the serine / threonine kinase akt ( pkb / c - akt ) promotes cell growth ( i.e. , accumulation of cell mass ) predominantly through activation of complex 1 of the mammalian target of rapamycin ( mtorc1 ) ( sarbassov et  al . , mtorc1 is regulated by both nutrients and growth factors and plays a conserved role in cell growth control ( wullschleger et  al . , 2006 ) . glucose uptake and induction of glycolysis by akt have been studied in the context of the metabolic effects of insulin signaling on cellular and organismal levels ( whiteman et  al . , 2002 ) . however , the contribution of anabolic pathways other than protein biosynthesis to cell growth have only been considered recently ( plas and thompson , 2005 ) . akt regulates cell metabolism through several downstream targets ( manning and cantley , 2007 ) . akt stimulates glucose uptake via induction of translocation of the glucose transporter 4 ( glut4 ) to the plasma membrane and by increasing expression of glut1 ( kohn et  al . , 1996 ; welsh et  al . , akt promotes conversion of glucose into pyruvate through the glycolytic pathway by increasing the expression of glycolytic enzymes ( semenza et  al . , 1996 ) and by phosphorylating and activating hexokinase 2 ( hk2 ) ( robey and hay , 2006 ) . pyruvate can either be converted into lactate , or it can enter the tricarboxylic acid cycle ( tca cycle ) , where it is fully oxidized to generate atp . in liver and adipose tissue , glucose is converted via pyruvate and citrate into lipids . phosphorylation of tsc2 by akt ( inoki et  al . , 2002 ; potter et  al . , 2002 ) results in activation of the rheb gtpase while phosphorylation of the pras40 induces the dissociation of this inhibitory component from the mtorc1 complex ( oshiro et  al . , 2007 ; sancak et  al . , 2007 ; vander haar et  al . , 2007 ) . there are several lines of evidence that the akt / mtorc1 axis is an essential regulator of cell growth in  vivo . akt1/akt2 double - knockout mice exhibit severe growth deficiency ( peng et  al . , 2003 ) and mice deficient in s6-kinase 1 ( s6k1 ) show a significant reduction in size accompanied by hypoinsulinaemia and glucose intolerance ( pende et  al . , 2004 ) . the family of sterol regulatory element binding proteins ( srebp ) consists of three closely related members : srebp1a , srebp1c , and srebp2 ( eberle et  al . , 2004 ) . they have been identified as mediators of the effect of sterols on expression of  enzymes involved in lipid and cholesterol homeostasis ( yokoyama et  al . , 1993 ) . srebps belong to the family of basic helix - loop - helix - leucine zipper ( bhlh - zip ) transcription factors . they are synthesized as inactive precursors and localize to the endoplasmic reticulum ( er ) , where they bind to the sterol cleavage activating protein ( scap ) . the srebp / scap complex binds to copii proteins and translocates to the golgi where a two - step proteolytic cleavage releases the n - terminal half of the srebp protein and allows entry into the nucleus ( rawson , 2003 ) . srebps bind to sterol regulatory element ( sre ) and e box sequences found in the promoter regions of genes involved in cholesterol and fatty acid biosynthesis . studies in knockout and transgenic mice have shown that srebp1 preferentially regulates genes involved in fatty acid biosynthesis while srebp2 mainly regulates genes of the cholesterol pathway ( horton et  al . , 2003 ) . two key enzymes that are required to divert glycolytic carbon flux into lipid biosynthesis are atp - citrate lyase ( acly ) and fatty acid synthase ( fasn ) . acly converts cytosolic citrate into acetyl - coa and oxaloacetate , thereby supplying the essential metabolite for lipid biosynthesis . akt phosphorylates acly ( berwick et  al . , 2002 ) , and acly activity is required for cell growth and tumorigenesis ( bauer et  al . , 2005 ; hatzivassiliou et  al . , fasn catalyzes the condensation of acetyl - coa and malonyl - coa to generate long - chain fatty acids . we have previously shown that activation of akt induces expression of a number of lipogenic genes , including acly and fasn ( porstmann et  al . , 2005 ) . we show here that activation of srebp and akt - dependent induction of lipid biosynthesis requires the activity of mtorc1 . furthermore , activation of srebp contributes to akt - dependent cell growth in mammalian cells in  vitro and in drosophila melanogaster in  vivo . our data demonstrate the importance of lipogenesis for cell growth in two distinct model systems . akt is involved in the regulation of cell growth in both mammalian and invertebrate systems by activation of protein biosynthesis