Dynamic and differential regulation of proteins that coat lipid droplets in fatty liver dystrophic mice.

Lipid droplet proteins (LDPs) coat the surface of triglyceride-rich lipid droplets and regulate their formation and lipolysis. We profiled hepatic LDP expression in fatty liver dystrophic (fld) mice, a unique model of neonatal hepatic steatosis that predictably resolves between postnatal day 14 (P14) and P17. Western blotting revealed that perilipin-2/ADRP and perilipin-5/OXPAT were markedly increased in steatotic fld liver but returned to normal by P17. However, the changes in perilipin-2 and perilipin-5 protein content in fld mice were exaggerated compared with relatively modest increases in corresponding mRNAs encoding these proteins, a phenomenon likely mediated by increased protein stability. Conversely, cell death-inducing DFFA-like effector (Cide) family genes were strongly induced at the level of mRNA expression in steatotic fld mouse liver. Surprisingly, levels of peroxisome proliferator-activated receptor gamma, which is known to regulate Cide expression, were unchanged in fld mice. However, sterol-regulatory element binding protein 1 (SREBP-1) was activated in fld liver and CideA was revealed as a new direct target gene of SREBP-1. In summary, LDP content is markedly increased in liver of fld mice. However, whereas perilipin-2 and perilipin-5 levels are primarily regulated posttranslationally, Cide family mRNA expression is induced, suggesting that these families of LDP are controlled at different regulatory checkpoints.

for tissue collection. All animal experiments were approved by the Washington University School of Medicine Animal Studies Committee and conformed to criteria outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals .

Tissue histology
Freshly obtained liver samples were fi xed overnight in 10% buffered formalin and embedded in paraffi n. Hematoxylin and eosin staining was carried out by the Digestive Disease Research Core Center at Washington University School of Medicine.

Determination of liver TG levels
TG concentration was determined by a commercial colorimetric method (Wako Chemicals, Richmond, VA) with solvent extracted lipid, as described ( 22 ). Total liver protein was extracted using tissue protein extraction reagent (number 78510; Pierce) and TG concentration is expressed as milligrams of TG/g of liver protein.

mRNA isolation and gene expression analyses
Liver RNA was extracted with RNAzol Bee (Isotexdiagnostics, Friendswood, TX) according to the manufacturer's instructions. Real time RT-PCR was performed using the ABI PRISM 7500 sequence detection system (Applied Biosystems, Foster City, CA) and the SYBR green kit. Using the standard curve method, the relative amount of specifi c PCR products for each primer set was generated. For normalization, 36B4 was amplifi ed from each sample, and arbitrary units of target mRNA were corrected to the corresponding level of the 36B4 mRNA. The sequences of the primers used in these studies can be found in supplementary  Table I .

Protein isolation and cellular fractionation of mouse livers
For Western blot studies in Fig. 2 , protein extracts were obtained from liver by using the following lysis buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EDTA, 0.1% sodium deoxycholate, and 1% Triton X-100) containing a protease inhibitor cocktail. For sterol-regulatory element binding protein (SREBP)-1 Western blots, nuclear proteins were isolated using the NXTRACT nuclear protein isolation kit (Sigma Chemical Co., St. Louis, MO). Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Scientifi c, Rockford, IL).
For subcellular fractionation studies, livers from 12-week-old fed fl d and wild-type (WT) mice were minced and then homogenized by a Tefl on pestle tissue homogenizer in lysis buffer (10 mM HEPES, 1 mM EDTA, pH 7.4). The nuclear fraction was obtained by 2,000 g centrifugation for 5 min. The 2,000 g supernatant was weighted with sucrose to 40% (w/v) with 65% sucrose and was then overlaid with successive layers of 5 ml 35% sucrose and 5 ml 10% sucrose. The tubes were then fi lled to capacity with lysis buffer. The gradients were centrifuged at 172,000 g for 3 h at 4°. Fractions were harvested as described previously and stored at Ϫ 80°C until Western blot analyses ( 23 ).

