adipose tissue functions as a repository for long - term energy storage and as a critical regulator of energy balance , satiety , glucose metabolism , and other physiological processes .
white adipose tissue ( wat ) stores and releases fatty acids and glycerol in response to the energetic needs of the organism , whereas brown adipose tissue ( bat ) produces heat to maintain thermostasis upon cold - exposure .
the most common form of lipodystrophy is congenital generalized lipodystrophy ( cgl ) , which affects approximately one in ten million individuals and is characterized by a near complete absence of adipose tissue .
cgl type 1 is caused by mutations in 1-acylglycerol-3-phosophate o - acyltransferase 2 ( agpat2 ) ,   and mice null for agpat2 exhibit a complete lipoatrophy .
mutations in seipin cause cgl type 2 , or berardinelli - seip congenital lipodystrophy type 2 ( bscl2 ) , and mice null for bscl2/seipin , or with adipose - tissue specific loss , are also lipodystrophic , , .
several additional genes have been implicated in cgl and familial partial lipodystrophy , including akt2 and a subunit of phosphatidylinositol 3 kinase ( pi3k ) , , , , , .
hepatomegaly from fatty liver and splenomegaly are common in lipodystrophic patients , with hyperinsulinemia and hypertriglyceridemia often present .
akt , a ser / thr kinase , is activated upon engagement of the insulin receptor and mediates hormonal regulation of many aspects of energy homeostasis in insulin - responsive tissues , including adipose tissue .
akt can also be activated by insulin growth factor 1 ( igf-1 ) , , .
three isoforms of akt have been identified , of which akt1 and akt2 account for the majority of akt in adipocytes , .
akt1 is expressed nearly ubiquitously and is critical in the control of growth , while the akt2 isoform is expressed predominantly in the adult insulin - responsive tissues , including liver , muscle , and adipose .
mice lacking akt2 are insulin - resistant   but spared from severe metabolic abnormalities due to compensation by akt1 , , . in this study , we deleted akt1 and akt2 selectively in adipocytes of mice , thus interrupting a critical insulin - signaling pathway .
we found that loss of these akt isoforms in adipocytes caused profound lipodystrophy , associated with fatty liver and systemic insulin resistance .
all mice were on a c57/b6 background and were maintained on normal chow diets from laboratory rodent diet , lab diet 5001 .
cre - positive male mice heterozygous for akt1 and akt2 floxed alleles were bred to females homozygous for akt1 and akt2 floxed alleles .
this breeding scheme generated cre - positive experimental mice homozygous floxed for both genes and cre - negative male mice homozygous floxed for both genes that served as controls .
cre - positive males heterozygous for both floxed genes were generated and exhibited a wild type phenotype ( data not shown ) .
all mouse experiments were reviewed and approved by the university of pennsylvania institutional animal care and use committee in accordance with the guidelines of the national institutes of health .
organs were harvested for histopathology from 12-week - old mice , fixed in formalin for 24  h , rinsed in 70% ethanol , and stored in 70% ethanol at 4  c overnight .
sectioning and staining of the liver , spleen , kidney , and pancreas were performed by the molecular pathology and imaging core at the university of pennsylvania , supported by nih grants dk050306 , ca098101 , and dk049210 .
sectioning of the epididymal fat pads was performed by the pathology core at the children 's hospital of philadelphia research institute .
slides were deparaffinized in xylene ( 3    3  min ) , then rehydrated in an ethanol series of 100% ( 3    3  min ) , 95% ( 1    3  min ) , 80% ( 1    3  min ) , dh2o ( 1    5  min ) .
slides were stained in hematoxylin for 30  min , rinsed in dh2o , and rinsed in tap water for 5  min .
slides were dipped in acid ethanol 812 to destain them , rinsed in tap water ( 2    1  min ) , and rinsed in dh2o ( 1    2  min ) .
slides were then dehydrated in an ethanol series : 95% ( 3    5  min ) , 100% ( 3    5  min ) , and then in xylene ( 3    15  min ) before drying and mounting a coverslip .
rat anti - mouse f4/80 monoclonal antibody c1:a3 - 1 from abd serotec was used for staining sections from the epididymal fat for macrophage , natural killer cells , and f4/80 positive antigen presenting cell infiltrate .
blood samples were taken from 8 to 12 week old mice between 12 and 2  pm .
adiponectin , leptin , and resistin levels were measured using elisa kits from linco by the radioimmunoassay and biomarkers core at the penn diabetes research center , supported by nih grant dk19525 .
