rictor mice ( 10 ) were crossed with ap2-cre transgenic mice [ strain , b6-cg - tg(fabp4-cre)1rev / j ; the jackson laboratory , bar harbor , me ] to obtain heterozygous ap2-cre;rictor offspring ( where wt refers to wild type ) in the f1 generation .
these heterozygous mice were crossed with rictor mice to obtain the fric mice with genotype ap2-cre;rictor and their rictor littermates lacking ap2-cre expression ( referred to here as fric ) .
age - matched fric and fric mice of both sexes were studied at 35 months of age ( young ) and when they were aged > 9 months ( old ) . for all mice used , the genotypes were determined by pcr analysis of tail genomic dna as described previously ( 10 ) .
both rictor mice as well as ap2-cre transgenic mice used for breeding had been backcrossed for 6 and 9 generations to the c57bl/6 strain , respectively .
the mice were maintained under temperature- and humidity - controlled conditions with a 12-h light / dark cycle and were allowed food ( total calories from fat 17% , lm-485 ; harlan - teklad ) and water ad libitum .
all animal studies performed in this investigation were approved by the university of virginia animal care and use committee . following an overnight fast ( 15 h ) , a 2-h hyperinsulinemic ( insulin at 150 mu / kg body wt priming followed by 2.5 mu / kg / min)-euglycemic clamp was conducted in awake mice using [ 3-h]-glucose and 2-deoxy - d-[1-c]-glucose to assess glucose metabolism in individual tissues as described in kim et al .
other experimental protocols are described in detail in the online appendix ( available at http://diabetes.diabetesjournals.org/cgi/content/full/dc09-1061/dc1 ) .
values given are means  se for the numbers of animals indicated in the figure legends .
rictor mice ( 10 ) were crossed with ap2-cre transgenic mice [ strain , b6-cg - tg(fabp4-cre)1rev / j ; the jackson laboratory , bar harbor , me ] to obtain heterozygous ap2-cre;rictor offspring ( where wt refers to wild type ) in the f1 generation .
these heterozygous mice were crossed with rictor mice to obtain the fric mice with genotype ap2-cre;rictor and their rictor littermates lacking ap2-cre expression ( referred to here as fric ) .
age - matched fric and fric mice of both sexes were studied at 35 months of age ( young ) and when they were aged > 9 months ( old ) . for all mice used , the genotypes were determined by pcr analysis of tail genomic dna as described previously ( 10 ) .
both rictor mice as well as ap2-cre transgenic mice used for breeding had been backcrossed for 6 and 9 generations to the c57bl/6 strain , respectively .
the mice were maintained under temperature- and humidity - controlled conditions with a 12-h light / dark cycle and were allowed food ( total calories from fat 17% , lm-485 ; harlan - teklad ) and water ad libitum .
all animal studies performed in this investigation were approved by the university of virginia animal care and use committee .
following an overnight fast ( 15 h ) , a 2-h hyperinsulinemic ( insulin at 150 mu / kg body wt priming followed by 2.5 mu / kg / min)-euglycemic clamp was conducted in awake mice using [ 3-h]-glucose and 2-deoxy - d-[1-c]-glucose to assess glucose metabolism in individual tissues as described in kim et al .
other experimental protocols are described in detail in the online appendix ( available at http://diabetes.diabetesjournals.org/cgi/content/full/dc09-1061/dc1 ) .
values given are means  se for the numbers of animals indicated in the figure legends .
fric mice were generated by breeding rictor mice with mice expressing cre recombinase under the control of the ap2 promoter .
rictor protein levels were measured in isolated fat cells , liver , and skeletal muscle from fric and fric littermates .
while fat cells from fric mice showed an 90% reduction ( p < 0.0001 ) in rictor protein levels when compared with fric fat cells , there was no change in rictor protein levels in liver and skeletal muscle of fric mice ( fig .
this result confirmed that the ap2-cre  mediated recombination event was restricted to fat cells .
mtor , the binding partner of rictor in mtorc2 , and akt , a known substrate of mtorc2 kinase , were expressed at similar levels in fric and fric fat cells ( fig .
analysis of rictor expression and insulin signaling in fric fat cells . a : lack of rictor protein in fric fat cells .
tissue extracts prepared from isolated fat cells , liver , and skeletal muscle were subjected to sds - page ( 6.5% gel ) and immunoblotted with anti - rictor antibody ( top ) .
the immunoblots for mtor , akt , and actin ( loading control ) are shown in the bottom panels .
immunoblot analysis of insulin ( ins)-stimulated phosphorylation of akt at s473 , akt at t308 , ir at y972 , foxo3a at t32 , gsk-3 at s9 , and as160 at t642 in fric and fric fat cells ( shown here are representative immunoblots , four mice for each genotype were analyzed ) . also shown
are immunoblots of total akt , foxo3a , gsk-3 , and as160 used for normalizing the corresponding anti - phospho immunoblots . to test the effect of loss of rictor on mtorc2 activity , we measured levels of akt phosphorylation at the s473 residue in insulin - stimulated fric and fric fat cells .
consistent with the data we obtained in mric muscles ( 11 ) , in fric fat cells insulin caused only a threefold increase in akt phosphorylation at s473 , whereas in fric fat cells an 10-fold increase above the basal level was observed ( fig .
this result suggests that mtorc2 is the major kinase responsible for phosphorylating akt at the s473 residue in response to insulin in fat cells .
similar to what we had previously reported in mric mice ( 11 ) , the level of akt phosphorylation at t308 ( a site phosphorylated by pdk1 ) in insulin - stimulated fric fat cells was comparable with that seen in fric fat cells ( fig .
1b ) . also , insulin - stimulated phosphorylation of the insulin receptor ( ir ) at the tyrosine ( y ) 972 residue was normal ( fig .
when evaluating phosphorylation of akt substrates in response to insulin , we found that in fric and fric fat cells phosphorylation of glycogen synthase kinase ( gsk ) 3 at s9 was similar between the two genotypes ( fig .