Western blot analysis
Western blotting studies were performed with whole-cell lysates (40 µg) or nuclear lysates (20 µg) as indicated. Proteins from sucrose gradient isolation were loaded by volume rather than protein content. Proteins were separated on 4%-12% gradient gels by SDS-PAGE. Proteins were then transferred onto nitrocellulose membranes that were then blocked with 5% (w/v) nonfat dry milk in TBS-Tween (20 mM Tris-HCl pH 7.5, 0.9% proteins, perilipin-4 (S3-12) and perilipin-5 (also known as OXPAT, MLDP, PAT-1, and LSDP5) ( 5,6 ), have now been added to the perilipin family based on sequence and functional similarities. Another family of proteins, known as the cell death-inducing DFFA-like effector (Cide) family of proteins (CideA, CideB, and CideC), has also recently emerged as LDPs that regulate LD metabolism ( 7 ). The mouse homolog of CideC is known as Fsp27 and will be referenced as such henceforth. Interestingly, work from several groups has convincingly demonstrated that Cide proteins are also physically associated with LDs and modulate LD size and metabolism ( 8,9 ).
LDPs allow LDs to maintain a dynamic communication with the endoplasmic reticulum and the plasma membrane ( 1 ). Several lines of evidence suggest that perilipin-1 and perilipin-2 are physical barriers to lipolytic enzymes under basal conditions yet can facilitate interactions with lipases and enhance lipolysis in response to lipolytic stimuli (10)(11)(12). The signifi cant alterations in lipid metabolism observed in mouse models with targeted deletion of LDPs are strong evidence for important roles for these proteins in regulating metabolism (13)(14)(15).
Although LDPs were originally studied from the perspective of their effects in adipose tissue, more recent work has shown critical roles for LDPs in regulating fat metabolism in liver, especially in the context of lipid overload such as occurs in obesity-related fatty liver disease. Specifically, the expression of perilipin-2, CideA, and Fsp27 is induced in liver of ob/ob mice, which exhibit marked hepatic steatosis (16)(17)(18). Moreover, mice defi cient in perilipin-2, CideB, or Fsp27 are protected from developing obesity-related fatty liver disease ( 15,19 ), suggesting that these proteins play a role in driving hepatic lipid accumulation or enhancing the capacity for storing lipid.
We sought to evaluate the expression of lipid droplet proteins in liver of fatty liver dystrophic ( fl d ) mice, a lipodystrophic model of fatty liver ( 20 ). The complex and severe metabolic phenotype of fl d mice is caused by a mutation in the gene encoding lipin 1 ( Lpin1 ) ( 21 ). The fl d mice appear normal at birth, but rapidly develop an enlarged fatty liver ( 20 ). Hepatic lipid accumulation in fl d mice spontaneously and rapidly resolves prior to weaning ( 20 ), making this model a unique and interesting system in which to study the expression of the LDP proteins. Herein, we demonstrate that expression of several LDPs is markedly increased in the steatotic liver of fl d mice, and decreases as the fatty liver phenotype resolves. Our studies also revealed that the PAT and Cide families of LDPs are controlled by distinct regulatory mechanisms in steatotic hepatocytes.

Animal studies
All studies were conducted with matched littermate mice. Homozygous fl d ( fl d/fl d ) mice were compared with littermate heterozygous ( fl d /+) or wild-type ( +/+ ) control mice. Eighteenweek-old female ob/ob and lean littermate (ob/+) were sacrifi ced tation and luciferase activity in cell lysates determined by Dual-Glo (Promega) assays 60 h after transfection. The Cidea promoter constructs have been previously described ( 29 ) and contain -1385 (pCID2), -888 (pCID3), or -578 (pCID4) of the 5 ′ -fl anking sequence of the Cidea gene (all notations are relative to the transcriptional start site). Cidea promoter constructs were cotransfected with expression constructs driving expression of the caSREBP-1 ((26), generous gift of Jay Horton) or empty vector control and SV40-driven renilla luciferase expression construct. All values are normalized to 1.0 and fi refl y luciferase values were corrected to renilla luciferase activity.