through the mtor / s6k pathway ( sarbassov et  al . , 2005 ) . to investigate the involvement of de novo lipid biosynthesis in the regulation of cell growth by akt , we used immortalized human retinal pigment epithelial cells ( rpe ) expressing a conditional allele of the akt1 kinase ( myrakt - er ) ( porstmann et  al . , 2005 ) . cells were cultured in medium supplemented with 1% lipoprotein deficient serum ( lpds ) to reduce activation of endogenous akt and limit the availability of exogenous lipids . activation of myrakt - er by addition of 4-hydroxytamoxifen ( 4-oht ) caused a 20%30% increase in median cell volume ( mcv ) ( figures 1a and 1b ) . simultaneous treatment with the specific mtorc1 inhibitor rapamycin abolished the akt - dependent increase in cell volume . rapamycin did not affect cell size in the absence of akt activation since the experiment was performed under serum starvation conditions . however , rapamycin markedly reduced the akt - dependent increase in cellular protein content ( figure  s1 ) . importantly , we did not observe significant changes in cell - cycle distribution upon akt activation under the conditions used here ( data not shown ) . we next used nuclear magnetic resonance spectroscopy ( nmr ) to measure changes in medium metabolite concentration before and after myrakt - er activation . akt activation induced an almost 2-fold increase in glucose uptake and lactate production in rpe cells ( figure  1c ) consistent with a role for akt in activation of glucose uptake and glycolysis . akt - dependent glucose uptake and lactate production were completely blocked in the presence of rapamycin ( figure  1c ) . likewise , we observed a reduction in akt - dependent amino acid uptake and cellular amino acid content by rapamycin treatment ( figure  s2 ) . analysis of incorporation of radioactive glucose , pyruvate , or acetate into cellular lipids showed that akt activation leads to a substantial increase in glucose - dependent lipogenesis . crucially , this was completely blocked by rapamycin treatment ( figure  1d ) , indicating that activation of de novo lipid synthesis by akt requires mtorc1 function . we have previously shown that akt induces accumulation of intracellular lipids including phosphoglycerides ( porstmann et  al . , 2005 ) . figure  1e shows that inhibition of mtorc1 by rapamycin prevents akt - dependent accumulation of unsaturated ( ufa ) and saturated fatty acids ( fa ) as well as phosphatidylcholine ( pc ) and phosphatidylglycerol ( pg ) levels ( figure  1e ) . we next asked whether induction of lipid biosynthesis is required for akt - dependent increase in cell size . fatty acid biosynthesis requires cytoplasmic acetyl - coa , which is generated by the enzyme atp - citrate lyase ( acly ) . acly function is required for tumor growth in  vivo ( bauer et  al . , 2005 ; hatzivassiliou et  al . , inhibition of acly using the inhibitor sb204990 attenuated akt - dependent increase in cell volume in rpe cells ( figures 1f and 1 g ) . studies in knockout mice have shown selective involvement of srebp1a and 1c in the regulation of lipid biosynthesis ( liang et  al . , 2002 ) and promoters of lipogenic genes are preferentially bound by srebp1a in  vivo ( bennett et  al . , 2004 ) . mature srebp1 can be detected in nuclear lysates as a diffuse band migrating between 60 and 65 kda . figures 2a and 2b show that accumulation of nuclear msrebp1 can be detected within 2 hr of akt activation . induction of msrebp1 precedes the increase in full - length srebp1 ( flsrebp1 ) , which can be detected after 8  hr of akt activation ( figures 2a , 2b , and s3 ) . an increase in fasn mrna and protein expression can be detected after 4  and 8 hr of akt activation , respectively ( figures 2c and 2d ) . induction of nuclear msrebp1 as well as fasn expression in response to akt activation is blocked by sterols ( figures 2e and 2f ) . sterols induce a conformational change in scap that induces insig binding , thereby preventing er / golgi translocation of the srebp / scap complex and srebp processing ( sun et  al . , 2005 ) . the observed induction of flsrebp in response to sterol treatment is most likely due to activation of the liver x receptor ( lxr ) ( repa et  al . , 2000 ) akt induces glucose uptake by inducing glucose transporter expression and membrane localization , and enhances glycolytic activity by activating hexokinase ( manning and cantley , 2007 ) . absence of glucose or treatment with 2-deoxyglucose , an inhibitor of glycolysis , blocked akt - dependent accumulation of msrebp1 ( figure  2 g ) and prevented induction of srebp target genes ( figure  