1b ) and as160 at t642 was dramatically reduced ( 69% reduction in insulin - stimulated as160 t642 phosphorylation in fric fat cells , p < 0.03 ) ( fig .
foxo3a is also shown to be phosphorylated at t32 by serum- and glucocorticoid - induced protein kinase ( sgk ) 1 , another target of mtorc2 kinase activity ( 18 ) . however , in insulin - stimulated fat cells , whether mtorc2 mediates sgk1 phosphorylation is not known .
recently , mtorc2 has been reported to promote the phosphorylation of turn motif sites in protein kinase c ( pkc )  , thus mediating its stabilization ( 19,20 ) . in mtorc2-null fric fat cells
, we observed a decrease in pkc levels ( p < 0.001 ; supplemental fig .
2a ) . also , fat cell sizes of fric and fric mice were not different ( supplemental fig .
2b and c ) . however , as previously reported ( 21 ) , parametrial fat pads ( 40% ) , pancreas ( 20% ) , kidney ( 24% ) , liver ( 19% ) , and heart ( 22% ) were heavier in fric mice compared with fric mice ( supplemental fig .
consistent with previous findings demonstrating decreased glucose transport in skeletal muscle lacking rictor ( 11 ) , glucose transport was reduced in fric fat cells . while insulin caused a three- to fourfold increase ( p < 0.004 ) in glucose uptake above basal levels in both fric and fric fat cells ( fig .
2a ) , in fric fat cells basal as well as insulin - stimulated glucose uptake were reduced by 75% ( p < 0.0002 ) and 65% ( p < 0.002 ) , respectively , when compared with fric fat cells .
glucose uptake in fric fat cells . a : [ uc]-glucose uptake in fat cells isolated from fric and fric mice ( 3- to 5-month - old female mice , n = 4 , * p < 0.004 ; * * p < 0.0002 ; * * * p < 0.0001 ) .
b : representative immunoblots of isolated fric and fric fat cell extracts from 3- to 5-month - old female mice , probed for glut4 , myo1c , and irap ( n = 5 ) .
c : immunoblots showing glut4 levels in plasma membrane isolated from basal and insulin - stimulated fat pads from fric and fric mice ( 3- to 5-month - old female , representative blots of two sets of fric and fric fat pads labeled as i and ii out of a total of three sets ) . cadherin levels in plasma membrane determined by immunoblotting with pan - cadherin antibody served as the loading control for plasma membrane preparations ( middle panel ) .
d : the glut4 quantification data ( means  se ) shows band intensity of glut4 after normalization to the levels of cadherin detected in the same sample ( n = 3 , * p < 0.05 ; * * p < 0.01 and * * * p < 0.006 ) . arb . , arbitrary .
ins , insulin . to elucidate the possible defect underlying the reduction in glucose uptake in fric fat cells
, we analyzed the expression of proteins known to be essential for insulin - stimulated glucose uptake .
2b ) , the insulin - regulated aminopeptidase ( irap ) , a protein associated with glut4 ( 22 ) , and myosin 1c ( myo1c ) , a protein involved in insulin - regulated glut4 translocation to the plasma membrane ( 23 ) , were similar in fric and fric fat cells ( fig .
however , the amount of glut4 in plasma membranes prepared from basal and insulin - stimulated fric fat pads was reduced by 40% when compared with plasma membranes prepared from fric fat pads ( n = 3 , p < 0.01 for basal and p < 0.05 for insulin stimulated ) ( fig . 2c , upper and lower panels , and d ) .
when measuring serum metabolite levels , we found normal fed and slightly decreased fasting triglyceride ( tag ) levels ( p < 0.04 ) ( table 1 ) in fric mice ( table 1 ) .
glycerol levels in serum from fasted ( p < 0.01 ) ( table 1 ) and fed ( p < 0.02 ) ( table 1 ) male fric mice were higher compared with fric mice . similarly , female fric mice had elevated glycerol levels ( 3- to 5-month - old fed mice , 12.9  2.4 g / ml in fric mice and 24.5  4.3 g / ml in fric mice , n = 56 , p = 0.057 ) . in addition , fasting nefa levels were increased significantly ( p < 0.04 ) ( table 1 ) in fric mice , while fed nefa levels were unchanged ( table 1 ) .
serum insulin , metabolite , and adipokine levels in fric mice data are means  se .
* p < 0.05 vs. fric mice of the same group . since serum levels of both nefas and glycerol , the products of lipolysis , were elevated in fric mice ( table 1 ) , we examined whether fric mice are resistant to insulin - induced inhibition of lipolysis . to test this in vivo
, we determined the levels of glycerol and nefas in serum taken from fric and fric mice before and 15 min after an intraperitoneal insulin injection ( 16 ) . in fric mice , insulin caused an 50% reduction in both glycerol and nefa levels , indicating inhibition of lipolysis by insulin ( fig .
3a ) , whereas in fric mice there was only a 13% reduction in glycerol levels ( n = 8 , p < 0.002 vs. fric mice ) ( fig .
serum nefa levels also declined significantly less after insulin injection in fric mice compared with fric mice ( n = 58 , p < 0.004 ) ( fig .
we next evaluated lipolysis ex vivo by determining glycerol release from isolated fric and fric fat cells in the presence of either cl316243 ( specific 3-adrenergic receptor agonist ) alone to induce lipolysis and cl316243 together with insulin to suppress lipolysis .
3c , fric fat cells showed twofold higher levels of basal lipolysis ( n = 4 , p < 0.01 ) when compared with fric fat cells , but the levels of cl316243-stimulated lipolysis was similar between fric and fric fat cells ( fig .
3c ) . insulin suppressed cl316243-induced lipolysis in fric fat cells by 50% ( n = 4 , p < 0.0001 ) ( fig .