ChIP analyses
Chromatin immunoprecipitation (ChIP) experiments were performed as previously described ( 27 ). Hepatocytes from WT mice were dissociated with collagenase, plated, and infected with adenovirus to express caSREBP-1 and/or GFP. After 24 h of infection, hepatocytes were cross-linked in 1% formaldehyde for 15 min. Chromatin-bound proteins were immunoprecipitated using antibodies directed against SREBP-1 or IgG control. PCR primers (supplementary Table I ) were designed to amplify a region of the Cidea gene promoter identifi ed in the promoter deletion series as being responsive to SREBP-1 or an exon of the Acadm gene ( 27,30 ).

Statistical analyses
Statistical comparisons were made using ANOVA or t -test. All data are presented as means ± SEM, with a statistically signifi cant difference defi ned as a P value < 0.05.

Time-course of hepatic steatosis in neonatal fl d mice
We sought to evaluate the hepatic histological and biochemical phenotype of young fl d mice at 3 day intervals. Histological examination of hepatic sections from WT and fl d mice at P8 and P11 revealed virtual replacement of the parenchyma by hepatocytes distended with tiny fat droplets. Hepatocytes had a "foamy" appearance, which is a morphologic appearance consistent with microvesicular steatosis ( Fig. 1 ). Hepatic triglyceride content was strikingly elevated 30-fold and 40-fold at P8 and P11, respectively, in fl d mice compared with WT mice ( Table 1 ). By P14, microvesicular LD were greatly reduced in fl d mice. However, occasional large lipid droplets remained present and hepatic TG content remained elevated 8-fold compared with WT. At P17, fl d and WT liver sections were indistinguishable in biochemical TG and histological analyses

PAT protein expression is increased in steatotic fl d mouse liver
LDP expression is induced in many models of hepatic steatosis secondary to obesity or lipodystrophy ( 16,18,(31)(32)(33), but whether these genes are regulated in fl d mice is unknown. Of the fi ve PAT genes, only the hepatic expression of perilipin-2 mRNA ( Plin2 ) was signifi cantly affected in fl d mice compared with control WT littermates ( Fig. 2A ). Plin2 mRNA expression was increased in fl d mice at P8 and P11 but then declined by P14 during the resolution of fatty liver. Perilipin-2 protein levels were also NaCl, 0.05% Tween 20) prior to immunoblotting. Generation of rabbit-derived antibodies directed to carboxyl-terminus of perilipin-5, the amino-terminus of perilipin-3, and the aminoterminus of perilipin-2 has been described previously ( 5,6 ). Antibodies to glycogen synthase (Proteintech Group, Chicago, IL), peroxisome proliferator-activated receptor ␥ (PPAR ␥ ) (Santa Cruz, San Diego, CA), SREBP-1 (Santa Cruz), CideA (Genway, San Diego, CA), Fsp27 (generous gift of Dr. Vishwajeet Puri), and actin (Sigma) were used according to the manufacturer's instructions.

Primary mouse hepatocyte isolation
Primary mouse hepatocytes were isolated from mice as previously described ( 24 ). Briefl y, mice were anesthetized and then perfused through the portal vein with warmed HBSS containing collagenase. Livers were mechanically disrupted with forceps. Released cells were washed extensively and plated onto collagen coated dishes and grown in culture in DMEM supplemented with 5% FBS.