liver triglycerides were measured by weighing 100  mg liver and lysing in 400  ul cell lysis buffer ( 50  mm tris , ph 7.4 , 140  mm nacl , 0.1% triton - x ) ( 250  mg / ml homogenate ) .
samples were diluted 10 fold and incubated in 1% deoxycholate at a 1:1 ratio for 10  min , then incubated for 10  min  in thermo - scientific infinity triglyceride reagent .
samples were read on a tecan plate reader at 500  nm . for glucose tolerance tests ,
812 week old male mice were fasted for 1618  h  overnight and injected with 1  g / kg glucose intraperitoneally .
blood samples were taken from the tail in sarstedt microvette lithium heparin tubes ( ref 16.433.100 ) at time 0 for fasting insulin measurements and 15  min  post injection .
blood glucose levels were taken from the tail on a onetouch ultra glucometer at fasting , and post - injection at 15 , 30 , 60 , and 120  min .
for the meal challenges , 812 week old male mice were fasted for 1618  h  overnight and then refed normal chow for 2  h. crystal chem ultra - sensitive mouse insulin elisa kits were used for all insulin measurements .
all mice were on a c57/b6 background and were maintained on normal chow diets from laboratory rodent diet , lab diet 5001 .
cre - positive male mice heterozygous for akt1 and akt2 floxed alleles were bred to females homozygous for akt1 and akt2 floxed alleles .
this breeding scheme generated cre - positive experimental mice homozygous floxed for both genes and cre - negative male mice homozygous floxed for both genes that served as controls .
cre - positive males heterozygous for both floxed genes were generated and exhibited a wild type phenotype ( data not shown ) .
all mouse experiments were reviewed and approved by the university of pennsylvania institutional animal care and use committee in accordance with the guidelines of the national institutes of health .
organs were harvested for histopathology from 12-week - old mice , fixed in formalin for 24  h , rinsed in 70% ethanol , and stored in 70% ethanol at 4  c overnight .
sectioning and staining of the liver , spleen , kidney , and pancreas were performed by the molecular pathology and imaging core at the university of pennsylvania , supported by nih grants dk050306 , ca098101 , and dk049210 .
sectioning of the epididymal fat pads was performed by the pathology core at the children 's hospital of philadelphia research institute .
slides were deparaffinized in xylene ( 3    3  min ) , then rehydrated in an ethanol series of 100% ( 3    3  min ) , 95% ( 1    3  min ) , 80% ( 1    3  min ) , dh2o ( 1    5  min ) .
slides were stained in hematoxylin for 30  min , rinsed in dh2o , and rinsed in tap water for 5  min .
slides were dipped in acid ethanol 812 to destain them , rinsed in tap water ( 2    1  min ) , and rinsed in dh2o ( 1    2  min ) .
slides were then dehydrated in an ethanol series : 95% ( 3    5  min ) , 100% ( 3    5  min ) , and then in xylene ( 3    15  min ) before drying and mounting a coverslip .
rat anti - mouse f4/80 monoclonal antibody c1:a3 - 1 from abd serotec was used for staining sections from the epididymal fat for macrophage , natural killer cells , and f4/80 positive antigen presenting cell infiltrate .
blood samples were taken from 8 to 12 week old mice between 12 and 2  pm .
adiponectin , leptin , and resistin levels were measured using elisa kits from linco by the radioimmunoassay and biomarkers core at the penn diabetes research center , supported by nih grant dk19525 .
liver triglycerides were measured by weighing 100  mg liver and lysing in 400  ul cell lysis buffer ( 50  mm tris , ph 7.4 , 140  mm nacl , 0.1% triton - x ) ( 250  mg / ml homogenate ) .
samples were diluted 10 fold and incubated in 1% deoxycholate at a 1:1 ratio for 10  min , then incubated for 10  min  in thermo - scientific infinity triglyceride reagent .
for glucose tolerance tests , 812 week old male mice were fasted for 1618  h  overnight and injected with 1  g / kg glucose intraperitoneally .
blood samples were taken from the tail in sarstedt microvette lithium heparin tubes ( ref 16.433.100 ) at time 0 for fasting insulin measurements and 15  min  post injection .
blood glucose levels were taken from the tail on a onetouch ultra glucometer at fasting , and post - injection at 15 , 30 , 60 , and 120  min .
for the meal challenges , 812 week old male mice were fasted for 1618  h  overnight and then refed normal chow for 2  h. crystal chem ultra - sensitive mouse insulin elisa kits were used for all insulin measurements .
to determine the role of akt in regulating adipocyte function and survival , we generated mice lacking both akt1 and akt2 using the adipoq - cre driver .