3c ) but in fric fat cells only by 15% ( n = 4 ) ( fig .
a and b : insulin - mediated inhibition of lipolysis was determined by measuring the levels of glycerol ( shown in a , n = 8 , * p < 0.002 ) and nefas ( shown in b , n = 58 , * p < 0.004 ) in serum before and 15 min after an intraperitoneal injection of insulin in fric and fric mice
c : lipolysis was determined ex vivo in fat cells isolated from fric and fric mice ( 3- to 5-month - old female mice , n = 4 , * p < 0.01 ; * * p < 0.001 ) in the absence or presence of cl316243 ( cl , 1 nmol / l ) without and with insulin ( 1 and 10 nmol / l ) for 30 min and measuring glycerol released into the medium .
d : phosphorylation of hsl at s563 in basal and insulin - stimulated fat pads from fric and fric mice ( representative immunoblots are shown , n = 3 ) .
e : pka activity in fric and fric fat cells incubated in the absence or presence of cl316243 ( cl , 1 nmol / l ) without and with insulin ( 1 nmol / l ) for 10 min ( n = 4 , * p < 0.04 ; * * p < 0.001 ) .
insulin suppresses lipolysis largely through the regulation of hormone - sensitive lipase ( hsl ) .
phosphorylation of hsl at s563 by camp - dependent protein kinase ( also known as protein kinase a , pka ) increases the lipolytic activity of hsl , and insulin suppresses hsl activity by inhibiting the activation of pka ( 24,25 ) .
3d ) , there was an increased basal level of phosphorylation of hsl at s563 in fric compared with fric fat pads ( fig .
3d ) . furthermore , whereas an 30% reduction in hsl s563 phosphorylation was observed in fric fat pads , no repression of hsl s563 phosphorylation was found in fric fat pads in response to insulin ( n = 3 ) ( fig .
3d ) . isoproterenol ( a -adrenergic receptor agonist ) and cl316243 are known to stimulate hsl s563 phosphorylation by inducing camp production and consequently activating pka . in isolated fat pads , isoproterenol stimulated hsl s563 phosphorylation in both fric and fric fat pads ( supplemental fig .
however , the insulin - mediated inhibition of isoproterenol - induced hsl s563 phosphorylation was seen only in fric fat pads ( supplemental fig .
consistent with these findings , basal pka activity in isolated fric fat cells was 50% higher ( n = 4 , p < 0.01 ) ( fig .
3e ) than in fric fat cells , but cl316243 stimulated pka activity in fric and fric fat cells to a similar level .
insulin suppressed cl316243-mediated activation of pka in fric fat cells by 50% ( n = 4 , p < 0.001 ) ( fig .
3e ) but had no effect in fric fat cells ( n = 4 ) ( fig .
this finding , together with reduced repression of lipolysis in insulin - stimulated fric fat cells , strongly supports a role for rictor / mtorc2 in the regulation of pka activity to suppress lipolysis in fat cells .
fat cell function affects whole - body glucose homeostasis and insulin sensitivity ( 26 ) . in fric mice ,
serum insulin levels were significantly higher than in fric mice ( for fasting , p < 0.008 ; for fed , p < 0.0001 ) ( table 1 ) .
furthermore , young fric mice showed impaired insulin sensitivity in insulin tolerance tests ( supplemental fig .
younger fric mice ( 35 months old ) of either sex showed slightly better glucose clearance during a glucose tolerance test when compared with fric controls ( supplemental fig .
however , old fric mice ( > 9 months old ) showed significantly higher blood glucose levels after an overnight fast and a dramatic impairment in glucose clearance after an intraperitoneal glucose load ( fig . 4a and supplemental fig .
these data indicate the presence of severe insulin resistance in young and old fric mice .
glucose homeostasis in male fric mice . a : intraperitoneal glucose tolerance tests in old male fric and fric mice ( > 9 months old , n = 4 , * p < 0.05 ) .
b  f : whole - body glucose homeostasis in young fric and fric mice as evaluated by hyperinsulinemic - euglycemic clamp .
h : hepatic glucose production ( 3- to 4-month - old male mice , n = 56 per group .
b  h : * p < 0.04 ; * * p < 0.001 ; * * * p < 0.0001 ) .
to measure insulin sensitivity in individual tissues , we performed a 2-h hyperinsulinemic - euglycemic clamp in conscious young male fric mice . during the clamp , the glucose infusion rate required to maintain euglycemia ( fig .
4b ) , and insulin - stimulated whole - body glucose turnover rates ( fig .
4c ) were significantly lower in the fric mice compared with fric mice ( glucose infusion rate , 80% reduction p < 0.0001 ; glucose turnover rate , 49% reduction , p < 0.0001 ) , indicating pronounced systemic insulin resistance in fric mice .
4e ) were also reduced in fric mice when compared with fric mice ( glycolysis , 44% reduction , p < 0.01 ; glycogen plus lipid synthesis , 59% reduction , p < 0.001 ) . as shown in fig . 4f and g , in vivo insulin - stimulated glucose uptake in adipose tissue and skeletal muscle during the hyperinsulinemic - euglycemic clamp
was also significantly reduced in fric mice ( 50% reduction in adipose tissue , p < 0.01 ; 33% reduction in skeletal muscle , p < 0.04 ) .
in addition , insulin induced a marked suppression of hepatic glucose production ( hgp ) during the clamp in fric mice ( 46% reduction compared with basal levels , p < 0.001 ) ( fig .
4h ) ; however , insulin completely failed to suppress hgp in fric mice ( fig .
these results point not only to severe insulin resistance in adipose tissue but also marked insulin resistance in skeletal muscle and liver of fric mice .
however , we found that serum levels of leptin , adiponectin , and resistin were similar in fric and fric mice ( table 1 ) .
this result shows that changes in at least these adipokines do not play a causative role in the reduced insulin sensitivity in fric mice .
since fat cell  specific loss of rictor / mtorc2 decreased skeletal muscle glucose transport in response to insulin , we measured insulin - stimulated glycogen synthesis in isolated soleus and extensor digitorum longus ( edl ) muscles .
we found that insulin - stimulated glycogen synthesis was markedly reduced in both soleus ( 41% reduction , p < 0.0001 ) ( fig .
5a ) and edl ( 27% reduction , p < 0.0004 ) ( supplemental fig .
the isolated edl muscle from fric mice showed normal insulin - stimulated phosphorylation of the ir at y972 .
however , a marked reduction in insulin - stimulated phosphorylations of insulin receptor substrate ( irs ) 1 at y896 and of akt at both t308 and s473 ( fig .
interestingly , basal phosphorylation of irs1 at s302 in isolated edl muscles from fric mice was slightly increased when compared with fric mice ( 30% , n = 4 , data not shown ) .
in addition , we found a twofold increase ( p < 0.03 ) in levels of irs1 phosphorylated at s302 ( fig . 5c , top panel ) and a 37% reduction ( p < 0.04 ) in the total irs1 ( fig .