Pulse-chase experiment
After plating and adherence, hepatocytes from adult WT mice were washed three times with PBS and incubated in Met-and Cysfree DMEM for 1 h to deplete the cellular pool of Met and Cys. Thereafter, the medium was replaced with 1 ml of Met-and Cysfree DMEM containing 200 µCi of [

Immunofl uorescence microscopy
Hepatocytes from P14 WT or fl d mice were isolated, plated, and then fi xed with formaldehyde after adhering for 1 h. Fixed hepatocytes were then stained as described previously ( 25 ) with antibodies below. The cells were stained with anti-perilipin-2 (Fitzgerald Industries International, Flanders NJ, CAT# 20R-AP002), perilipin-3 ( 6 ), or perilipin-5 ( 5 ) antibodies. The secondary antibodies were goat Anti-Guinea Pig Alexa 488 and Donkey Anti-Rabbit Alexa 594 (Invitrogen, CAT# A-11073, CAT #A-21207), respectively. Images were captured on a Nikon Eclipse TE2000U by using a 60× oil objective lens on a photometric coolsnap camera (Nikon Instruments) driven by Metamorph software (UIC, Downingtown, PA). Appropriate fi lters were used to image the signals from the two secondary antibodies separately.

Adenovirus studies
To produce an adenovirus expressing a constitutively active form of human SREBP-1a ( 26 ), a cDNA fragment ( ‫ف‬ 1.5 kb) encoding the amino acids 1-460 of human SREBP-1a was generated from poly-A RNA isolated from HepG2 cells by RT-PCR using the following primers: 5 ′ primer, 5 ′ -GGGAAGCTTGCTCCC-TAGGAAGGGCCGTACGAGGCG-3 ′ ; and 3 ′ primer, 5 ′ -GTCTA-GACTACTAGTCAGGCTCCGAGTCACTGCCACTGCCAC-3 ′ . The resultant PCR product was subcloned into a TA-cloning vector and subcloned into the Ad-track shuttle vector. For overexpression of PPAR ␥ , a full-length, FLAG-tagged, mouse PPAR ␥ 1 cDNA was cloned into the Adtrack vector. The fi nal adenovirus constructs were produced using the AdEasy system as described ( 27 ). Mouse hepatocytes were infected with the specifi ed adenovirus at an MOI of 8 as described previously ( 28 ). RNA was isolated 48 h after adenoviral infection.

Promoter-luciferase reporter studies
HEK-293 cells were maintained in DMEM-10% fetal calf serum. Cells were transfected using calcium-phosphate coprecipi-levels declined coincident with the resolution of hepatic steatosis. Perilipin-1 and perlipin-4 protein and mRNA were undetectable in all livers (data not shown), consistent with low expression of these proteins in liver. These data suggest that levels of the PAT proteins, perilipin-2, perilipin-5, and to a lesser extent, perilipin-3, are increased in fl d liver coincident with elevated hepatic TG levels, but are regulated independent of changes in their corresponding RNAs.
To confi rm that the observed disconnect between mRNA protein was applicable to other models of hepatic steatosis, we measured the expression of Plin2 and Plin5 mRNA in ob/ob mouse liver and compared the levels of the corresponding proteins. We found that perilipin-2 and perilipin-5 protein levels were markedly induced in ob/ob liver compared with littermate controls (supplementary Fig. I ). Although Plin2 and Plin5 mRNA expression was also upregulated, the observed increase in protein was out of proportion to the mRNA induction. These data also suggest posttranscriptional regulation of PAT proteins possibly related to the increased stability with fatty acid overabundance.

Fat loading increases PAT protein stability in hepatocytes
Previous work has shown that fatty acid abundance can increase the stability of perilipin-2 and perilipin-1 in immortalized cell lines ( 34,35 ). Given the disconnect between PAT mRNA and protein in steatotic liver, we sought to evaluate protein stability of perilipin-2, perilipin-3, and perilipin-5 after loading WT hepatocytes with 400 uM oleate for 1 h. As we hypothesized, oleate loading increased the quantity of 35 S-labeled perilipin-2 6-fold 12 h after chasing with cold methionine ( Fig. 2C ). Similarly, oleate loading increased perilipin-5 content (3-fold) after a 12 h chase, whereas perilipin-3 content was not signifi cantly increased ( Fig. 2C ). These data suggest that lipid overabundance stabilizes perilipin-2 and perilipin-5 and enhances their half-lives.

