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 .
 objectiveinsulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity . given the emerging role of signal transduction in these syndromes , 
 we set out to explore the possible role that g protein  coupled receptor kinase 2 ( grk2 ) , first identified as a g protein  coupled receptor regulator , could have as a modulator of insulin responses.research design and methodswe analyzed the influence of grk2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of grk2 , as well as in grk2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance : tumor necrosis factor- ( tnf- ) infusion , aging , and high - fat diet ( hfd ) . 
 glucose transport , whole - body glucose and insulin tolerance , the activation status of insulin pathway components , and the circulating levels of important mediators were measured . 
 the development of obesity and adipocyte size with age and hfd was analyzed.resultsaltering grk2 levels markedly modifies insulin - mediated signaling in cultured adipocytes and myocytes . 
 grk2 levels are increased by 2-fold in muscle and adipose tissue in the animal models tested , as well as in lymphocytes from metabolic syndrome patients . 
 in contrast , hemizygous grk2 mice show enhanced insulin sensitivity and do not develop insulin resistance by tnf- , aging , or hfd . 
 furthermore , reduced grk2 levels induce a lean phenotype and decrease age - related adiposity.conclusionsoverall , our data identify grk2 as an important negative regulator of insulin effects , key to the etiopathogenesis of insulin resistance and obesity , which uncovers this protein as a potential therapeutic target in the treatment of these disorders . 
RESEARCH DESIGN AND METHODS
Cell culture and transfections.
Metabolic assays.
Glucose uptake assay.
In vivo signaling assay.
RESULTS
Insulin signaling is regulated by GRK2 levels in adipocytes and myocytes.
GRK2 protein expression is enhanced under insulin-resistant conditions.
GRK2
GRK2 downregulation prevents the development of aging-associated insulin resistance.
GRK2
GRK2
DISCUSSION
Supplementary Material