5c , middle panel ) levels in tibialis anterior ( ta ) muscles from fric mice . glucose and lipid metabolism and insulin signaling in skeletal muscles from fric mice . a : insulin - stimulated glycogen synthesis in ex vivo  incubated soleus muscles ( female , 35 months old , n = 45 , * p < 0.02 ; * * p <
b : insulin signaling in isolated edl muscles from fric and fric mice . protein extracts prepared from edl muscles stimulated without or with 20 mu of insulin for 30 min were immunoblotted with phospho - specific antibodies to the y-972 residue of ir , the y896 residue of irs1 , and the t308 and s473 residues of akt ( representative blot is shown , n = 34 ) .
protein extracts prepared from ta muscles of fed fric and fric mice were immunoblotted using antibodies against phospho - s302 irs1 ( p - s302 irs1 ) , total irs1 , and actin ( female , 35 months old , representative blots of two sets of fric and fric mice labeled as i and ii out of a total of eight sets ) .
d : triglyceride levels in skeletal muscle homogenates prepared from fed fric and fric mice ( 3- to 5-month - old female , n = 56 , * p < 0.04 ) .
e : protein extracts prepared from ta muscles of fed fric and fric mice were immunoblotted for lipin1 and actin ( 3- to 5-month - old female mice , representative blots of two sets of fric and fric mice labeled as i and ii out of a total of eight sets ) .
ins , insulin . since the fric mice showed increased lipolysis and muscle is known to take up nefas to store as tag , we measured tag levels in ta muscle homogenates .
indeed tag levels in muscles from fric mice were significantly increased when compared with fric mice ( p < 0.04 ) ( fig .
consistent with the increase in tag levels , we found that lipin 1 , an enzyme involved in the synthesis of tag ( 27 ) , was increased ( 1.4-fold , p < 0.002 ) in the ta muscles of fric mice ( fig .
thus , the loss of rictor in fat cells leads to increased lipid deposition and impaired insulin signaling in skeletal muscle .
livers from both young and old fric mice showed increased lipid accumulation ( hepatic steatosis ) when compared with age - matched fricmice ( fig .
consistent with this finding , gene expression of two important hepatic lipogenic enzymes , l - type pyruvate kinase ( l - pk ) and fatty acid synthase ( fas ) were increased two- and threefold , respectively , in livers from old fric mice ( p < 0.05 ) ( fig .
a : young ( 25 months old ) and old ( > 9 months old ) female fric or fric mice liver sections stained with hematoxylin and eosin ( h & e ) ( upper panels ) , or oil red o ( lower panels ) ( representative images are shown out of four to five for each age - group ) .
b : triglyceride concentration in liver homogenates of fric and fric mice ( > 6-month - old female , n = 89 , * p
c : l - type pyruvate kinase gene expression ( > 6-month - old male mice , n = 5 , * p < 0.03 ) .
d : fas gene expression ( > 6-month - old male mice , n = 5 , * p < 0.02 ) .
( a high - quality digital representation of this figure is available in the online issue . )
fric mice were generated by breeding rictor mice with mice expressing cre recombinase under the control of the ap2 promoter .
rictor protein levels were measured in isolated fat cells , liver , and skeletal muscle from fric and fric littermates .
while fat cells from fric mice showed an 90% reduction ( p < 0.0001 ) in rictor protein levels when compared with fric fat cells , there was no change in rictor protein levels in liver and skeletal muscle of fric mice ( fig .
this result confirmed that the ap2-cre  mediated recombination event was restricted to fat cells .
mtor , the binding partner of rictor in mtorc2 , and akt , a known substrate of mtorc2 kinase , were expressed at similar levels in fric and fric fat cells ( fig .
analysis of rictor expression and insulin signaling in fric fat cells . a : lack of rictor protein in fric fat cells .
tissue extracts prepared from isolated fat cells , liver , and skeletal muscle were subjected to sds - page ( 6.5% gel ) and immunoblotted with anti - rictor antibody ( top ) .
the immunoblots for mtor , akt , and actin ( loading control ) are shown in the bottom panels .
immunoblot analysis of insulin ( ins)-stimulated phosphorylation of akt at s473 , akt at t308 , ir at y972 , foxo3a at t32 , gsk-3 at s9 , and as160 at t642 in fric and fric fat cells ( shown here are representative immunoblots , four mice for each genotype were analyzed ) . also shown
are immunoblots of total akt , foxo3a , gsk-3 , and as160 used for normalizing the corresponding anti - phospho immunoblots . to test the effect of loss of rictor on mtorc2 activity , we measured levels of akt phosphorylation at the s473 residue in insulin - stimulated fric and fric fat cells .
consistent with the data we obtained in mric muscles ( 11 ) , in fric fat cells insulin caused only a threefold increase in akt phosphorylation at s473 , whereas in fric fat cells an 10-fold increase above the basal level was observed ( fig .
this result suggests that mtorc2 is the major kinase responsible for phosphorylating akt at the s473 residue in response to insulin in fat cells .
similar to what we had previously reported in mric mice ( 11 ) , the level of akt phosphorylation at t308 ( a site phosphorylated by pdk1 ) in insulin - stimulated fric fat cells was comparable with that seen in fric fat cells ( fig .
1b ) . also , insulin - stimulated phosphorylation of the insulin receptor ( ir ) at the tyrosine ( y ) 972 residue was normal ( fig .
when evaluating phosphorylation of akt substrates in response to insulin , we found that in fric and fric fat cells phosphorylation of glycogen synthase kinase ( gsk ) 3 at s9 was similar between the two genotypes ( fig .
1b ) and as160 at t642 was dramatically reduced ( 69% reduction in insulin - stimulated as160 t642 phosphorylation in fric fat cells , p < 0.03 ) ( fig .
foxo3a is also shown to be phosphorylated at t32 by serum- and glucocorticoid - induced protein kinase ( sgk ) 1 , another target of mtorc2 kinase activity ( 18 ) . however , in insulin - stimulated fat cells , whether mtorc2 mediates sgk1 phosphorylation is not known .
recently , mtorc2 has been reported to promote the phosphorylation of turn motif sites in protein kinase c ( pkc )  , thus mediating its stabilization ( 19,20 ) . in mtorc2-null fric fat cells
, we observed a decrease in pkc levels ( p < 0.001 ; supplemental fig .