Perilipin-2, perilipin-3, and perilipin-5 coat LDs but may mark distinct lipid pools
We next determined the subcellular distribution of the PAT proteins that were induced in fl d liver by using isolated hepatocytes from WT and fl d mice and performing immunofl uorescent staining. Perilipin-2 was localized primarily to large LDs in both WT and fl d hepatocytes ( Fig. 3 ). An increase in the quantity of perilipin-2 in fl d hepatocytes compared with WT cells was also evident. In contrast, perilipin-5 and perilipin-3 are observed in a diffuse pattern throughout the cytosol in both fl d and WT hepatocytes with more intense staining in the fl d hepatocytes ( Fig. 3 ). Perilipin-5 antibodies also illuminate a punctate pattern in both WT and fl d hepatocytes.
Although the identity of the structures marked by perilipin-5 and perilipin-3 is beyond the technical capability of this technique to visualize, based on the biochemical studies below, we believe that these two proteins are soluble in WT mice and may be marking small LDs in fl d hepatocytes.
vigorously increased in fl d mice at P8, P11, and P14, but declined by P17 ( Fig. 2B ). Compared with the increase in Plin2 mRNA, the increase in perilipin-2 protein was more robust, suggesting that posttranscriptional mechanisms also impact perilipin-2 protein content in lipid-laden liver.
The expression of Plin3 and Plin5 mRNA was unchanged between fl d and WT animals at all ages examined ( Fig.  2A ). Conversely, perilipin-5 protein levels were induced markedly in fl d liver compared with littermate controls at P8, P11, and P14 ( Fig. 2B ), suggesting posttranscriptional regulation. Perilipin-3 protein was also increased modestly in steatotic fl d livers. Perilipin-3 and perilipin-5 protein  P11, and P14 but then declined to control levels at P17 ( Fig. 5 ). Cideb expression was unchanged between WT and fl d livers ( Fig. 5 ). These data parallel observed increases in Cidea and Fsp27 expression in ob/ob liver (supplementary Fig. I ). Western blotting analyses using antibodies against CideA and Fsp27 and lysates from LD fractions from P8 WT and fl d mice demonstrated that changes in Cide mRNA levels were accompanied by coordinate increases in protein levels.

PPAR ␥ levels are not increased in fl d liver
The induction of PPAR ␥ is believed to be a primary driving force in the development of hepatic steatosis as well as the increased expression of perilipin-2 and Cide genes ( 18 ). PPAR ␥ is expressed at very low levels in normal liver, but the hepatic expression of this transcription factor is induced in nearly every animal and human model of fatty liver disease examined thus far ( 36 ). Interestingly, PPAR ␥ mRNA ( Pparg ) and protein were not increased in fl d mice at any time point and were actually reduced in P8 fl d mice compared with littermate controls ( Fig. 6 ). This is a unique feature of fl d liver that differentiates it from other fatty liver models.
PAT protein localization was confi rmed by subcellular fractionation and Western blotting. We subjected fl d or WT liver homogenates to sucrose gradient centrifugation and determined the distribution of endogenous PAT proteins across the gradient ( Fig. 4 ). In the WT liver homogenates, perilipin-2 was primarily detected in the LD fraction whereas perilipin-3 and perilipin-5 were detected primarily in the cytosolic fractions, which were marked by glycogen synthase ( Fig. 4 ). Importantly, we also observed redistribution of perilipin-5 and perilipin-3 protein to the LD fraction in fl d liver homogenates compared with WT controls. Together with the immunofl uorescence data ( Fig. 3 ), these data suggest that perilipin-3 and perilipin-5 proteins are primarily soluble in normal mouse liver, but a signifi cant portion of the protein redistributes to coat small LDs in steatotic liver.