2h ) . energy depletion after glucose starvation or inhibition of glycolysis is likely to lead to activation of ampk ( hardie and carling , 1997 ) . activation of ampk by aicar also blocked accumulation of msrebp1 in response to akt activation ( figure  2i ) and prevented induction of srebp target genes ( figure  s4 ) . these results indicate that activation of srebp1 by akt is regulated by cellular energy status . the activity of mtorc1 is regulated by growth factor signaling as well as nutrient availability and energy homeostasis ( sarbassov et  al . , 2005 ) . nuclear accumulation of msrebp1 in response to akt activation was prevented by rapamycin ( figure  3a ) . likewise , induction of fasn and acly mrna and protein levels was blocked by rapamycin treatment ( figures 3b and 3c ) . importantly , treatment with rapamycin completely abolished induction of phosphorylation of the ribosomal protein s6 , but only had a minor effect on phosphorylation of gsk3 or acly , indicating that the activity of the myrakt - er fusion protein is not affected by rapamycin ( figure  3c ) . furthermore , rapamycin treatment also blocked insulin - dependent induction of fasn protein and mrna ( figures s5a and s5b ) and ectopic expression of mtor was sufficient to induce msrebp1 as well as fasn expression in the  absence of myrakt - er activation in u2os cells ( figure  s5c ) . mature srebp proteins are highly unstable and are rapidly degraded through an ubiquitin - dependent pathway ( hirano et  al . , 2001 ) . this phosphorylation enables binding of the fbw7 ubiquitin ligase and induces degradation of the srebp protein . activation of akt increased levels of transfected myc - tagged wild - type msrebp1 ( amino acids 1490 ) ( figure  3d ) . interestingly , akt - dependent accumulation of exogenous wild - type msrebp could still be observed in the presence of rapamycin , indicating that mtorc1 function is dispensable for stabilization of msrebp1 in response to akt activation . furthermore , inhibition of gsk3 using the chemical inhibitor sb216763 did not block akt - dependent accumulation of endogenous msrebp1 , induction of fasn protein , or fasn promoter activity in response to akt activation ( figure  s6 ) . these results indicate that akt - dependent activation of endogenous srebp does not solely involve regulation of the stability of srebp but suggest an additional , mtorc1-dependent pathway that contributes to srebp activation . it has previously been suggested that rapamycin treatment can also affect the activity of mtorc2 in certain cell types ( sarbassov et  al . , 2006 ) . our own results show that long - term treatment with rapamycin leads to activation of akt phosphorylation by inhibition of a negative feedback loop in the cell line used here ( figure  s7 ) . to address any potential involvement of mtorc2 in the regulation of lipogenic gene expression , we used sirna to selectively abolish expression of raptor or rictor , components of mtorc1 or mtorc2 , respectively . silencing of mtor and raptor significantly reduces s6 phosphorylation ( figure  3e ) , while rictor silencing results in reduction of akt phosphorylation on threonine 450 ( figure  s8 ) , which has been shown to be a substrate for mtorc2 ( facchinetti et  al . , 2008 ; ikenoue et  al . , 2008 ) . silencing of mtor or raptor , but not rictor , prevents akt - dependent induction of fasn and srebp1 expression ( figure  3e ) . silencing of mtor also blocked akt- dependent fasn and acly induction in u2os cells ( figure  s9 ) . we also addressed the role of srebp in the induction of de novo lipid biosynthesis in response to akt activation . figure  3f shows that silencing of srebp1 and 2 blocks incorporation of glucose , pyruvate , or acetate into cellular lipids . furthermore , silencing of mtor or raptor , but not rictor , abolished induction of lipogenesis in response to akt activation ( figure  3f ) . we next asked whether induction of lipogenic gene expression by srebp is involved in the regulation of cell growth in mammalian cells . transfection of sirna oligonucleotides targeting both srebp1 and 2 significantly reduced the increase in cell size observed in response to akt activation in rpe cells in a dose - dependent manner ( figures 4a and 4b ) . likewise , induction of fasn expression in response to akt activation was reduced when srebp1 and 2 were silenced ( figure  4c ) . similar results were obtained when srebp1 and 2 were silenced using different sirna sequences ( data not shown ) . these results indicate that activation of srebp and expression of srebp target genes is required for efficient cell growth in