wat : retroperitoneal , epididymal , and inguinal fat pads from 9-month - old male wt and grk2 mice stained with hematoxylin and eosin . ( a high - quality color representation of this figure is available in the online issue . ) in this report , we uncover grk2 as a key modulator of insulin sensitivity in vivo . in cultured adipocytes and myoblasts , increased grk2 or grk2-k220r levels inhibit insulin - stimulated glucose uptake and signaling in a kinase - activity independent manner , by mechanisms involving the formation of dynamic grk2/irs1 complexes . grk2 expression is enhanced by 2-fold in insulin - resistant human adipocytes , in blood mononuclear cells from insulin - resistant patients , and in adipose and muscle tissues in either tnf- , aging- , or hfd - induced insulin - resistance models . importantly , grk2 mice maintain glucose tolerance and insulin signaling in the major insulin - responsive tissues under such experimental conditions , suggesting that enhanced grk2 expression above a certain threshold markedly impairs insulin sensitivity in vivo . because grk2 haploinsufficient animals show a phenotype quite similar to mice deficient in other well established negative regulators of insulin signaling such as ptp1b ( 24,33,34 ) , grk2 can be regarded as a bona fide novel physiological regulator of insulin action and leanness . previous reports have indicated that grk2 could act as an inhibitor of insulin action in cellular models . binding of grk2 to gq/11 impairs insulin - stimulated glucose uptake in 3t3l1 preadipocytes ( 19 ) , and enhanced grk2 levels favor insulin resistance induced by endothelin-1 in 3t3l1 cells ( 29 ) or by chronic -adrenergic receptor stimulation in hek-293 cells ( 28 ) . gpcr agonists promote grk2 interaction with gq and also with irs1 , resulting in decreased insulin - stimulated glucose transport , irs - serine phosphorylation , and irs1 degradation ( 29 ) . our data in cultured cells as well as in adipose tissue in vivo show that irs1 levels as well as the amount of basal grk2-irs1 complexes depend on grk2 expression and that insulin stimulation rapidly disrupts basal irs1/grk2 complexes , which is hampered in the presence of elevated levels of grk2 . overall , these data suggest that altered grk2 levels could lead to modulation of insulin signals through grk2-gq/11 binding , grk2-irs1 association , and altered endothelin-1 or -adrenergic - mediated transmodulation of the insulin pathway . interestingly , -arrestin2 , another member of the gpcr signal transduction pathway , positively regulates insulin sensitivity by serving as a scaffold of akt and src to the insulin receptor ( 35 ) . -arrestin2 levels are downregulated in liver and muscle in animal models and patients with insulin resistance . therefore , simultaneous upregulation of grk2 and downregulation of -arrestin2 in insulin - resistant conditions could lead to major alterations in the insulin signaling pathways and in gpcr  insulin receptor cross - talk . it is worth noting that elevated levels of insulin and igf-1 have been shown to exert differential effects on grk2 and -arrestin2 expression ( 36,37 ) . enhanced grk2 levels could alter the proposed novel insulin receptor  arrestin signaling pathway by redirecting arrestin to other interactors , such as gpcr , or , given the reported direct interactions of the kinase with akt or src , by inhibiting the formation of the arrestin / akt / src complexes . grk2 is degraded by the proteasome pathway by associating with the mdm2 e3-ubiquitin ligase , and activation of the pi3k / akt pathway by igf-1 blocks mdm2-mediated grk2 degradation , leading to enhanced grk2 levels ( 38 ) . consistently , chronic insulin has been recently shown to increase grk2 levels in hek-293 and liver fl83b cells ( 20,28 ) , which is in agreement with the upregulation of grk2 we observe after insulin treatment in human adipocytic cells . thus , it is tempting to suggest that the hyperinsulinemia associated with aging- , hfd- , or tnf-induced insulin - resistant conditions , and clinically associated with obesity and type 2 diabetes , would trigger the observed upregulation of grk2 in such conditions . consistent with our findings , enhanced grk2 expression has been reported in tissues from obese zucker rats , a model of insulin resistance and hyperinsulinemia ( 39 ) . an altered cytokine expression pattern typical of insulin - resistant states could also contribute to enhance grk2 expression levels . tnf- or il-6 increase the expression of other negative modulators of the insulin signaling cascade like ptp1b ( 24,40 ) , and we find that tnf- also enhances grk2 protein content in adipose tissue and muscle of wild - type mice in the presence of mild hyperinsulinemia . as in grk2 mice , ptp1b - deficient animals also exhibit protection against insulin resistance induced by tnf- ( 24 ) or il-6 ( 40 ) . proinflammatory mediators can modulate endogenous grk2 expression in a cell - type specific fashion ( 41 ) , and an increase in grk2 protein is found upon chronic il-1 treatment ( 26,42 ) . interestingly , grk2 appears to have a relevant role in the etiology and/or in the development of several inflammatory diseases such as multiple sclerosis and autoimmune arthritis ( 43 ) . a role for grk2 in states of insulin resistance associated with inflammatory diseases deserves further investigation . the fact that the 50% downregulation of grk2 levels is sufficient per se to protect against tnf-- , aging- , or hfd - induced alterations in glucose homeostasis and insulin signaling strongly supports that this kinase is a key modulator of insulin sensitivity in vivo and suggests new therapeutic strategies against type 2 diabetes and obesity . in fact , in animal models of type 2 diabetes , grk2 inhibition through systemic delivery of small peptides derived from its catalytic domain results in improved glucose homeostasis ( 28,44 ) . it remains to be established whether inhibition of catalytic activity , downregulation of protein expression , or targeted disruption of the specific interaction of grk2 with insulin signaling pathway components are the more appropriate strategies for specifically improving insulin sensitivity . the grk2 mice constitute a good model that recapitulates a sustained systemic inhibition of kinase functionality . we report here that grk2 levels are important modulators of age- and diet - induced adiposity . grk2 mice gained less weight and showed diminished adipocyte size with a hfd , and adult hemizygous animals displayed reduced adiposity and lower circulating levels of insulin and leptin . aging is associated with fat mass accretion and with decreased peripheral insulin sensitivity in humans and rodents ( 45 ) , and impaired insulin actions in adipose tissue could represent a key step leading to the overall insulin - resistance characteristic of adult and/or obese animals ( 46 ) . adipocyte - conditioned medium impairs insulin signaling in muscle cells ( 22 ) and hepatocytes ( 47 ) , and mice that exhibit reduced adiposity typically display improved glucose tolerance and increased insulin sensitivity ( 46 ) . therefore , it is possible that part of the insulin hypersensitivity detected in grk2 mice and the protection afforded to the development of insulin - resistant conditions in these animals could be related to the observed changes in adiposity . future experiments involving tissue - specific downregulation will provide new insight into the understanding of the role of grk2 in obesity and insulin resistance .