Cide mRNA expression is increased in fl d liver
Given emerging evidence that the Cide family of LDPs plays important roles in regulating hepatic fat metabolism, we also examined the expression of this family of genes. Cidea and Fsp27 were strongly induced in fl d livers at P8,  35 Slabeled perilipin-2, perilipin-3, or perilipin-5 from pulse-chase studies are shown. Hepatocytes isolated from adult WT mice were cultured in the presence or absence of 0.4M oleic acid (OA) and labeled for 1 h with 35 S-methionine. Labeled methionine was then chased with medium containing 1000× cold methionine for 12 h before cell lysates were collected. Perilipin-2 and perilipin-5 were then immunoprecipitated, purifi ed by washing, separated by SDS-PAGE, and the fi xed and dried gels exposed to autoradiographic fi lm.
Gpam, Acacb ( 38 )] were increased in P8 fl d mice compared with littermate controls ( Fig. 7B ). Collectively, this evidence supports the idea that SREBP-1 is activated in fl d mouse liver.

Cidea is a direct target gene of SREBP-1
Although SREBP-1 regulates many genes involved in lipid metabolism, to our knowledge, Cide genes have not been identifi ed as SREBP-1 targets. To address this, we overexpressed a constitutively active form of SREBP-1a ( 26 ) in WT hepatocytes using an adenovirus and assessed Cide gene expression. Cidea expression was robustly induced, whereas Fsp27 and Cideb were not signifi cantly affected by constitutively-active SREBP-1 ( Fig. 8 ). In addition, expression of Plin2 , Plin3 , and Plin5 were not induced by SREBP-1 ( Fig. 8 ).
To determine whether SREBP-1 directly regulates Cidea transcription, a series of Cidea promoter-luciferase reporter constructs ( 29 ) was transfected into 293 cells, which are virtually null for SREBP-1, with a caSREBP-1 expression construct. As predicted, SREBP-1a overexpression induced Cidea promoter activity more than 30-fold in 293 cells ( Fig. 9 ). A deletion series of Cidea promoter constructs was used to map the SREBP1-responsive regions of the Cidea promoter. Loss of nucleotides -888 to -578 relative to the transcriptional start site signifi cantly blunted the SREBP-1 response. However, the previously defi ned minimal promoter remained responsive to SREBP-1 and was activated 10-fold. This is consistent with the presence of a canonical SREBP-1 response element (SRE) in this region. Congruent with this, ChIP studies demonstrated that endogenous SREBP-1 protein was directly associated with chromatin in the Cidea promoter ( Fig. 9 ). Collectively, these data identify Cidea as a direct target of SREBP-1 signaling.