adipo - akt knockout ( ko ) mice were severely lipodystrophic at 812 weeks of age such that all subcutaneous and cutaneous fat depots , including interscapular brown adipose tissue , were completely absent ( figure  1a ) .
the epididymal wat was about 10 smaller ( as a percentage of body weight ) in adipo - akt ko mice as compared to control mice ( figure  1b ) .
hematoxylin and eosin staining of tissue sections showed that the residual epididymal wat ( ewat ) in ko mice contained a modest number of enlarged adipocytes ( figure  1c ) .
the ko ewat also displayed increased infiltration of f4/80 +  macrophages ( figure  1d ) .
the levels of important adipokines , such as leptin , adiponectin , and resistin , were dramatically decreased in adipo - akt ko mice relative to controls ( figure  1e ) .
the body weight of adipo - akt ko mice was increased about 15% by 5 weeks of age , with controls weighing 17.3  g and adipo - akt ko mice weighing 20.5  g ( figure  1f ) .
this increase in body weight was maintained at 9 weeks of age with control mice weighing 21.8  g and adipo - akt ko mice weighing 26.0  g ( figure 1f ) .
adipo - akt ko mice had livers weighing 2.7 times that of control animals when normalized for body weight at 812 weeks of age .
livers in adipo - akt mice constituted 13% of body weight , and were 3.5  g on average , as compared to 4.9% of body weight and about 1  g in controls ( figure  2a , b ) .
the greater size of ko livers was associated with a large increase in liver triglyceride levels .
control mice had an average of 9.2  mg triglyceride / g of liver whereas adipo - akt ko livers had 169.1  mg triglyceride / g of liver ( figure  2c ) .
histopathological analysis of livers from adipo - akt ko mice revealed a disorganized morphology with an increase in both the number and size of lipid droplets ( figure  2d ) .
in addition to hepatomegaly , the adipo - akt ko mice displayed enlarged pancreases and kidneys ( figure  3a ) . on the other hand , adipo - akt ko mice did not exhibit splenomegaly , which is often associated with lipodystrophy , , ,   ( figure  3a ) .
histopathology of both the spleen and kidney revealed no obvious abnormalities ( figure  3b , c ) .
in contrast , the pancreases of adipo - akt ko mice had enlarged islets ( figure  3d ) .
adipo - akt ko mice had glucose tolerance comparable to control mice at 812 weeks of age ( figure  4a ) . however , while control mice had fasting insulin levels of 0.5  ng / ml , levels in the ko mice were appreciably higher at 2.5  ng / ml , indicative of significant insulin resistance ( figure  4b ) . to further investigate the ability of adipo - akt ko mice to regulate blood glucose in the face of insulin resistance , we fasted mice overnight and challenged them with a meal of normal chow .
two hours after refeeding , blood glucose levels were significantly greater in the adipo - akt ko mice than the controls ( figure  4c ) .
consistent with the results from gtt , the insulin levels were elevated in fasted adipo - akt ko and increased even further following feeding ( figure  4d ) .
plasma non - esterified fatty acids ( nefa ) levels were also measured during fasting and after a 2  h  feeding challenge on normal chow .
adipo - akt ko mice had low basal nefa levels of 0.7  mmol / l compared to control mice that had fasting nefa of 1.6  mmol / l .
postprandially , nefa levels in control mice are suppressed to 0.68  mmol / l while in adipo - akt ko mice nefa levels were not changed ( figure  4e ) .
to determine the role of akt in regulating adipocyte function and survival , we generated mice lacking both akt1 and akt2 using the adipoq - cre driver .
adipo - akt knockout ( ko ) mice were severely lipodystrophic at 812 weeks of age such that all subcutaneous and cutaneous fat depots , including interscapular brown adipose tissue , were completely absent ( figure  1a ) .
the epididymal wat was about 10 smaller ( as a percentage of body weight ) in adipo - akt ko mice as compared to control mice ( figure  1b ) .
hematoxylin and eosin staining of tissue sections showed that the residual epididymal wat ( ewat ) in ko mice contained a modest number of enlarged adipocytes ( figure  1c ) .
the ko ewat also displayed increased infiltration of f4/80 +  macrophages ( figure  1d ) .
the levels of important adipokines , such as leptin , adiponectin , and resistin , were dramatically decreased in adipo - akt ko mice relative to controls ( figure  1e ) .
the body weight of adipo - akt ko mice was increased about 15% by 5 weeks of age , with controls weighing 17.3  g and adipo - akt ko mice weighing 20.5  g ( figure  1f ) .
this increase in body weight was maintained at 9 weeks of age with control mice weighing 21.8  g and adipo - akt ko mice weighing 26.0  g ( figure 1f ) .