mammalian cells in  vitro . hlh106 has been identified as the gene coding for a single srebp isoform in flies , and dsrebp shows significant sequence homology to human srebp1 within its dna binding domain ( seegmiller et  al . we first measured the effect of transient silencing of dsrebp or dfas on cell volume in the drosophila cell line kc167 . figures 5a and 5b show that silencing of dsrebp or dfas results in a significant reduction in mean cell volume and fsc of g1 cells . interestingly , insulin treatment of kc167 cells resulted in increased levels of fatty acids and phosphoglycerides similar to the results obtained upon akt activation in mammalian cells ( figure  s10 ) . to address the role of dsrebp in the regulation of cell growth in  vivo , we generated a series of transgenic flies expressing an inverted repeat rna under the control of the uas promoter . three different fly lines ( rnai - s1 , rnai - s2 , and rnai - s3 ) were used for the analysis . induction of dsrebp rnai expression using the daughterless ( da)-gal4 driver resulted in reduced expression of dsrebp and dfas in larvae ( figure  5c ) . the three rnai lines showed differences in silencing efficiency with rnai - s3 having the strongest effect ( figure  5c ) . silencing of dsrebp during embryogenesis caused a dose - dependent developmental delay and lethality during larval and pupal stages ( figure  s11 ) . flies reaching adulthood showed a significant reduction in body size , reduced body weight , and wing area ( figures 5d and s12 ) . the rnai - s3 fly line did not produce any viable offspring when crossed to the da - gal4 driver . this is consistent with an essential role of dsrebp during larval development ( kunte et  al . , 2006 ) . deletion or overexpression of components of the insulin signaling pathway affects cell size in a cell - autonomous manner ( weinkove et  al . , 1999 ) . we therefore asked whether silencing of dsrebp in specific compartments of the drosophila wing could affect cell and organ size . figures 5e and 5f show that expression of dsrebp rnai cassette in the dorsal wing compartment ( ms1096-gal4 ) caused a dose - dependent reduction in adult wing area . similarly , silencing of dsrebp using the engrailed ( en)-gal4 driver caused a specific reduction in the area of the posterior compartment of the wing ( figures 5 g and 5h ) . furthermore , reduced size of the posterior compartment was accompanied by an increase in density of wing epithelial cells ( figures 5i and 5j ) indicating that silencing of dsrebp causes a reduction in cell size . we also investigated whether inhibition of dsrebp function by means other than gene silencing would cause similar growth inhibition . deletion of the n - terminal transactivation domain in mammalian srebp results in dominant inhibition of srebp function ( sato et  al . , 1994 ) . ms1096-gal4-driven expression of n - terminally deleted dsrebp ( dn75 ) or a mutant in which the transmembrane domain has been deleted in addition to the n terminus ( dn75 t ) resulted in a substantial reduction in wing area ( figure  s13 ) . we next asked whether the pi3k / akt pathway regulates the activity of srebp in flies . transient silencing of the catalytic subunit of pi3k , dp110 , or dakt reduced mrna abundance of dfas and dsrebp in kc167 cells ( figure  6a ) . conversely , silencing of dpten resulted in increased expression of both transcripts ( figure 6a ) . ubiquitous expression of dp110 using the da - gal4 driver resulted in enhanced dsrebp and dfas expression in second instar larvae compared to controls ( figure  6b ) , indicating that the pi3k / akt pathway activates dsrebp function . expression of mature dsrebp ( m - dsrebp ) using the ms1096-gal4 driver resulted in significant lethality when flies were reared at 25c ( data not shown ) . when flies were reared at 18c ( resulting in a reduced activity of the gal4/uas system ) , severe deformation of the wing was observed ( figure  6c ) . expression of the full - length form of dsrebp ( fl - dsrebp ) in the wing caused less severely misshapen wings even when flies were reared at 25c . however , coexpression of dp110 and full - length dsrebp resulted in severely deformed wings , a phenotype that is similar to that caused by expression of m - dsrebp ( figure  6c ) . this result suggests that the activity of full - length dsrebp is enhanced by pi3k signaling . expression of dp110 causes an overgrowth phenotype in the wing , indicated by a 15%20% increase in surface area of the wing ( leevers et  al . silencing of dsrebp attenuated the increase in wing size induced by expression of dp110 ( figure  6d ) . similar