DISCUSSION
The profi le of proteins that coat LDs plays important roles in regulating LD formation, morphology, and lipolysis. Herein, we characterized the expression of LDP in fl d mice; a unique model of lipodystrophy-related fatty liver that spontaneously and rapidly resolves. A loss of function mutation in the gene encoding lipin 1 causes the conspicuous hepatic steatosis and global metabolic abnormalities of fl d mice ( 20,21 ). The physiologic mechanisms whereby lipin 1 defi ciency causes hepatic steatosis are still unclear, but the molecular functions of lipin 1 provide some possible explanations. Lipin 1 acts in the nucleus to coactivate PPAR ␣ to stimulate expression of genes involved in fatty acid oxidation ( 27 ). Steatotic hepatocytes from fl d mice exhibit diminished rates of fatty acid catabolism ( 39 ) and mitochondrial dysfunction (our unpublished observations), suggesting that insuffi cient capacity for fatty acid oxidation leads to lipid accretion. Interestingly, the microvesicular pattern of LD partitioning observed in fl d mice is consistent with microvesicular steatosis caused by pharmacological inhibitors of fatty acid oxidation ( 40 ) or inborn errors in mitochondrial metabolism ( 41 ). In contrast, obesity-related We also evaluated the nuclear content of SREBP-1, which is known to be activated in steatotic liver and to regulate lipogenic gene expression ( 37 ). Nuclear content of the active SREBP-1 cleavage product was increased in P8 fl d mice ( Fig. 7A ), suggesting that SREBP-1 is activated in fl d mouse liver. Consistent with an activation of SREBP-1, several well-defi ned SREBP-1 target genes [ Scd1, Elovl6,   Fig. 3. Perilipin-2, perilipin-3, and perilipin-5 mark distinct subcellular structures in primary hepatocytes. Images were captured from isolated P14 WT (A, C, E) or fl d (B, D, F) hepatocytes fi xed and then stained with antibodies directed against perilipin-2, perilipin-3, or perilipin-5. Proteins were then visualized using immunofl uorecent microscopy. (reviewed in ref. 36 ). The evidence for a key role for PPAR ␥ in the development of hepatic steatosis and ectopic induction of lipogenic gene expression is extensive. Liverspecifi c inactivation of PPAR ␥ markedly ameliorates fatty liver in several obesity-related fatty liver models ( 46,47 ) whereas PPAR ␥ overexpression drives hepatic steatosis hepatic steatosis is usually characterized by macrovesicular fat droplets. Thus, the microvesicular morphology of the LD may be another indication of severe defects in hepatic fatty acid oxidation and/or mitochondrial dysfunction in fl d liver that result in hepatic steatosis.
On the other hand, the lack of adipose tissue (lipodystrophy) is also likely to play a role in the development of hepatic steatosis in these mice. Most models of lipodystrophy are accompanied by hepatic steatosis (reviewed in ref. 42 ) probably because there is insuffi cient capacity to store TG in adipose tissue, which increases the fl ux of fatty acids to the liver. Lipin 1 is a bi-functional protein that also catalyzes a key step in TG synthesis [phosphatidate phosphohydrolase ( 43 )]. Fld mice completely lack Mg 2+ -dependent phosphatidate phosphohydrolase activity in adipose tissue ( 44 ) leading to a failure of adipocytes to differentiate and store TG ( 45 ). It is our opinion that the hepatic steatosis in fl d mice is a product of both the hepatic oxidative insuffi ciency and lipodystrophy, which synergize to cause severe hepatic steatosis. The explanation for the recovery remains unclear. Because lipodystrophy is a lifelong aspect of the fl d mouse, the resolution suggests compensatory changes in liver metabolism. A developmental and adaptive normalization of oxidative capacity (our unpublished observations) or increased VLDL triglyceride export ( 28 ) could explain the resolution of the neonatal fatty liver. However, additional mouse models are needed to probe this idea further and to understand the sudden, predictable, and apparently geneticallyprogrammed resolution of the fatty liver in this model.
The fl d mouse model is also unique in that PPAR ␥ levels are not increased as has been reported in nearly every model of obesity-or lipodystrophy-related hepatic steatosis  genic gene expression in fl d liver. We found that the nuclear content of SREBP-1 and the expression of multiple known SREBP-1 target genes was induced in liver of P8 fl d mice, which is strong evidence for increased SREBP-1 activity. We show herein that Cidea expression is induced by SREBP-1 via a direct effect of SREBP-1 occupying the Cidea promoter. To our knowledge, Cidea has not previously been identifi ed as a target of SREBP-1 transcriptional control. Based on consensus sequence matching, the original characterization of the Cidea promoter identifi ed two putative canonical SREBP-1 response elements, including a site within the minimal promoter (pL-Cid4) ( 29 ). Indeed the pL-Cid4 construct responds 10-fold to SREBP-1 overexpression and this region would be immunoprecipitated in our ChIP studies. The promoter deletion studies also suggest an SREBP-1-responsive region between -888 and -578. However, our searches have not revealed a canonical SREBP-1 response element within this region. Due to the proximity of the canonical site in the minimal promoter, ChIP studies cannot distinguish whether SREBP-1 binds directly to DNA in the -888/-578 region. Nevertheless, our studies indicate that Cidea is a new and direct target of SREBP-1 signaling and future work will be needed to determine the metabolic consequences of SREBP-1-mediated Cidea activation.
Whereas the Cide genes are regulated at the transcriptional level, PAT protein levels were governed by posttranslational mechanisms. Although Plin5 mRNA expression was unchanged, levels of its corresponding protein were increased several-fold in liver of fl d mice compared with controls. Perilipin-2 was also upregulated at the level of its mRNA but the increase in its protein levels was signifi cantly greater. Degradation of perilipin proteins can occur by proteosomal and lysosomal pathways ( 35,49,50 ). In immortalized cell lines, previous work has shown that perilipin-1 and perilipin-2 protein stability is enhanced by the presence of fatty acids and that the increased stability is due to inhibition of ubiquitin-mediated degradation ( 35,49 ). Herein, we demonstrate that perilipin-2 stability is regulated by fat loading in primary hepatocytes and present data that perilipin-5 stability is ("hepatic adiposis") in normal mice ( 17,18,48 ). It is believed that this transcription factor plays a primary role in the development of fatty liver by promoting the expression of adipogenic gene expression ( 17,48 ). PPAR ␥ is required for the induction of perilipin-2 ( 17,32 ), Cidea , and Fsp27 ( 18 ) expression in ob/ob mice. The lack of increase in PPAR ␥ in fl d mice was unexpected and is challenging to explain. As with other forms of microvesicular steatosis, it is possible that the acute nature of fatty liver in fl d mice may be insuffi cient to cause induction of PPAR ␥ . As a caveat to this, because the transcriptional activity of PPAR ␥ is highly dependent on ligand availability, we cannot say with certainty that PPAR ␥ activity is unaffected in fl d liver. Indeed, the availability of endogenous ligands for PPAR ␥ , which are lipid derivatives, is probably high.
We also evaluated SREBP-1, which is activated in some steatotic liver models ( 37 ), as a potential mediator of lipo-  also subject to regulation by fatty acid availability. This seems to be a key mechanism of regulation and demonstrates the importance of quantifying PAT protein levels, rather than mRNA content, in future studies.