adipo - akt ko mice had livers weighing 2.7 times that of control animals when normalized for body weight at 812 weeks of age .
livers in adipo - akt mice constituted 13% of body weight , and were 3.5  g on average , as compared to 4.9% of body weight and about 1  g in controls ( figure  2a , b ) .
the greater size of ko livers was associated with a large increase in liver triglyceride levels .
control mice had an average of 9.2  mg triglyceride / g of liver whereas adipo - akt ko livers had 169.1  mg triglyceride / g of liver ( figure  2c ) .
histopathological analysis of livers from adipo - akt ko mice revealed a disorganized morphology with an increase in both the number and size of lipid droplets ( figure  2d ) .
in addition to hepatomegaly , the adipo - akt ko mice displayed enlarged pancreases and kidneys ( figure  3a ) . on the other hand , adipo - akt ko mice did not exhibit splenomegaly , which is often associated with lipodystrophy , ,
histopathology of both the spleen and kidney revealed no obvious abnormalities ( figure  3b , c ) .
in contrast , the pancreases of adipo - akt ko mice had enlarged islets ( figure  3d ) .
adipo - akt ko mice had glucose tolerance comparable to control mice at 812 weeks of age ( figure  4a ) . however , while control mice had fasting insulin levels of 0.5  ng / ml , levels in the ko mice were appreciably higher at 2.5  ng / ml , indicative of significant insulin resistance ( figure  4b ) . to further investigate the ability of adipo - akt ko mice to regulate blood glucose in the face of insulin resistance , we fasted mice overnight and challenged them with a meal of normal chow .
two hours after refeeding , blood glucose levels were significantly greater in the adipo - akt ko mice than the controls ( figure  4c ) .
consistent with the results from gtt , the insulin levels were elevated in fasted adipo - akt ko and increased even further following feeding ( figure  4d ) .
plasma non - esterified fatty acids ( nefa ) levels were also measured during fasting and after a 2  h  feeding challenge on normal chow .
adipo - akt ko mice had low basal nefa levels of 0.7  mmol / l compared to control mice that had fasting nefa of 1.6  mmol / l .
mmol / l while in adipo - akt ko mice nefa levels were not changed ( figure  4e ) .
our findings indicate that akt1 and/or akt2 are required for adipose maintenance in  vivo .
the severity of insulin resistance in the adipo - akt ko mice and their inability to regulate blood glucose postprandially follows the pattern observed in human patients with lipodystrophy , many of whom develop frank diabetes following puberty , , .
mutations in agpat-2 and bscl-2/seipin account for most human lipodystrophy patients , cgl type i and cgl type ii , respectively , , and mice null for these genes have lipodystrophic and insulin resistant phenotypes , .
several other genes have been manipulated in mice resulting in lipodystrophy , notably the azip / f-1 dominant negative mouse , which interferes with adipocyte development , the lamin a ( lmna ) dominant negative mouse that inhibits adipose renewal , and adipocyte - specific loss of peroxisome proliferator - activated receptor gamma ( ppar ) , a transcription factor required for adipocyte differentiation and maintenance , , , .
studies performed in  vitro established that akt signaling is necessary for adipogenesis in cultured cells , , , , .
akt1 is required for adipogenesis in mouse embryo fibroblasts ( mefs ) and 3t3l1 preadipocytes , while akt2 is dispensable , , .
the isoform specificity in  vitro are in conflict with in  vivo data from akt1 null mice , that have an overall smaller body size but do not exhibit a lipodystrophic phenotype , .
this suggests that either non - autonomous factors influence adipogenesis , or that akt2 can support adipogenesis in the whole animal .
additionally , akt2 null mice have been suggested to develop an age - dependent lipodystrophy .
our data expand on these studies and document a critical role for akt signaling in maintaining mature adipocytes .
the adipoq - cre transgene is thought to be mainly expressed in mature adipocytes , , , .