2a ) . also , fat cell sizes of fric and fric mice were not different ( supplemental fig .
2b and c ) . however , as previously reported ( 21 ) , parametrial fat pads ( 40% ) , pancreas ( 20% ) , kidney ( 24% ) , liver ( 19% ) , and heart ( 22% ) were heavier in fric mice compared with fric mice ( supplemental fig .
consistent with previous findings demonstrating decreased glucose transport in skeletal muscle lacking rictor ( 11 ) , glucose transport was reduced in fric fat cells . while insulin caused a three- to fourfold increase ( p < 0.004 ) in glucose uptake above basal levels in both fric and fric fat cells ( fig .
2a ) , in fric fat cells basal as well as insulin - stimulated glucose uptake were reduced by 75% ( p < 0.0002 ) and 65% ( p < 0.002 ) , respectively , when compared with fric fat cells . glucose uptake in fric fat cells . a : [ uc]-glucose uptake in fat cells isolated from fric and fric mice ( 3- to 5-month - old female mice , n = 4 , * p < 0.004 ; * * p < 0.0002 ; * * * p < 0.0001 ) .
b : representative immunoblots of isolated fric and fric fat cell extracts from 3- to 5-month - old female mice , probed for glut4 , myo1c , and irap ( n = 5 ) .
c : immunoblots showing glut4 levels in plasma membrane isolated from basal and insulin - stimulated fat pads from fric and fric mice ( 3- to 5-month - old female , representative blots of two sets of fric and fric fat pads labeled as i and ii out of a total of three sets ) .
cadherin levels in plasma membrane determined by immunoblotting with pan - cadherin antibody served as the loading control for plasma membrane preparations ( middle panel ) .
d : the glut4 quantification data ( means  se ) shows band intensity of glut4 after normalization to the levels of cadherin detected in the same sample ( n = 3 , * p < 0.05 ; * * p < 0.01 and * * * p < 0.006 ) . arb . , arbitrary .
ins , insulin . to elucidate the possible defect underlying the reduction in glucose uptake in fric fat cells
, we analyzed the expression of proteins known to be essential for insulin - stimulated glucose uptake .
2b ) , the insulin - regulated aminopeptidase ( irap ) , a protein associated with glut4 ( 22 ) , and myosin 1c ( myo1c ) , a protein involved in insulin - regulated glut4 translocation to the plasma membrane ( 23 ) , were similar in fric and fric fat cells ( fig .
however , the amount of glut4 in plasma membranes prepared from basal and insulin - stimulated fric fat pads was reduced by 40% when compared with plasma membranes prepared from fric fat pads ( n = 3 , p < 0.01 for basal and p < 0.05 for insulin stimulated ) ( fig . 2c , upper and lower panels , and d ) .
when measuring serum metabolite levels , we found normal fed and slightly decreased fasting triglyceride ( tag ) levels ( p < 0.04 ) ( table 1 ) in fric mice ( table 1 ) .
glycerol levels in serum from fasted ( p < 0.01 ) ( table 1 ) and fed ( p < 0.02 ) ( table 1 ) male fric mice were higher compared with fric mice .
similarly , female fric mice had elevated glycerol levels ( 3- to 5-month - old fed mice , 12.9  2.4 g / ml in fric mice and 24.5  4.3 g / ml in fric mice , n = 56 , p = 0.057 ) .
in addition , fasting nefa levels were increased significantly ( p < 0.04 ) ( table 1 ) in fric mice , while fed nefa levels were unchanged ( table 1 ) .
serum insulin , metabolite , and adipokine levels in fric mice data are means  se .
* p < 0.05 vs. fric mice of the same group . since serum levels of both nefas and glycerol , the products of lipolysis , were elevated in fric mice ( table 1 ) , we examined whether fric mice are resistant to insulin - induced inhibition of lipolysis . to test this in vivo
, we determined the levels of glycerol and nefas in serum taken from fric and fric mice before and 15 min after an intraperitoneal insulin injection ( 16 ) .
in fric mice , insulin caused an 50% reduction in both glycerol and nefa levels , indicating inhibition of lipolysis by insulin ( fig .
3a ) , whereas in fric mice there was only a 13% reduction in glycerol levels ( n = 8 , p < 0.002 vs. fric mice ) ( fig .
serum nefa levels also declined significantly less after insulin injection in fric mice compared with fric mice ( n = 58 , p < 0.004 ) ( fig .
we next evaluated lipolysis ex vivo by determining glycerol release from isolated fric and fric fat cells in the presence of either cl316243 ( specific 3-adrenergic receptor agonist ) alone to induce lipolysis and cl316243 together with insulin to suppress lipolysis .
3c , fric fat cells showed twofold higher levels of basal lipolysis ( n = 4 , p < 0.01 ) when compared with fric fat cells , but the levels of cl316243-stimulated lipolysis was similar between fric and fric fat cells ( fig .
3c ) . insulin suppressed cl316243-induced lipolysis in fric fat cells by 50% ( n = 4 , p < 0.0001 ) ( fig .
3c ) but in fric fat cells only by 15% ( n = 4 ) ( fig .
resistance to insulin - mediated inhibition of lipolysis in fric mice . a and b : insulin - mediated inhibition of lipolysis was determined by measuring the levels of glycerol ( shown in a ,
n = 8 , * p < 0.002 ) and nefas ( shown in b , n = 58 , * p < 0.004 ) in serum before and 15 min after an intraperitoneal injection of insulin in fric and fric mice .
c : lipolysis was determined ex vivo in fat cells isolated from fric and fric mice ( 3- to 5-month - old female mice , n = 4 , * p < 0.01 ; * * p < 0.001 ) in the absence or presence of cl316243 ( cl , 1 nmol / l ) without and with insulin ( 1 and 10 nmol / l ) for 30 min and measuring glycerol released into the medium .
d : phosphorylation of hsl at s563 in basal and insulin - stimulated fat pads from fric and fric mice ( representative immunoblots are shown , n = 3 ) .
e : pka activity in fric and fric fat cells incubated in the absence or presence of cl316243 ( cl , 1 nmol / l ) without and with insulin ( 1 nmol / l ) for 10 min ( n = 4 , * p < 0.04 ; * * p < 0.001 ) .
insulin suppresses lipolysis largely through the regulation of hormone - sensitive lipase ( hsl ) .
phosphorylation of hsl at s563 by camp - dependent protein kinase ( also known as protein kinase a , pka ) increases the lipolytic activity of hsl , and insulin suppresses hsl activity by inhibiting the activation of pka ( 24,25 ) .