CONCLUSIONS
Our work has shown that the microvesicular hepatic steatosis of fl d mice is accompanied by increased levels of several LDPs. These studies also reveal that the PAT and Cide families of LDPs are controlled at different regulatory levels and that the acute hepatic steatosis in fl d mice is not accompanied by an increased in PPAR ␥ , a putative master regulator of hepatic steatosis. Further study of the mechanisms that regulate LD levels in steatotic liver, in particular the marked and rapid egress of steatosis, is needed to determine whether targeting these pathways might be a viable treatment for fatty liver disease. Fig. 9. SREBP-1 directly activates Cidea gene transcription. Graphs represent mean (± SEM) luciferase activity in relative luciferase units (RLU) corrected for renilla luciferase activity and normalized (=1.0) to the value of empty expression vector-transfected cells. The results of studies using 293 cells cotransfected with a deletion series of Cidea promoter-luciferase reporter constructs and expression vectors driving expression of ca-SREBP-1 or empty vector control. Schematics of the various reporter constructs are shown at left. The location of canonical SREBP-1 response elements is denoted (SRE). TSS, transcriptional start site. * P <0.05 versus the value of empty vector control. ** P <0.05 versus caSREBP-1-stimulated pCID2 and pCID3. The images depict the results of chromatin immunoprecipitation (ChIP) studies using chromatin from hepatocytes isolated from WT mice infected with adenovirus to overexpress caSREBP-1 (abbreviated "S") and/or GFP (abbreviated "G"). Crosslinked proteins were immunoprecipitated with SREBP-1 antibody or IgG control. Input represents 0.2% of the total chromatin used in the IP reactions. Primers specifi c for the Cidea promoter (The general annealing site of primers used is shown in A) or an exon of Acadm (negative control) were used to detect immunoprecipitated DNA.