results were obtained using a nonoverlapping rnai sequence targeting dsrebp or by heterozygous deletion of the dsrebp gene ( figure  s14 ) . expression of a kinase domain mutant of pi3k ( dp110[kd ] ) decreases cell and organ size in drosophila ( leevers et  al . , 1996 ) . expression of dp110[kd ] using the decapentaplegic ( dpp)-gal4 driver resulted in a 20%30% reduction in the size of the dpp compartment . expression of the dsrebp rnai hairpin resulted in a small but significant decrease in wing area ( figure  6e ) . however , coexpression of dsrebp rnai with dp110kd did not further decrease the size of this compartment . , these results suggest that dp110 and dsrebp are components of the same pathway in the regulation of cell growth in drosophila . cell growth is tightly controlled by mitogenic signaling pathways and requires the activation of biosynthetic pathways for the generation of macromolecules , including proteins and lipids . activation of the mtor complex 1 ( mtorc1 ) increases translation and ribosome biogenesis ( wullschleger et  al . , 2006 ) , but effects of mtorc1 on other anabolic processes are less well understood . we show here that ectopic activation of akt induces a significant increase in cell volume in the absence of mitogens and that akt - dependent size increase is blocked by the mtorc1 inhibitor rapamycin . analysis of medium metabolites by nmr revealed that akt activation results in increased uptake of glucose and amino acids as well as secretion of lactate . the involvement of mtorc1 in akt - dependent translocation of glucose and amino acid transporters to the plasma membrane has been demonstrated before ( edinger and thompson , 2002 ) . we observed significant accumulation of fatty acids and phosphoglycerides in response to akt activation and enhanced incorporation of labeled glucose , pyruvate , or acetate into the lipid fraction in cells with activated akt , all of which was blocked by rapamycin . these results show that akt activates de novo lipogenesis and that mtorc1 activity is required for this effect . hepatocytes show increased srebp1c mrna levels in response to insulin treatment or akt expression ( azzout - marniche et  al . , 2000 ; we have previously shown that akt activation results in nuclear accumulation of msrebp1 and enhances expression of a number of srebp target genes ( porstmann et  al . , , we show that nuclear accumulation of msrebp1 is rapid and precedes the induction of transcription of srebp target genes as well as the increase in flsrebp1 . it should be noted that the srebf1 gene is a target of positive feed - forward regulation , since a functional sre has been identified in its promoter region ( amemiya - kudo et  al . , 2000 ) . thus , induction of flsrebp after akt activation could be due to enhanced sre - dependent transcription of the gene . activation of srebp1 by akt was completely blocked by glucose starvation , inhibition of glycolysis , or ampk activation by aicar . ampk regulates lipid metabolism by phosphorylating acc and induces a switch from fatty acid biosynthesis to fatty acid oxidation ( hardie and carling , 1997 ) . thus , regulation of srebp activity by ampk could reflect an additional adaptation to energy restriction . activity of mtorc1 is regulated by mitogens but also responds to cellular energy status ( sarbassov et  al . , 2005 ) . ampk activates tsc2 resulting in inhibition of mtorc1 ( inoki et  al . , 2003 ) . it seems likely that inhibition of srebp1 activity following glucose deprivation or ampk activation is mediated by mtorc1 . our results show that nuclear accumulation of srebp1 and induction of srebp target genes requires mtorc1 activity . it has been suggested that induction of fasn and acc expression by her2 in breast cancer cells involves increased protein translation ( yoon et  al . , 2007 ) . however , we observed an increase in fasn and acly mrna abundance in response to akt activation . induction of fasn and acly mrna was blocked in the presence of rapamycin indicating that mtorc1 is involved in the regulation of srebp - dependent transcription . furthermore , silencing of mtor or raptor , but not rictor , abolished activation of expression of srebp1 , fasn , and acly , thus confirming that regulation of srebp - dependent gene expression is exclusive to mtorc1 . it has been shown that gsk3 regulates the stability of msrebp1 by inducing the association of the ubiquitin ligase fbw7 resulting in rapid degradation of srebp1 ( sundqvist et  al . , 2005 ) . we observed an increase in the stability of exogenous msrebp in response to akt activation . this was dependent on the