3d ) , there was an increased basal level of phosphorylation of hsl at s563 in fric compared with fric fat pads ( fig .
3d ) . furthermore , whereas an 30% reduction in hsl s563 phosphorylation was observed in fric fat pads , no repression of hsl s563 phosphorylation was found in fric fat pads in response to insulin ( n = 3 ) ( fig .
3d ) . isoproterenol ( a -adrenergic receptor agonist ) and cl316243 are known to stimulate hsl s563 phosphorylation by inducing camp production and consequently activating pka . in isolated fat pads ,
isoproterenol stimulated hsl s563 phosphorylation in both fric and fric fat pads ( supplemental fig .
however , the insulin - mediated inhibition of isoproterenol - induced hsl s563 phosphorylation was seen only in fric fat pads ( supplemental fig .
consistent with these findings , basal pka activity in isolated fric fat cells was 50% higher ( n = 4 , p < 0.01 ) ( fig .
3e ) than in fric fat cells , but cl316243 stimulated pka activity in fric and fric fat cells to a similar level .
insulin suppressed cl316243-mediated activation of pka in fric fat cells by 50% ( n = 4 , p
3e ) but had no effect in fric fat cells ( n = 4 ) ( fig .
this finding , together with reduced repression of lipolysis in insulin - stimulated fric fat cells , strongly supports a role for rictor / mtorc2 in the regulation of pka activity to suppress lipolysis in fat cells .
fat cell function affects whole - body glucose homeostasis and insulin sensitivity ( 26 ) . in fric mice ,
serum insulin levels were significantly higher than in fric mice ( for fasting , p <
0.008 ; for fed , p < 0.0001 ) ( table 1 ) . furthermore , young fric mice showed impaired insulin sensitivity in insulin tolerance tests ( supplemental fig .
younger fric mice ( 35 months old ) of either sex showed slightly better glucose clearance during a glucose tolerance test when compared with fric controls ( supplemental fig .
5a ) as previously reported ( 21 ) . however , old fric mice ( > 9 months old ) showed significantly higher blood glucose levels after an overnight fast and a dramatic impairment in glucose clearance after an intraperitoneal glucose load ( fig . 4a and supplemental fig .
these data indicate the presence of severe insulin resistance in young and old fric mice .
glucose homeostasis in male fric mice . a : intraperitoneal glucose tolerance tests in old male fric and fric mice ( > 9 months old , n = 4 , * p < 0.05 ) . b  f : whole - body glucose homeostasis in young fric and fric mice as evaluated by hyperinsulinemic - euglycemic clamp .
h : hepatic glucose production ( 3- to 4-month - old male mice , n = 56 per group .
, we performed a 2-h hyperinsulinemic - euglycemic clamp in conscious young male fric mice . during the clamp , the glucose infusion rate required to maintain euglycemia ( fig .
4b ) , and insulin - stimulated whole - body glucose turnover rates ( fig .
4c ) were significantly lower in the fric mice compared with fric mice ( glucose infusion rate , 80% reduction p < 0.0001 ; glucose turnover rate , 49% reduction , p < 0.0001 ) , indicating pronounced systemic insulin resistance in fric mice .
4e ) were also reduced in fric mice when compared with fric mice ( glycolysis , 44% reduction , p < 0.01 ; glycogen plus lipid synthesis , 59% reduction , p < 0.001 ) . as shown in fig . 4f and g , in vivo insulin - stimulated glucose uptake in adipose tissue and skeletal muscle during the hyperinsulinemic - euglycemic clamp
was also significantly reduced in fric mice ( 50% reduction in adipose tissue , p < 0.01 ; 33% reduction in skeletal muscle , p < 0.04 ) .
in addition , insulin induced a marked suppression of hepatic glucose production ( hgp ) during the clamp in fric mice ( 46% reduction compared with basal levels , p < 0.001 ) ( fig .
4h ) ; however , insulin completely failed to suppress hgp in fric mice ( fig .
these results point not only to severe insulin resistance in adipose tissue but also marked insulin resistance in skeletal muscle and liver of fric mice .
however , we found that serum levels of leptin , adiponectin , and resistin were similar in fric and fric mice ( table 1 ) .
this result shows that changes in at least these adipokines do not play a causative role in the reduced insulin sensitivity in fric mice .
since fat cell  specific loss of rictor / mtorc2 decreased skeletal muscle glucose transport in response to insulin , we measured insulin - stimulated glycogen synthesis in isolated soleus and extensor digitorum longus ( edl ) muscles .
we found that insulin - stimulated glycogen synthesis was markedly reduced in both soleus ( 41% reduction , p < 0.0001 ) ( fig .
5a ) and edl ( 27% reduction , p < 0.0004 ) ( supplemental fig .
the isolated edl muscle from fric mice showed normal insulin - stimulated phosphorylation of the ir at y972 .
however , a marked reduction in insulin - stimulated phosphorylations of insulin receptor substrate ( irs ) 1 at y896 and of akt at both t308 and s473 ( fig .
interestingly , basal phosphorylation of irs1 at s302 in isolated edl muscles from fric mice was slightly increased when compared with fric mice ( 30% , n = 4 , data not shown ) .
in addition , we found a twofold increase ( p < 0.03 ) in levels of irs1 phosphorylated at s302 ( fig .