( 2013 ) showed that recombination does not occur in the stromal vascular fraction of adipose tissue depots , .
consistent with this , berry and rodeheffer ( 2013 ) found that highly purified adipogenic precursor cells in wat do not express adipoq - cre 
 .
interestingly , a recent study shows that adiponectin is expressed at the embryonic stage in adipocyte precursors of inguinal wat but not in adult preadipocytes .
it is unclear if adipoq - cre is also expressed at sufficient levels in this population to induce efficient recombination .
moreover , it is not known if adipose precursors of other depots , such as the epididymal wat similarly express adiponectin during embryogenesis .
the adipoq - cre mediated deletion of akt in our model may thus reflect defects in adipocyte differentiation and maintenance , depending on the depot .
there are three akt isoforms , akt1 - 3 , that function in the insulin signaling pathway .
each of these isoforms is expressed in adipocytes , but akt2 is the most abundant isoform , .
mice null for akt1 and akt2 are born with dwarfism , are not viable , and mefs derived from these animals are unable to undergo adipogenesis .
akt3 is expressed in differentiated adipocytes , but it is clearly not sufficient to preserve adipocyte insulin / igf signaling in the absence of both akt1 and akt2 .
the insulin and insulin - like growth factor 1 ( igf-1 ) pathways converge on the insulin receptor substrate and phosphoinositide-3-kinase ( pi3k ) , upstream of akt , , .
loss of akt1 and akt2 in adipocytes likely ablates both insulin signaling and igf-1 signaling , potentially contributing to the severity of the lipodystrophy .
concurrent deletion of the insulin receptor and insulin growth factor 1 receptor ( igf1r ) using ap2-cre causes lipodystrophy with a greater reduction in bat size than wat when compared to the ir knockout alone , suggesting that igf1r signaling has a more important role in bat than in wat , .
interpretation of these data is confounded by the use of the ap2-cre . while the requirement for ir and igf1r signaling may differ between depots , all depots depend on akt - function .
the adipo - akt ko mice display profound hepatomegaly and a small but significant renomegaly along with increased pancreas weight ( figure  2 , figure  3d ) .
this organomegaly is also observed in some human lipodystrophic patients , with hepatomegaly often being most prominent , , , .
hyperinsulinemia potentially contributes to the increased body size and organomegaly of adipo - akt ko mice through enhanced ir / igfr-1 signaling .
another possible contributor to organomegaly is increased food intake as a result of reduced circulating levels of leptin ( figure  1e ) , a critical regulator of energy homeostasis , , , , .
increased food intake and decreased movement were observed in the agpat  / lipodystrophy model and normalized with leptin supplementation . though not measured in this study , it is likely the adipo - akt ko mice would exhibit a similar phenotype . surprisingly , adipo - akt ko mice had low plasma nefa levels , a phenomenon also observed in some human lipodystrophic patients .
similarly , the adipo - akt ko mice had refed nefa levels that were not significantly differently from either their fasted levels or the fed levels in control mice . in many mouse models of lipodystrophy
interestingly , genetic background influences lipid profiles in lipodystrophic azip / f-1 mice . on a fvb
background azip / f-1 mice have abnormally high nefa levels , whereas nefa levels are similar between control and azip / f-1 mice on a c57/b6 background .
similarly , agpat2 and bscl-2/seipin null mice , both in the c57/b6 background , have normal nefa levels , . these mice , and the c57/b6 azip / f-1 mice , have significantly decreased fasting nefa levels , similar to the adipo - akt ko mice . by contrast ,
adipocyte - specific deletion of bscl-2/seipin results in a milder lipodystrophy and these mice do not have reduced fasting nefa levels .
given the severity of lipodystrophy in the adipo - akt ko mice , it follows that they have no available adipose stores to undergo lipolysis and release fatty acids in response to a fasting state .
our mouse model recapitulates many of the features observed in a familial lipodystrophy syndrome caused by loss of function mutations in akt1 and akt2 , including insulin resistance and hepatomegaly .
our study also demonstrates that akt signaling , likely acting downstream of the insulin and igf-1 receptors , is critically required for proper adipose expansion and/or maintenance .
 objectiveadipose depot mass is tightly regulated to maintain energy homeostasis . 
 akt is a critical kinase in the insulin - signaling cascade that is required for the process of adipogenesis in  vitro . 
 however , the role of akt in the maintenance and/or function of mature adipocytes in  vivo had not been examined.methodsto study this , we deleted akt1 and akt2 in adipocytes of mice using the adipoq - cre driver.resultsstrikingly , mice lacking adipocyte akt were severely lipodystrophic , having dramatically reduced gonadal adipose and no discernible subcutaneous or brown adipose tissue . as a result , these mice developed severe insulin resistance accompanied by fatty liver , hepatomegaly and with enlarged islets of langerhans.conclusionsthese data reveal the critical role of adipocyte akt and insulin signaling for maintaining adipose tissue mass . 
Introduction
Materials and methods
Adipo-AKT KO mice
Histopathology
F4/80 staining
Adipokines
Liver triglyceride
Glucose homeostasis
Results
Loss of AKT1 and AKT2 in adipocytes causes lipodystrophy
Hepatomegaly
Organomegaly
Glucose homeostasis
Discussion
Conclusions
Conflict of interest