presence of two gsk3 phosphorylation sites but was still observed in the presence of rapamycin . however , our results indicate that akt also regulates srebp1 activity in a gsk3-independent , mtorc1-dependent manner . it has been shown that dominant negative akt prevents er / golgi translocation of scap ( du et  al . , 2006 ) . tor has been implicated in vesicle transport in yeast ( neufeld , 2007 ) and localization of mtor to the er and the golgi plays an essential role in its regulation and downstream signaling ( drenan et  al . , 2004 ) . thus , it seems possible that mtorc1 , or proteins downstream of mtorc1 signaling such as s6k or 4-ebp , could be involved in the regulation of srebp processing within these compartments . silencing mtor or raptor , but not rictor , is sufficient to abolish akt - dependent incorporation of glucose , pyruvate , or acetate into cellular lipids . this effect seems to be most pronounced for the incorporation of acetate , which only requires the activity of acc and fasn . an involvement of raptor in the regulation of lipogenesis is also consistent with its essential function during mouse development ( guertin et  al . , 2006 ) . interestingly , silencing of srebp1 and 2 was sufficient to prevent akt - dependent lipogenesis and significantly reduced cell growth in response to akt activation . this result confirms that srebp - dependent gene expression represents an important mechanism in the regulation of lipogenesis during cell growth . consistent with a role for srebp in cell size regulation in mammalian cells , we observed that silencing of dsrebp or its target gene dfas resulted in a significant decrease in cell size in drosophila kc167 cells . interestingly , dsrebp or dfas silencing completely blocked the increase in cell volume caused by silencing of tsc2 . silencing of dsrebp in different compartments of the developing wing imaginal disc caused a reduction of the respective compartment in adult flies and was caused by a reduction in cell size rather than cell number . it is somewhat surprising that expression of m - dsrebp in the wing causes a severe growth defect . it has been shown before that overexpression of msrebp1a causes cell - cycle arrest through induction of the cdk inhibitors p21 , p27 , and p16 ( inoue et  al . , 2005 ; it is also possible that overactivation of srebp causes an imbalance between protein and lipid biosynthesis and suggests that srebp activity needs to be tightly controlled to allow normal tissue development . kunte and coworkers have detected dsrebp activation in the developing fat body , the midgut as well as in oenocytes , a specialized cell type that have hepatocyte - like functions in lipid metabolism ( gutierrez et  al . , 2007 ; kunte et  al . , this expression pattern suggests a role in lipid metabolism similar to the function of srebp1c in mammalian liver . our results are consistent with a cell - autonomous role for dsrebp and suggest that dsrebp is required for normal growth of peripheral larval tissue . dsrebp silencing restricted tissue growth induced by overexpression of dp110 in the wing and did not enhance the reduction in size of a wing compartment expressing kinase - dead dp110 . these results suggest that both factors are components of the same pathway and indicate that activation of dsrebp is required for the induction of cell growth by the pi3k pathway in flies . it will be interesting to investigate whether regulation of dsrebp downstream of pi3k requires tor . further investigations are required to elucidate the exact mechanism of regulation of dsrebp activity by signaling pathways involved in growth regulation in flies . taken together , our data are consistent with a model that places srebp activation downstream of the intracellular signaling pathway that integrates mitogenic signaling with energy status and nutrient availability ( figure  7 ) . it also suggests that protein biosynthesis and lipogenesis are regulated in a concerted manner during cell growth . rpe myrakt - er and u2os myrakt - er cells have been described before ( porstmann et  al . , 2005 ) . rpe myrakt - er cells were maintained in dmem / hams f12 ( 1:1 ) medium with 10% fcs , glutamine , and sodium bicarbonate . u2os myrakt - er cells were grown in dmem with 10% fcs and glutamine . for culture at low sterol concentration , cells were grown in medium with 1% lipoprotein deficient serum ( lpds , intracel ) . kc167 cells were cultured in schneider 's medium ( gibco ) with 10% fcs , penicillin / streptomycin , and glutamine . a list of antibodies , chemicals , and plasmids is provided in the supplemental experimental