5c , middle panel ) levels in tibialis anterior ( ta ) muscles from fric mice . glucose and lipid metabolism and insulin signaling in skeletal muscles from fric mice . a : insulin - stimulated glycogen synthesis in ex vivo  incubated soleus muscles ( female , 35 months old , n = 45 , * p < 0.02 ; * * p < 0.0001 ) from fricmice using [ uc]-glucose .
b : insulin signaling in isolated edl muscles from fric and fric mice . protein extracts prepared from edl muscles stimulated without or with 20 mu of insulin for 30 min were immunoblotted with phospho - specific antibodies to the y-972 residue of ir , the y896 residue of irs1 , and the t308 and s473 residues of akt ( representative blot is shown , n = 34 ) .
protein extracts prepared from ta muscles of fed fric and fric mice were immunoblotted using antibodies against phospho - s302 irs1 ( p - s302 irs1 ) , total irs1 , and actin ( female , 35 months old , representative blots of two sets of fric and fric mice labeled as i and ii out of a total of eight sets ) .
d : triglyceride levels in skeletal muscle homogenates prepared from fed fric and fric mice ( 3- to 5-month - old female , n = 56 , * p < 0.04 ) .
e : protein extracts prepared from ta muscles of fed fric and fric mice were immunoblotted for lipin1 and actin ( 3- to 5-month - old female mice , representative blots of two sets of fric and fric mice labeled as i and ii out of a total of eight sets ) .
ins , insulin . since the fric mice showed increased lipolysis and muscle is known to take up nefas to store as tag , we measured tag levels in ta muscle homogenates .
indeed tag levels in muscles from fric mice were significantly increased when compared with fric mice ( p < 0.04 ) ( fig .
consistent with the increase in tag levels , we found that lipin 1 , an enzyme involved in the synthesis of tag ( 27 ) , was increased ( 1.4-fold , p < 0.002 ) in the ta muscles of fric mice ( fig . 5e , top panel ) .
thus , the loss of rictor in fat cells leads to increased lipid deposition and impaired insulin signaling in skeletal muscle .
livers from both young and old fric mice showed increased lipid accumulation ( hepatic steatosis ) when compared with age - matched fricmice ( fig .
consistent with this finding , gene expression of two important hepatic lipogenic enzymes , l - type pyruvate kinase ( l - pk ) and fatty acid synthase ( fas ) were increased two- and threefold , respectively , in livers from old fric mice ( p < 0.05 ) ( fig . 6c and d ) .
a : young ( 25 months old ) and old ( > 9 months old ) female fric or fric mice liver sections stained with hematoxylin and eosin ( h & e ) ( upper panels ) , or oil red o ( lower panels ) ( representative images are shown out of four to five for each age - group ) .
b : triglyceride concentration in liver homogenates of fric and fric mice ( > 6-month - old female , n = 89 , * p < 0.01 ) . c and d : lipogenic gene expression in liver normalized to gapdh .
c : l - type pyruvate kinase gene expression ( > 6-month - old male mice , n = 5 , * p < 0.03 ) .
d : fas gene expression ( > 6-month - old male mice , n = 5 , * p < 0.02 ) . data shown
( a high - quality digital representation of this figure is available in the online issue . )
the loss of fat cell rictor expression in mice results in a metabolic phenotype similar to type 2 diabetes with impaired fat cell function associated with severe insulin resistance in adipose tissue , skeletal muscle , and liver .
this suggests that rictor plays an important role in regulating fat cell metabolism and , consequently , whole - body glucose and lipid homeostasis and insulin sensitivity .
fric fat cells showed impaired regulation of glucose transport and lipolysis in response to insulin .
defective insulin signaling in fric fat cells most likely causes these metabolic abnormalities . our analyses of insulin signaling events that lie downstream of mtorc2 and
mediate metabolic responses revealed a dramatic reduction in phosphorylation of akt at s473 and , consequently , foxo3a at t32 and as160 at t642 . at this time
, it is not clear whether foxo3a has a role in the observed metabolic defects in fric fat cells .
however , the akt - mediated as160 phosphorylation at t642 and consequent inhibition of the rab - gap ( gtpase activating protein ) activity of as160 is essential for insulin - stimulated glut4 translocation to the plasma membrane ( 28,29 ) .
consistent with decreased phosphorylation of as160 , glut4 translocation to the plasma membrane in response to insulin is impaired and glucose uptake is decreased in fric fat cells .
rictor / mtorc2 activity has not been linked to regulation of either basal lipolysis or insulin - mediated suppression of lipolysis . however , our observations that in fric fat cells increased phosphorylation of hsl at s563 concomitant with upregulated pka activity and that insulin failed to inhibit these events suggests a role for mtorc2 in the regulation of lipolysis by modifying pka and hsl activities .
it is not clear how mtorc2 regulates basal pka activity . in response to insulin ,
akt directly phosphorylates and activates phosphodiesterase 3b ( pde3b ) ( 30 ) increasing camp to amp conversion and thereby decreasing pka activity ( 31 ) .
since pka activates lipolysis via phosphorylation of hsl , a defect in insulin activation of pde3b would impair downregulation of lipolysis .
in fact , defective regulation of lipolysis in response to insulin is reported in pde3b knockout mice ( 32 ) .
while our data supports a model whereby mtorc2 activity controls akt - mediated activation of pde3b by insulin , this remains to be established .
rapamycin - mediated inhibition of mtor has implicated mtorc1 in the regulation of lipolysis ( 14,15 ) ; however , eliminating mtorc1 by ablating raptor in fat cells does not affect lipolysis ( 33 ) . considering that prolonged rapamycin treatment inhibits the formation of mtorc2 in some cell types ( 4 ) , our studies suggest that clinical observations of elevated nefa levels during rapamycin treatment ( 14,15 ) could be due to inhibition of mtorc2 .
how do fat cell  specific genetic alterations in the insulin signaling pathway lead to impairment of whole - body glucose homeostasis ?
although fric mice show a profound reduction in glucose transport in fat cells , this defect is likely to be only partly responsible for impaired glucose homeostasis in the fric mice .
the major contributors to impaired glucose homeostasis in fric mice are reduced insulin - stimulated skeletal muscle glucose transport and impaired suppression of hepatic glucose production by insulin .
we propose that elevated circulating nefas due to upregulation of lipolysis in fat cells cause the insulin resistance in skeletal muscle and liver observed in the fric mice .
the increased serum nefa levels in fric mice are most likely responsible for the elevated intramuscular accumulation of tag , which in turn may be mediated by increased lipin 1 levels in fric skeletal muscles . increased nefas flux into skeletal muscle leads to elevated intramuscular levels of lipid metabolites , such as fatty acyl coa and diacylglycerol ( 35 ) .
these lipids induce insulin resistance in skeletal muscle by causing defects in insulin signaling ( 36 ) .
previously , nefas have been shown to activate serine / threonine kinases such as ib kinase-2 ( 37 ) , jun nh2-terminal kinase ( 38 ) , and protein kinase c ( 39 ) .
all of these kinases can phosphorylate irs1 on critical serine residues , thereby blocking phosphorylation of irs1 by ir kinase on tyrosine sites that are required for pi3k association and activation ( 40 ) .
irs1 s302 phosphorylation through ib kinase-2 ( 41 ) or jun nh2-terminal kinase ( 42 ) can lead to irs1 degradation . since we see an increase in phosphorylation of irs1 at s302 in fric muscle , a decrease in irs1 y896 phosphorylation , as well as a reduction in total irs1 levels
, we suggest that elevated lipids are responsible for the insulin resistance observed in vivo and ex vivo in the skeletal muscles of fric mice .
the development of hepatic steatosis in fric mice may be due to the prolonged elevation of nefas and glycerol in serum , as both are used for synthesis of tag in the liver .
furthermore , under hyperglycemic conditions , an increased glucose flux into the liver in fric mice could compensate for skeletal muscle insulin resistance to maintain normoglycemia ( 43 ) .
the surplus glucose is converted to tag and contributes to the development of hepatic steatosis .
thus , liver function also becomes affected in fric animals after chronic exposure to elevated circulating nefas , glycerol , and glucose .
a different line of fat cell  specific rictor knockout mice ( referred to as rictor ) has recently been described ( 21 ) .
rictor mice showed that visceral organs such as liver , pancreas , kidney , fat pads , spleen , and heart increased in size with concomitantly increased lean mass that was more apparent on a high - fat diet .
this finding suggests that rictor / mtorc2 in fat cells is important for organismal growth ( 21 ) .
the rictor mice were hyperinsulinemic but were normoglycemic in the fed and fasting state , similar to young fric mice . increased pancreatic -cell mass
could contribute to hyperinsulinemia in fric mice , as suggested for the rictor mice . however , increased -cell mass alone is not sufficient to induce hyperinsulinemia ( 44 ) but requires a factor that would induce insulin secretion .
it is well known that insulin resistance is compensated by increased -cell mass and insulin secretion ( 45 ) .
our results from hyperinsulinemic - euglycemic clamp studies in fric mice show severe whole - body insulin resistance including adipose tissue , skeletal muscle , and liver . while the insulin sensitivity in rictor mice was not directly measured , results from insulin tolerance tests suggest that these mice are also insulin resistant .
( 21 ) ascribe the insulin resistance in the rictormice to decreased circulating adiponectin levels .
there was no change in adiponectin levels in the fric animals on a standard diet .
the reason for this discrepancy is not clear , as both fat cell  specific rictor knockout models have very similar genetic backgrounds of 129 crossed to c57bl/6j , although the 129 substrains differ ( 129s6 for fric mice and 129s1/svlmj for rictor mice ) .
however , since the genetic loss of adiponectin does not affect insulin sensitivity of standard diet  fed mice ( 46 ) , it is unlikely that decreased adiponectin is the cause for decreased insulin sensitivity arising from fat cell  specific loss of rictor in mice .
the elevated circulating nefa levels that we observe in the fric mice could not only contribute to peripheral insulin resistance but also directly stimulate insulin secretion from -cells ( data not shown ) as well as increase pancreatic -cell mass ( 47 ) .
therefore , we propose that hyperinsulinemia and increased -cell mass in fric mice is predominantly a consequence of insulin resistance and increased nefas . in conclusion
, we demonstrate that mtorc2 is essential for insulin action on glucose transport and lipolysis in fat cells .
our studies further demonstrate the important role for mtorc2 in the regulation of fat cell function and control of whole - body glucose and lipid homeostasis and insulin sensitivity .
thus , mtorc2 may serve as a drug target for the treatment of obesity and type 2 diabetes .
 objectiverictor is an essential component of mammalian target of rapamycin ( mtor ) complex ( mtorc ) 2 , a kinase that phosphorylates and activates akt , an insulin signaling intermediary that regulates glucose and lipid metabolism in adipose tissue , skeletal muscle , and liver . 
 to determine the physiological role of rictor / mtorc2 in insulin signaling and action in fat cells , we developed fat cell  specific rictor knockout ( fric/ ) mice.research design and methodsinsulin signaling and glucose and lipid metabolism were studied in fric/ fat cells . in vivo glucose metabolism was evaluated by hyperinsulinemic - euglycemic clamp.resultsloss of rictor in fat cells prevents insulin - stimulated phosphorylation of akt at s473 , which , in turn , impairs the phosphorylation of downstream targets such as foxo3a at t32 and as160 at t642 . 
 however , glycogen synthase kinase-3 phosphorylation at s9 is not affected . 
 the signaling defects in fric/ fat cells lead to impaired insulin - stimulated glut4 translocation to the plasma membrane and decreased glucose transport . 
 furthermore , rictor - null fat cells are unable to suppress lipolysis in response to insulin , leading to elevated circulating free fatty acids and glycerol . 
 these metabolic perturbations are likely to account for defects observed at the whole - body level of fric/ mice , including glucose intolerance , marked hyperinsulinemia , insulin resistance in skeletal muscle and liver , and hepatic steatosis.conclusionsrictor/mtorc2 in fat cells plays an important role in whole - body energy homeostasis by mediating signaling necessary for the regulation of glucose and lipid metabolism in fat cells . 
RESEARCH DESIGN AND METHODS
Generation of FRic
Hyperinsulinemic-euglycemic clamps.
Statistics.
RESULTS
Ablation of 
Loss of 
Loss of 
Dysregulation of lipolysis in FRic
Rictor/mTORC2 in fat cells regulates whole-body glucose homeostasis.
Decreased glucose metabolism, impaired insulin signaling, and increased lipid accumulation in skeletal muscles of FRic
FRic
DISCUSSION
Supplementary Material