procedures . rpe myrakt - er cells were detached using trypsin , washed , and resuspended in pbs . median electronic cell volumes were determined in triplicate using a z2 coulter counter ( multisizer ii , beckman - coulter ) . kc167 cells were collected by centrifugation and analyzed as above . for g1 specific cell size , four hundred and fifty microliters of medium samples were mixed with 50 l d2o , and 25 l of 10 mm sodium tsp were added for chemical shift calibration and quantification . intracellular metabolites were extracted using a dual - phase - extraction method as previously described ( tyagi et  al . , 1996 ) . lipid extracts were reconstituted in 600 l deuterated chloroform , and 0.008% v / v tetramethylsilane was used as a chemical shift reference ( 0  ppm ) . water - soluble metabolites were reconstituted in 600 l d2o , and 25  l of 1 mm tsp was added for quantitation and chemical shift calibration . proton nmr spectra were acquired using a bruker 600 mhz ( bruker avance ) nmr system . spectral peaks for water - soluble and lipid metabolites were assigned according to sitter et  al . metabolite levels were normalized to cell number , and each experiment was repeated at least six times . cells were grown in medium with 1% lpds in the presence of 100 nm 4-oht or ethanol for 24 hr . 2.5 ci / ml d-[6-c]glucose ( 45 m final concentration , amersham ) , 2.5 ci / ml [ 2-c]-pyruvate ( 166 m final concentration ) or 10  ci / ml [ 1-c]-acetate ( 85 m final concentration , both perkin elmer ) was added , and cells were incubated for 4 hr at 37c . lipids were extracted by successive addition of 2 ml methanol , 2 ml chloroform , and 1 ml dh2o . lipids were dissolved in ultima gold lsc cocktail ( perkin elmer ) and counted on a beckman ls 6500 scintillation counter . each measurement was performed in duplicate , results were normalized to total protein content , and experiments were repeated at least twice . for total cell lysis , cells were washed with ice - cold pbs and dissolved in lysis buffer ( 1% triton x-100 , 50 mm tris ph 7.5 , 300 mm nacl , 1 mm egta , 1 mm dtt , 1 mm navo4 , and protease - inhibitor [ roche ] ) . nuclear lysates were prepared as described ( porstmann et  al . , 2005 ) . for detection of msrebp , cells were treated with 25 g / ml alln for 1 hr prior to lysis . equal amounts of cell lysates were separated on sds - page and blotted onto pvdf membrane ( immobilon ) . total rna was isolated from rpe myrakt - er or kc267 cells using the rneasy kit ( qiagen ) and treated with dnase i ( qiagen ) . for preparation of rna from drosophila larvae , 1020 s instar larvae were homogenized in liquid nitrogen and dissolved in buffer rlt ( qiagen ) followed by rna purification according to manufacturer 's protocol . two - and - a - half micrograms of total rna was used for first - strand cdna synthesis with superscript ii rt and oligo dt primer ( invitrogen ) . real - time pcr was performed with sybr green pcr master mix ( applied biosystems ) . rpe - myrakt - er cells were transfected using fugene ( roche ) according to manufacturer 's instructions . twenty - four hours post transfection , cells were placed into medium containing 1% lpds and induced with 100 nm 4-oht as indicated . for gene - silencing experiments , cells were transfected using dharmafect reagent 1 ( dharmacon ) following a reverse transfection protocol . twenty - four hours post transfection , cells were split and divided into parallel cultures for different treatments . sirna sequences are provided in the supplemental experimental procedures . for depletion experiments in kc167 cells , dsrnas were generated by in  vitro transcription using the megascript t7 transcription kit ( ambion ) . silencing was performed using 25 g of dsrna , and cells were analyzed after 5 days . a sequence comprising 700 bp of the 5 end of the dsrebp gene was amplified by pcr from genomic dna and inserted as an inverted repeat into pwiz - uast ( lee and carthew , 2003 ) . uas - dp110wt , uas - dp110[kd ] , and dpp - gal4/uas - dp110[kd ] fly lines were described previously ( weinkove et  al . , 1999 ) . ms1096-gal4/uas - dp110wt flies were a gift from ernst hafen ( eth , zuerich ) . dsrebp flies were a gift from robert b. rawson ( university of texas southwestern medical center , dallas ) . wings of male flies were dehydrated in ethanol , mounted in euparal ( agar scientific ) , and photographed . cell number was determined by counting wing hairs in an area of fixed size in anterior or posterior wing compartments . unless stated , p values were calculated using a two - tailed student 's t test assuming equal variance .